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Getting Started with

DNASTAR® Lasergene®

For Macintosh® and Windows®
Version 7.2
DNASTAR, Inc. 2007

Contents
Copyright © 2007 by DNASTAR, Inc.

1

Integration

3

Overview ............................................................................................... 3
Synchronous updating ........................................................................... 3
Open and modify a file in two applications............................. 4
Add a feature to a file in two applications............................... 6
Working with files in multiple applications ............................ 8
Saving documents in cross-application formats..................................... 9
Modifying a text format file .................................................... 9
Modifying a multi-sequence file............................................ 10
Modifying a project file......................................................... 10

Online database searches

13

Overview ............................................................................................. 13
Setting up preferences.......................................................................... 13
Specifying online server addresses........................................ 14
Specifying a download folder................................................ 14
Performing a BLAST search................................................................ 15
BLAST search results window .............................................. 16
Performing a text search ...................................................................... 17
Changing a text query............................................................ 19
Text results search window ................................................... 19
Searching text search query results ....................................... 20
Working with search results windows ................................................. 21
Selecting one or more results................................................. 21
Opening results in a Lasergene application ........................... 22
Viewing results in a web browser.......................................... 23
Saving multiple results .......................................................... 24
Printing results....................................................................... 24
Copying results...................................................................... 25
Getting Started with Lasergene

Contents • iii

.................................... 95 Negating weights................................................................ 37 Working with SeqBuilder Cloning Projects ...................Saving results as text files ................. 61 Assembling Trace Data in SeqMan Pro........................................................................................................................... 32 Entering an Entrez sequence............... 56 Viewing & editing restriction site information.......................................................... 62 Assembling 454® Data in SeqMan Pro.................. 99 Opening an Existing Project and Organizing Methods ............................................ 96 GeneQuest 97 Overview ....................................................................................... 55 Create and remove panes and curtains...................................................................................................................... 57 Searching BLAST and text databases........ 35 Displaying manually chosen restriction sites ....................... 98 GeneQuest tutorials ............................ 29 Display restriction enzyme sites .............................. 29 Create a new feature ................................................................................................................................................................ 57 SeqMan Pro 59 Overview ......................................................................... 60 SeqMan tutorials..................... 34 Displaying restriction sites based on frequency .................................. 27 10 Quick-Start Steps for Using SeqBuilder ............................................................. 86 Further exploration ...................... 95 Handling repetitive & contaminant sequences .................. 99 Applying Methods to the Sequence.............. 95 Importing Phrap data ................................................... 96 Searching BLAST and text databases............................................................................... 80 Discovering SNPs.................... 59 10 Quick-Start Steps for Using SeqMan........ 95 Using the vector catalog ...... 28 SeqBuilder tutorials ...................................................................... 26 SeqBuilder 27 Overview . 39 Further exploration ........................................................ 56 Using the seven SeqBuilder views .............................................. 100 iv • Contents Getting Started with Lasergene . 97 10 Quick-Start Steps for Using GeneQuest ................................................................................. 55 Searching the sequence.................................................................................................................................... 96 Exporting data ............................................................................ 74 Designing sequencing primers and improving the coverage ............................................

................................................................................................................................. 108 Checking Predictions against External Data..................................................... 125 MegAlign tutorials.................................................... 122 Using method outlines............................ 116 Calculating methods with different parameters ...................................................... 124 Searching BLAST and text databases........................ 103 Corroborating Predictions by BLAST Search ...................................................................................................................... 129 Performing a Dot Plot pairwise alignment ........ 133 Creating an alignment report ..... 115 Protean tutorials ... 139 Exporting data .............................................. 139 Creating an alignment using a portion of the sequences.............................. 119 Further exploration ............................................................. 126 Entering sequences using drag and drop ................................ 139 Realigning residues ................................................... 139 Working with the Alignment Report ............................... 128 Performing a pairwise alignment................................................................................................................................................................................................................................... 125 10 Quick-Start Steps for Using MegAlign.. 112 Protean 113 Overview ..................... 139 BLAST and Entrez Searching .............................. 127 Setting ends using features ............................Predicting Coding Regions and Creating Features .......... 110 Using the microscope view .............. 118 Predicting proteolytic cleavage sites & performing SDS PAGE simulations ..................... 112 Using method outlines................................................ 109 Further exploration ....... 111 Changing formatting options ..... 115 Correlating annotations with predicted structures ................................................................................................ 110 Additional analyses ..... 122 Additional analyses ............................ 111 Changing method parameters .................. 124 MegAlign 125 Overview .................. 131 Performing a multiple alignment........... 124 Using the microscope view ....................................................................................................... 112 Searching BLAST and text databases.................................................... 113 10 Quick-Start Steps for Using Protean............................ 136 Further exploration ........................................................................................................................................................................................................... 140 Getting Started with Lasergene Contents • v ...................................................................

.......................................................... 153 BLAST and Entrez Searching ..................................................................................................... 164 Further exploration ... 145 Viewing composition & hairpin reports .................................................. 142 Defining primer design criteria......... 165 Changing layout options............ 158 Adding a feature with a translation.................. 160 Searching a BLAST database ...... 153 Selecting a primer pair.................. reversing and translating sequence . 142 Searching for primer pairs ........................... 161 Viewing sequences located by online searches ..... 155 EditSeq tutorials............................ 165 Proofreading a sequence............................................ 153 Creating an oligo request form .....................PrimerSelect 141 Overview .................................................................................................................................................................................................................................................... 166 Searching BLAST and text databases........................................................................ 165 Importing and exporting sequences.............................................................................. 141 10 Quick-Start Steps for using PrimerSelect ............................................................................................ 153 Using only cataloged primers ............................................................................................... 153 EditSeq 155 Overview ....... 159 Copying and translating sequences................. 151 Creating a new primer .................................................. 149 Introducing a primer mutation.............................................. 156 Opening a subset of a sequence............................................. 166 Complementing....................... 156 Locating Open Reading Frames ................................. 152 Further exploration ............ 163 Saving sequences located by online searches ........................... 166 vi • Contents Getting Started with Lasergene .................................... 153 Sorting primers by characteristic......................................................................... 141 PrimerSelect tutorials........................................................................................................ 155 10 Quick-Start Steps for using EditSeq ............. 147 Changing primer length........................................................

All rights reserved. Inc. Lumio.invitrogen. Because some states do not allow the exclusion or limitation of liability for consequential or incidental damages. Inc. expressed or implied. trademarks. Getting Started with Lasergene Copyright © 2007 by DNASTAR. Invitrogen Corporation is the assignee of numerous patents related to aspects of GateWay® and TOPO® cloning technologies. the above limitations may not apply to you. Inc. reserves the right to revise this publication and to make changes to it from time to time without obligation of DNASTAR. adaptation. 377 and 3700 are registered trademarks of Applera Corp. The following products are either registered trademarks. DNASTAR does not warrant. All other copyrights. makes no warranties. Lasergene.. Windows Vista™ is a trademark of Microsoft Corporation. trademarks or copyrighted materials of Invitrogen Corp: TOPO. pCR. KeyAccess®. In no event will DNASTAR. employees. or otherwise. BioEase. including without limitation the implied warranties of merchantability and fitness for a particular purpose. does not encourage the infringement of any patents owned by any other party. SeqMan. Intel® and Pentium® are registered trademarks of the Intel Corporation. Inc. Sixth Edition. to notify any person or organization of such revision or changes. Inc. Inc. GeneMan. KeyServer®. Inc. loss of business information and the like) arising out of the use of. The screen and other illustrations in this publication are meant to be representative of those that appear on your monitor or printer. Protean. reliability. Printed in Madison. up-to-date status. Lasergene99. HisMax. • 1 . business interruption. pGEM is a registered trademark of Promega Corporation. Xpress.B. Inc. officers. MacVector® and GCG® are registered trademarks of Pharmacopeia Inc. SeqBuilder. except as allowed under the copyright laws or with the permission of DNASTAR. GeneBLAzer. Sentinel is a registered trademark® of SafeNet. pDONR. incidental or indirect damages (including damages for loss of business profits. Zeocin. Gus. pENTR.com to obtain more information regarding their requirements in this regard. All rights reserved.Copyright © 2007 by DNASTAR. or translation without prior written permission is prohibited. MegAlign. The license management portion of this Licensed Technology is based on: SentinelLM®. GeneFont. guaranty. ALF is a registered trademark of Pharmacia Biotech A. June 2007.. 454® is a registered trademark of the 454 Life Sciences™ Corporation. Inc. Reproduction. EditSeq. BLOCK-iT. Inc. or the inability to use the software even if DNASTAR. accuracy. DNASTAR. and the Method Curtain are trademarks or registered trademarks of DNASTAR. The A List © 1997-2001 Kyle Hammond. regarding the software. and KeyVerify® are registered trademarks of Sassafras Software Inc. PrimerSelect. MapDraw. ABI Prism 373. or make any representation regarding the use or the results of the use of the software in terms of correctness. Inc. You should contact Invitrogen Corporation at (760) 6037200 in the United States or via their website at www. The exclusion of implied warranties is not permitted by some states.2. DNASTAR. Disclaimer & Liability: DNASTAR. Lasergene 7. GeneQuest. © 1989-2005 SafeNet. Windows® is a registered trademark of Microsoft Corp. SeqMan Pro. CAT. and their directors. Inc. USA Trademark Information: DNASTAR. SeqMan II. The entire risk as to the results and performance of the software is assumed by you. Use of GateWay® and TOPO® vectors and reagents to perform in vitro or in vivo experiments that are simulated using DNASTAR’s Lasergene software may require a license from Invitrogen. DEST. Wisconsin. pcDNA. or agents (collectively DNASTAR) be liable to you for any consequential. and registered trademarks are the property of their respective owners. Macintosh® is a registered trademark of Apple Computers. Inc. The above exclusion may not apply to you. has been advised of the possibility of such damages.

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Protean. • Opening and saving allows you to open a sequence file in any of the applications named above and save that file in a format that is usable by any of the other named applications. PrimerSelect. To use synchronous updating. simply open the exact same file in more than one application. • Synchronous updating allows you to work on the same file in any or all of the five Lasergene applications at the same time. Use synchronous updating with SeqBuilder. and MegAlign. GeneQuest. Getting Started with Lasergene Integration • 3 . Synchronous updating Synchronous updating allows you to work on the same file in any or all of five Lasergene applications at the same time. Changes made in one application will automatically be applied to the same data in the other shared applications. Protean and MegAlign are able to share much of the same data by means of synchronous updating and by opening and saving documents in cross-application formats. • The screenshots in this tutorial are from Windows and Macintosh platforms. Changes made in one application will automatically be applied to the same data in the other shared applications.Integration Overview SeqBuilder. GeneQuest. Edits that would affect the presentation of that sequence in the other application will appear when you bring the focus to the other application. PrimerSelect. The following examples demonstrate some of the functionality that you can access using this integration feature.

If. The remaining steps in this example will edit 2 residues in the Black Mold Calmodulin sequence. Highlight the Black Mold Calmodulin sequence in the list of Sequence Names. without closing the file. Select Calmodulin Alignment.Open and modify a file in two applications In this example. 2. 4 ● Synchronous updating Getting Started with Lasergene . and Protean in a manner similar to the following example. in this case. you could do so using SeqBuilder. The Open dialog appears. GeneQuest. while working on this file. MegAlign and SeqBuilder. you decided that you needed to edit one of the sequences. However.meg from the Demo MegAlign folder. PrimerSelect. Select FILE > Open. you may use any two of SeqBuilder. 3. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo MegAlign 1. • 4. Launch MegAlign. 2. you will see how you can open the same file in two applications. MegAlign.

File in MegAlign showing modifications made in SeqBuilder Note: Only the sequence has been updated.5. Select FILE > Send Sequence To > SeqBuilder. you will save the changes in the original MegAlign document. This may affect the alignment. SeqBuilder opens and displays the Black Mold Calmodulin sequence. • If you save the changes made in either application using FILE > Save As and you rename the file or change the file format. The alignment is not automatically updated. Modified file in SeqBuilder 7. • If you save the changes made in either application using FILE > Save. Note that the change has been made in the file in MegAlign. Modify the sequence by changing the 4th residue from an S to a Q and the 10th residue from a V to an I. Move the focus to MegAlign. running the alignment again is recommended. Therefore after editing. the link between the two applications will be broken and synchronous updating will no longer occur. 6. Getting Started with Lasergene Integration • 5 .

dad From the Demo Data folder. Launch GeneQuest. you will see how adding a feature to a sequence in one application will result in adding the same feature to the sequence in another application. Select FILE > Open to open the project Nematode R01H10 Assay. MegAlign. PrimerSelect. 2. However. and Protean for this example. This example uses GeneQuest and PrimerSelect. Note: Click to view the image as it appears below. 6 ● Synchronous updating Getting Started with Lasergene . GeneQuest.Add a feature to a file in two applications In this example. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo Data Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo Data 1. you may use any two of SeqBuilder.

• In the Title field. 6. The Feature Editor appears. 7. Return to GeneQuest and create a new feature. Note that the new feature appears at the bottom of the list. Click From Feature Table. Select CONDITIONS > Sequence Positions and Limits. • Select SITES & FEATURES> New Feature. Return the focus to PrimerSelect. 4. The Sequence Positions and Limits dialog appears.3. Select FILE > Send Sequence to > PrimerSelect to open the file in PrimerSelect. • Click and then select a range of bases. Getting Started with Lasergene Integration • 7 . Click on to display the Tm plot view. name the new feature Test feature. • Click OK. 5.

See the next section to learn how to save documents in other formats while retaining modifications made to your data. Working with files in multiple applications The previous examples demonstrate how to open the same file in two Lasergene applications simultaneously. • And in PrimerSelect to develop primers for that region. • In MegAlign to perform a sequence alignment. Refer to the next section.dad file. For example. you could open the same document simultaneously: • In SeqBuilder to edit the sequences. You may open the same file in up to four different applications simultaneously. the synchronous updating between GeneQuest and PrimerSelect will be lost. if you were working with an alignment of DNA sequences.Note: If you save this sequence file from either application as anything but the original . 8 ● Synchronous updating Getting Started with Lasergene . • In GeneQuest to further analyze a region of conservation. Saving documents in cross-application formats to learn which menu options you should use to save the data you desire in the most appropriate format.

.pad (Protean) as file extensions. Using Save As Using the Save As option from the FILE menu will save the application-specific data into a project file. formatting. these three programs can continue to use synchronous updating. and SeqBuilder.seq or .seq or . As long as you only use Save. Using Save Using the Save option from the FILE menu will save the sequence information. Modifying a text format file Sequences saved in a text format (like .sbd (SeqBuilder). There are three main situations that may occur.dad (GeneQuest). However. you can no longer use synchronous updating to the original text file because newly-saved project file has copied the information from that sequence file into the new project file along with additional project information. layout. once you use Save As. features. or GeneQuest by opening the newly created project file in the other application(s). Protean.pro). You do not need to close the original Getting Started with Lasergene Integration • 9 .pro files) may be opened simultaneously in GeneQuest.Saving documents in cross-application formats GeneQuest. All application-specific information like methods. and comments back to the original text file (. and so on will not be saved. Protean. MegAlign and PrimerSelect can open project files created by each other. or . as described in Synchronous updating. FILE > Save and FILE > Save As work differently. You can reestablish a synchronous updating link between SeqBuilder. When working with a text file. Protean. SeqBuilder. Project files have .

10 ● Saving documents in cross-application formats Getting Started with Lasergene . when you use either FILE > Save or Save As. PrimerSelect and MegAlign projects contain multiple sequences. if you are currently using PrimerSelect or MegAlign (or both) to modify a project file that is not either a .pcr or . and Protean open one sequence per project. Any of the first three (GeneQuest. These three can save the project file in the format of any other Lasergene application file. File types other than each application’s own project file (. However. The text formatted sequence file will not be written over other than by using an Export menu option. SeqBuilder. Modifying a multi-sequence file PrimerSelect and MegAlign can each have multiple sequences within a single project.meg) should be entered into the application by using the FILE > Enter Sequence(s) option. Thus. The sequence. because Save As has been used in another open application. using FILE > Save will create a project file. that file will no longer be updated when you make changes in the application that you last used Save As.meg file format.pcr or . or Protean) can open and modify a project file from the other applications’ project files. When using these applications. if you start with anything other than the application's project file. features.project file. you will need to open the new project file in any other applications with which you will use synchronous updating as described in Synchronous updating. and comments (if available) will be read into the application. SeqBuilder. Modifying a project file GeneQuest.

Just as when working with a text file, Save and Save As work somewhat differently. For example, if
you have opened a GeneQuest project file in SeqBuilder:

Using FILE > Save in SeqBuilder saves the project in the original GeneQuest document, including
the modified sequence information, GeneQuest methods, and SeqBuilder layout information.

Using FILE > Save As in SeqBuilder saves the project file as a SeqBuilder (.sbd) document, and it
contains the modified sequence information, the GeneQuest methods, and the SeqBuilder layout
and formatting information. However, it breaks the current synchronous updating link between the
two applications.

To re-connect the synchronous updating link between the two applications, you will have to open
the new project file in GeneQuest.

In other words,

Using FILE > Save maintains the connection between the two applications so that synchronous
updating could continue.

Using FILE > Save As creates a new document that breaks the current connection between
applications and prevents synchronous updating until both applications once again have the same
project file opened.

Getting Started with Lasergene

Integration • 11

Online database searches

Overview
Lasergene allows you to search online databases, such as NCBI’s (National Center for Biotechnology
Information) BLAST and Entrez servers, for sequences or proteins that match all or part of the current
file. Perform all online searches from the NET SEARCH menu in any Lasergene application. Some
options vary from application to application, as noted in each section below. Additionally, applications
may be able to use different types of results, or to use similar results in different manners.

Setting up preferences
Before performing an online database search, you must setup Lasergene’s online search preferences.
This may be done in any Lasergene application. Once preferences are setup in one Lasergene
application, they are shared by all other Lasergene applications.
To access online preferences, choose an appropriate option from the list below.
Windows

SeqMan: Select PROJECT > Parameters > Servers or Internet.

All other Lasergene applications: Select EDIT > Preferences.

Macintosh

SeqMan: Select SEQMAN > Preferences > Servers or Internet.

Getting Started with Lasergene

Online database searches • 13

Select the Server tab. • To add a new address to either box. click the address and click Delete.gov/entrez/eutils.nih. repeat this process but choose a different server in step 2. use http://www.dnastar. To specify a default download folder and web browser: 14 ● Setting up preferences Getting Started with Lasergene . • For NCBI’s Entrez server.html or check DNASTAR’s website (http://www. • To delete an address.• All other Lasergene applications: Select [NAME] > Preferences. in SeqBuilder.nlm.dnastar. please consult your system administrator. use http://www. 1. The current addresses appear below the words “BLAST Server” and “Entrez Server.0. 3. Note: To change the default server later. Click Cancel to leave settings as they were prior to making changes. To set the default server that Lasergene will use when performing a BLAST or text search. or check the DNASTAR website. The upper portion of the ensuing dialog allows you to store BLAST Server addresses. This is where Lasergene applications will save files downloaded from an online database. click the associated Add button and type in the address. 2.) These menus appear between the FILE menu and the Apple (Finder) menu on the menu bar. while the lower portion lets you store Entrez Server addresses.com/blast/ncbi-blast. Click OK to accept the changes or Restore to reset the default parameters. Specifying online server addresses Use Preferences (Parameters – Servers in SeqMan) to enter or change your BLAST and Entrez (text) server addresses. but you may use the save process from a Lasergene application to save projects that you have studied or modified using that application. where [NAME] is the menu that shares the same name as the Lasergene application. this menu is called SEQBUILDER. Note: For other servers. click the name of the server in either box and then click Choose. (For example.” respectively. Server URLs • For NCBI’s BLAST 2.ncbi.com) for updated BLAST information. Specifying a download folder Use Preferences (Parameters – Internet in SeqMan) to select a default folder for storing downloaded sequences.

When the server returns results. Databases: Select from the databases available on the server by selecting one from this list box. Select a portion of the current DNA or protein sequence by highlighting it. 2. Note: When using BLAST ORF in GeneQuest. 1. The Blast Query dialog appears. or select New Server and then type a new URL into the Server field. Programs: The following programs are available through this list box. the BLAST search results window appears. A dialog will appear where you may specify the desired folder. Click OK.1. Click OK to accept the change or Restore to reset the default parameter. Click Set Folder. You may continue to work Getting Started with Lasergene Online database searches • 15 . click OK. 3. Performing a BLAST search You can search NCBI’s BLAST databases for a sequence that contains a section that matches the current selected portion of the sequence. When you are satisfied with your choice. the program is automatically selected for you as blastp. You must select a portion of the current sequence before beginning a BLAST search. You may change to a different server by choosing one from the list box. tblastx (searches the six-frame translation of a nucleotide sequence database with the sixframe translation of a nucleotide query sequence). Select the Internet tab. • 3. This dialog contains the following options: • • • • • • • Server: BLAST Servers that have been setup using Preferences appear here. Select NET SEARCH > BLAST Selection (or BLAST ORF in GeneQuest). After initiating a BLAST search. though some servers may not offer all of these programs: blastn (searches a nucleotide sequence database with a nucleotide query sequence) blastp (searches a protein sequence database with an amino acid query sequence) blastx (searches a protein sequence database with the six-frame translation of a nucleotide query sequence) tblastn (searches the six-frame translation of a nucleotide sequence database with a protein query sequence). Click Cancel to leave settings as they were prior to making changes. this window lists any sequences that match the current sequence. 2.

but you cannot launch another BLAST search until you close the current BLAST window.nlm. In general. BLAST results window showing sample results This two-paned window presents the results of a BLAST search.nih.with other Lasergene application activities while waiting for BLAST search results. 16 ● Performing a BLAST search Getting Started with Lasergene . while the lower pane contains the alignment of the query (upper sequence) to the highlighted database entry (lower sequence). the BLAST results window appears. a higher score and lower expectation connote a better match. BLAST search results window Upon completing a BLAST search. The upper pane contains the name of possible matches in order of probability. see Working with search results windows.ncbi. Detailed information about how "score" and "expectation" are derived is available at http://www. For additional information on using the BLAST results window.gov/BLAST.

ncbi. This menu will reflect the available databases on the selected server. 1. • Lasergene's default Internet text server points to the NCBI’s Entrez server. enter a new URL into the New Entrez Server dialog and click Connect. On NCBI’s Entrez database. • Text servers may be accessed via the Internet or. Detailed information about NCBI's Entrez database is available at http://www.gov/entrez/eutils/. Create the search criteria.Performing a text search Lasergene provides a graphical interface for constructing Boolean queries of text databases like NCBI’s Entrez database. 4.nih. You may click Cancel to return to the Entrez Query dialog without creating a new server URL. Use the default server as set in Preferences. if available. Select NET SEARCH > New Text Search. Nucleotide or Genome. • 5. Then. If you have not already done so. or to select another server or • To select another server. select A New Server from the popup menu that appears when you click the button.nlm. Getting Started with Lasergene Online database searches • 17 . an Intranet. 2. • To create a new server. or click to create a new server. the choices are Protein. Search results are returned as a list from which you may select any or all sequences. The Entrez Query dialog appears. Use the pull-down menu on the lower right to choose the database to search. then save them or open them as documents in the current application. specify the database URL using Preferences in any Lasergene application. select it from the popup menu that appears when you click the button. See Setting up preferences for additional information. 3.

7. click – in the row that you want to remove. the text search results window appears. use Search These Results and build another query with one or more new search terms. The criterion row disappears. or And Not from the list box on the left of the new row. 18 ● Performing a text search Getting Started with Lasergene . • And requires both the first and the second criterion to be present. • Or will look for matches containing either criterion. • To add a criterion to the query. The default will search every word of every listing for the query term in the database. choose from the available list of text search options. 6. • And Not will search for matches containing the first criterion but not the second. If you add more terms than can be displayed on the dialog. the dialog scrolls to accommodate the additional terms. Or. Repeat step 5 for each additional criterion that you will add to your query. • Select And. The field headings vary depending upon the online database that you are using. A second row appears for you to enter search criteria. 8. showing a list of sequences that match your query.” To change the search field heading.A sample text search criteria • Enter the first (or only) criterion into the text box on the left. • To remove a criterion. To search within the current set of results in the text search results window. Click Search to send the query to the database. • You may change the search field heading from the pull-down menu to the right of that text box. click +. The default is "All terms from all searches. When the search is complete.

showing a list of sequences that match your query. 2. and/or add new terms using the method described in Performing a text search. In a single pane. Text results search window Upon completing a text search. Getting Started with Lasergene Online database searches • 19 . The Entrez Query dialog appears containing the same search criteria that you last used. You may edit the current search term(s). it lists all of the results that match your search criteria. see Working with search results windows. To do this. Click Search to send the query to the online database. Text results window showing sample results This window presents the results of the text search.Changing a text query You may modify a text query to obtain different results without having to completely recreate a complex query. For additional information on using the text search query results window. You can search the results of a text search to reduce the number of matches using the technique described in Searching text search query results. Select NET SEARCH > Change This Query. a text results window appears. When the search is complete. 1. below. use the following method. 3. the text search results window appears.

3. • You may change the search field heading from the pull-down menu to the right of that text box. click +. • Select And. 2. • And requires both the first and the second criterion to be present. • To remove a criterion. A sample text search criteria • Enter the first (or only) criterion into the text box on the left. 1. The field headings vary depending upon the online database that was used for the original search. If you have several text results windows open. The default is "All terms from all searches. Or. • To add a criterion to the query. • And Not will search for matches containing the first criterion but not the second. 20 ● Performing a text search Getting Started with Lasergene .” To change the search field heading. Enter the search criteria. • Or will look for matches containing either criterion. use NET SEARCH > Current Results to locate the desired window. or And Not from the list box on the left of the new row. click – in the row that you want to remove.Searching text search query results You may search the results of a text query rather than re-searching the entire text database. choose from the available list of text search options. The default will search every word of every listing for the query term in the results. The criterion row disappears. Activate a text search results window. This can help you identify the most useful results of a previously-performed query. Select NET SEARCH > Search These Results to open a text query window. A second row appears for you to enter search criteria.

See Saving multiple results for additional information on using this option. 6. you may then open. • Batch Save: Click this option to save a group of results as sequence files. 5. view. Click Search to use the query to search the current results. Select results from the top pane of the BLAST search results window or from the list of results in a text search results window. See Printing results for additional information on using this option. Repeat steps 2 through 5 to further narrow the results. Once selected. If you add more terms than can be displayed on the dialog. Use this method to select a group of consecutive or non-consecutive results. the text search results window appears. When the search is complete. This method has an advantage over using Ctrl+click as you will not lose selected results if you forget to hold down the button when choosing a result. Getting Started with Lasergene Online database searches • 21 . • Launch browser: Click this option to open one or more results in your default web browser. • Click a result to select only that result. Repeat step 3 for each additional criterion that you will add to your query. Working with search results windows The BLAST and text search results windows permit many similar functions and have the same buttons. Selecting one or more results You can select one or more results from a results window by using any of the following methods.4. save. showing a list of sequences that match your query. See Viewing results in a web browser for additional information on using this option. or print that selection of results. See Opening results in a Lasergene application for additional information on using this option. This section describes features and activities that are common to both results windows. the dialog scrolls to accommodate the additional terms. • Print: Click this option to print the one or more results. • Check each result by clicking to the left of the result in the results window. Common buttons • Open With and Create Document/Add to Project: Use this to open one or more results in the selected or current Lasergene application.

and then press and hold Shift while clicking another result to select those two results and all of the intervening results. • Click a result. Opening results in a Lasergene application You can view results by opening one or more in a Lasergene application. You may also select FILE > Send Sequence To > [Program Name]. 2. The Save dialog appears. (The name of the button is relative to application you are currently using). Use this method to select a group of consecutive or non-consecutive results. 3. Select one or more results using a method described in Selecting one or more results. The Put in Document dialog appears.Checked results in a BLAST results window • Press and hold Ctrl/Cmd (Win/Mac) while clicking each desired result on the results window. • Or • To open the results in the current Lasergene application. select a Lasergene application from the list box above the words Open With. Do one of the following: • To open the results in a different Lasergene application than the current one. click Put in Document/Create Document/Add to Project. Use the following method to open results in a Lasergene application. 1. or choose results in step 3. You may also this option from the NET SEARCH menu. Select an option from the Save list in the Save or Put in Document dialog to select affected results. 22 ● Working with search results windows Getting Started with Lasergene .

• Next opens the specified number of sequences. • Selected opens only highlighted sequences. 2. click Set Location. Specify a number in the text box (default is 10). simply go on to step 5. Lasergene automatically saves and opens only unique sequences. Lasergene starting with the first entry on the list. Specify a number in the text box (default is 10). • To save the files in a new location. Do this using the following method. Click Launch Browser or select NET SEARCH > Launch Browser. Click OK again to save the sequence files and either add them to the current project or open them in a new project file. only the last selected match will appear in the browser window. 1. 5. Each selected match will be displayed in a separate browser window. Consult the documentation for your web browser for additional information. starting with the first one currently highlighted. • Checked opens sequences with a checkmark beside them. you may change the total number of sequences to open by entering a new number in the Sequences field. You can save the files to the default folder set in Preferences or to a new folder. Getting Started with Lasergene Online database searches • 23 .• Top opens the specified number of matches. Lasergene begins with the first highlighted sequence in the list. select where to store the files. starting with the first sequence on the list. If using Next. Note: Certain web browser settings may prevent multiple browser windows from opening. If using Top. In this case. Viewing results in a web browser You can view detailed information about your search results using a web browser. • To use the default folder. 4. • All opens all sequences. Select one or more results by pressing and holding Shift or Ctrl/Cmd (Win/Mac) while clicking several results. Note: If you select Top or Next. and then click OK or Choose.

Specify a number in the text box (default is 10). and then click OK or Choose. If using Next. or choose results in step 3. 2. 4. click Set Location. • Checked opens sequences with a checkmark beside them. If using Top. Specify a number in the text box (default is 10). choose where to store the files. 1. 3. Select one or more results using a method described in Selecting one or more results. • Top opens the specified number of matches. Note: If you select Top or Next. Select an option from the Save list in the Batch Save dialog to select affected results. starting with the first sequence on the list.Saving multiple results You may save multiple search results as sequence files with the following method. 5. • Selected opens only highlighted sequences. Lasergene starting with the first entry on the list. You can save the files to the default folder set in Preferences or to a new folder. Printing results You may print one or more search results from a results window by using the following method. • Next opens the specified number of sequences. starting with the first one currently highlighted. Click Batch Save. • All opens all sequences. Lasergene begins with the first highlighted sequence in the list. you may change the total number of sequences to open by entering a new number in the Sequences field. simply go on to step 5. Lasergene automatically saves and opens only unique sequences. The Batch Save dialog opens. Click OK again to save the results as sequence files in the specified save location. • To use the default folder. • To save the files in a new location. 24 ● Working with search results windows Getting Started with Lasergene .

Lasergene starting with the first entry on the list. Note: If you select Top or Next. • Next opens the specified number of sequences. the information includes identifier information and a description for each selected result. click OK or Print. 1. • In a text search results window. • Checked opens sequences with a checkmark beside them. You may select one of the following options from the Print list to specify which matches to print from a BLAST search results window. Specify a number in the text box (default is 10). Click Print. starting with the first sequence on the list. • Click a result to select only that result. specify items such as the number of copies to print and whether you wish to print a header. In the Print dialog.Note: In a text results window only. 2. Copying results When you copy or save match information and alignments from the upper pane of the BLAST search results window or the text search results window. 4. 3. this button opens a Batch Print dialog. If using Next. Proceed to step 4. Specify a number in the text box (default is 10). • Top opens the specified number of matches. starting with the first one currently highlighted. you may change the total number of sequences to open in the Sequences field. • All opens all BLAST matches. You may select results to copy to the clipboard from this list using one of these methods. Click Print to open the standard Print dialog for your computer’s operating system. • In a BLAST search results window. • Selected opens only highlighted sequences. If using Top. After you have made your selections. Proceed to step 2. this button opens the standard Print dialog for your computer’s operating system. Getting Started with Lasergene Online database searches • 25 . Lasergene begins with the first highlighted sequence in the list. to print out multiple results you will have to select one or more results using a method described in Selecting one or more results before beginning.

Press and hold Ctrl/Cmd (Win/Mac) while clicking each desired result on the results window. Use
this method to select a group of consecutive or non-consecutive results.

Click a result, and then press and hold Shift while clicking another result to select those two results
and all of the intervening results.

You may also copy text from the bottom pane of the window from a BLAST search results
window. In this case, the text you select is exactly what will be copied.

After selecting the result(s), select EDIT > Copy. You may now paste the result(s) into either a
Lasergene or other application.
Note: When pasting data to a word processor, choose a mono-spaced font such as Courier so that the
alignments display correctly.

Saving results as text files
You can save search results as a text file by using the following method.
1.

Make the results window the active window.

2.

Select FILE > Save As.

3.

Find a location to save the document using the Save In menu.

4.

Enter a new name in the File Name window (the default name is "Untitled").

5.

Click Save. Lasergene will automatically add the correct text file extension (*.txt) when saving
the file.

26 ● Working with search results windows

Getting Started with Lasergene

SeqBuilder

Overview
SeqBuilder enables you to edit nucleic acid and amino acid sequences and view the sequences and
related items in multiple ways. You can split SeqBuilder windows into multiple panes and view a
sequence several ways simultaneously:
Sequence view
Feature List view
Comment view
Linear map
Circular map
Minimap
Site Summary
Within a view, you can show or hide many meaningful display elements, including features, ORFs,
translations, restriction sites, and comments. While you work, SeqBuilder can keep the views
synchronized, or you can view different parts of the same sequence with different display elements by
utilizing the unsynchronized viewing option.
SeqBuilder uses dynamic links between the sequence and its annotations to automatically update
feature coordinates as you edit a sequence. Additional functionality includes the ability to reverse
complement, translate and back-translate sequences, identify ORFs and perform BLAST and Entrez
Getting Started with Lasergene

SeqBuilder • 27

text searches directly to NCBI. In addition to restriction cloning, SeqBuilder also supports TA Cloning,
Gateway® Cloning and Directional TOPO® Cloning.
SeqBuilder’s editing capabilities are integrated into most other Lasergene modules. SeqBuilder edits are
shared automatically with other Lasergene applications that analyze the same data, making SeqBuilder
an integral member of the powerful Lasergene Suite.

10 Quick-Start Steps for Using SeqBuilder
Here are ten basic steps for using SeqBuilder:
1.

Create a new SeqBuilder project or open an existing project.

2.

Choose from among the seven views available for DNA sequences or from the three views
available for protein sequences.

3.

Modify the views by resizing panes and curtains, opening multiple views simultaneously,
dividing panes, or closing panes and curtains so that your SeqBuilder workspace is more
efficient.

4.

Select the restriction sites to display or setup selectors to show cut sites for multiple restriction
enzymes using the options in the Enzymes Displayed folder located in the curtain (DNA
Sequences only).

5.

Customize views by turning elements on or off, rearranging elements on the display, or
changing colors and styles of features so that they are easier to differentiate.

6.

Use the graphic results to form a hypothesis about your sequence, and then annotate any
interesting features.

7.

Search the BLAST or Entrez databases for matches to your sequence or other interesting
sequences.

8.

Print your results in any of the available views and save your results

9.

Open SeqBuilder files in another Lasergene application. Edits made in SeqBuilder, Protean,
GeneQuest, PrimerSelect, or MegAlign will be shared universally between those programs.

10. Close this file and open another to begin again, or exit the program.

28 ● 10 Quick-Start Steps for Using SeqBuilder

Getting Started with Lasergene

com). Click Open. 3. as they are designed to walk you through an example SeqBuilder project from start to finish. Launch SeqBuilder. Locate pGEM-T Easy. 1. If you have any difficulties or questions. Select FILE > Open. Getting Started with Lasergene SeqBuilder • 29 . please contact a DNASTAR representative via telephone (608) 258-7420 or e-mail (support@dnastar. The Further Exploration section at the end of the chapter highlights optional features you may wish to use as you gain experience with the software.SeqBuilder tutorials The tutorials in this chapter should be followed in order. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo Data Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo Data Note: You will be working with the same sequence for this tutorial and the next (Display restriction enzyme sites). The sequence appears as follows. 2. Create a new feature Objectives: To open a sequence file and then to create and modify annotated features in a sequence.seq in the Demo Data folder.

By default. 30 ● SeqBuilder tutorials Getting Started with Lasergene . Features are annotations that provide additional information about a particular section of DNA or protein. 5. enter 10. and Circular Map views. In the Position box.. 4. This view displays the base level DNA sequence. ORFs. or they may be part of the input file from GenBank or from other Lasergene applications.• The upper pane of the window contains the Sequence view. this view also shows full translations. Features may be user-defined. • The lower pane of the window contains the Feature List view. The region from base pair 10 to base pair 128 is now highlighted in the Sequence view. this view displays the ruler and protein sequence. Click OK.128. This view displays features that have previously been identified in the DNA or protein sequence. the Linear Map. Select EDIT > Go To Position. and features in this view by using options from the curtain. For protein sequences. You can also display restriction sites. The Go To Position dialog appears.

Select FEATURES > New Feature. select Filled Arrow. The Feature Style panel should appear as below. 9. 7. If you do not see a blue box. The Feature Style panel appears. A feature appears in the Sequence view in the top pane as an orange box and in the Feature List view as a new line entry. Getting Started with Lasergene SeqBuilder • 31 . double-click the new feature to select it. 8. as shown below. A blue box outlining the feature (the orange box) indicates that the feature has been selected. Select FEATURES > Edit Feature Style. uncheck Shadow.6. On the Feature Style panel: from the Shape list.

click to the right of the equal sign next to the word /note on the new feature. you should complete the previous tutorial (Create a new feature). Type MCR for multiple cloning region and press Enter/Return. then click OK. 32 ● SeqBuilder tutorials Getting Started with Lasergene . Proceed to the next tutorial (Display restriction enzyme sites). Display restriction enzyme sites Objective: To display restriction enzyme sites in a sequence. 11. as shown below. Click Apply.10. Note: Before beginning this tutorial. 1. Click in the upper pane (Sequence view) to make it the active pane. this notation will now be the label for the feature in the Sequence view. In the Feature List view.

You can now see the Selectors and Alphabetical List folders under the Enzymes Displayed folder.2. • it. The Sequence view should now appear as follows: 4. Note that both of the EcoR1 sites fall within the feature defined in the previous tutorial. • Next to the Enzymes Displayed folder in the upper pane’s curtain. Uncheck (hide) the box to the left of the Full Translations folder in the upper pane’s curtain. Click • Navigate through the alphabetically named folders to find EcoRI. Locate the EcoRI enzyme by using the following technique. (Mac) or (Win) next to the All Enzymes-Alphabetical folder to expand Check the box next to the EcoRI enzyme to display it. Getting Started with Lasergene SeqBuilder • 33 . as shown below. click (Mac) or (Win) to expand the folder. 3. 5.

skip this tutorial and instead use FILE > Open to open tethis21. 8. Note that the selected enzyme EcoRI remains displayed in the Sequence view. (See Create a new feature for the location of the folder). (Mac) or (Win) next to the Enzymes Displayed folder in the curtain to collapse the Click folder. SeqBuilder will ask if you would like to save the file. Go to FILE>Close. Select FILE > Open Entrez Sequence. The Open Entrez dialog appears.6. Proceed to the next tutorial (Entering an Entrez sequence) Entering an Entrez sequence Objective: To enter a histone DNA sequence from NCBI’s Entrez database. Launch SeqBuilder if it is not already open. 2. 34 ● SeqBuilder tutorials Getting Started with Lasergene .seq from the Demo Data folder. Click No. Note: If your computer is not connected to the internet. 7. 1.

Select ENZYMES > New Selector. Click OK. 1. Getting Started with Lasergene SeqBuilder • 35 .3. The Enzyme Selector Manager appears. Note: Before beginning this tutorial. Under Enter Sequence ID or Locus Name. The sequence appears. you should complete the previous tutorial (Entering an Entrez sequence). 6. Displaying restriction sites based on frequency Objective: To create and apply a frequency selector that displays only enzymes that cut the sequence twice or less. 5. In the list box under that field. enter tethis21ma. A Save dialog appears. select Nucleotide (if it is not already selected). Proceed to the next tutorial (Displaying restriction sites based on frequency). Find a location to store the sequence and click Save. 4.

Check the box next to the Two cuts max selector to view the restriction enzymes that will cut this sequence two times or less. Click (Mac) or (Mac) or (Win) next to the Filter by Selectors folder to expand it. 8. 5. select Frequency. From the Type list box.Alphabetical folders under the Enzymes Displayed folder. 3. Leave all other parameters at their default settings. Overwrite the number 1 in the Max box with the number 2. Proceed to the next tutorial (Displaying manually chosen restriction sites). 4. 10. 36 ● SeqBuilder tutorials Getting Started with Lasergene . You can now see the Filter by Selectors and All Enzymes . 6. In the Name box. (Win) next to the Enzymes Displayed folder to collapse the hierarchy. click (Mac) or (Win) to expand the folder. Click OK to create the new selector and return to the main view. 7.Note: New Selector is highlighted in the Selector list and the Name is New Selector. 2. Locate the new selector in the upper pane’s curtain. Click 9. type Two cuts max. Next to the Enzymes Displayed folder in the upper pane’s curtain.

Double-click the feature to highlight the range defined by the feature in the Minimap view. and those cutting inside the feature’s range appear at the bottom. At the top of the Minimap view. This will sort the enzymes in order of those with cuts closest to the selection. 4. the CDS feature. In the Feature List view (the lower pane).Displaying manually chosen restriction sites Many restriction sites remain. you should complete the previous tutorial (Displaying restriction sites based on frequency). select Minimap. Each row in this view shows where a particular restriction site falls along the sequence. in this case. 1. 3. Note: The restriction sites are sorted so that those cutting closest to and outside the feature appear near the top. Getting Started with Lasergene SeqBuilder • 37 . Objective: To use one of the remaining sites to excise the histone-coding region from the Tethis21 sequence. 2. The information for the feature was imported with the sequence when you opened it. scroll down until you see the CDS feature named histone H2B-1. click on the ruler. Note: Before beginning this tutorial. This is a sequence feature that was annotated in the original GenBank entry. In the upper pane’s curtain. The Minimap is scaled to show the whole sequence in a single line. You can now set some more stringent criteria. those cutting further away from the outside appear further down. Minimap: This view displays a smaller version of the current DNA sequence.

select Pick. scroll to the top of the window. Select ENZYMES > New Selector. In the Type box. Click OK.5. as seen below. In the Remaining Enzymes list. 38 ● SeqBuilder tutorials Getting Started with Lasergene . ApoI now appears in the Selected Enzymes list. Note that the ApoI restriction site cuts just to the left and right of the histone gene. Leaving the feature highlighted. 11. 10. and thus may be used for excising the gene. 12. Click . scroll down to and select ApoI. 6. The Enzyme Selector Manager appears. enter ApoI selector. as shown below. 9. In the Name field. 7. 8. Apply the new selector using the following technique.

Alphabetical folders under the Enzymes Displayed folder. You can now see the Filter by Selectors and All Enzymes . click (Mac) or (Win) to expand the folder. 13.• Next to the Enzymes Displayed folder in the upper pane’s curtain. and can be accessed later either from the curtain or by going to ENZYMES>Selector Manager. SeqBuilder will ask if you would like to save the file. The Minimap view now shows only the ApoI restriction sites. • Click • Uncheck the box next to Two cuts max to unselect it. • Check the box next to the ApoI selector to display it. (Mac) or (Win) next to the Filter by Selectors folder to expand it. Select FILE > Close. SeqBuilder supports these three following commonly used methods: * Gateway® Cloning * Directional TOPO® Cloning * TA Cloning Beginning a New Cloning Project To start a new cloning project. Click No. select FILE > New Cloning Project: Getting Started with Lasergene SeqBuilder • 39 . 14. Working with SeqBuilder Cloning Projects In addition to restriction enzyme cloning. Note: The Enzyme Selectors that you created will be saved.

When you create a project.This New Project window is initially named Untitled Cloning Project. but you can rename it when you select FILE > Save Project As. All cloning projects contain three folders: Inserts. With vectors. it is best to move any custom vectors into the Vector Catalog prior to including them in a project. Here is an example of what the folder looks like after cloning a PCR fragment: 40 ● SeqBuilder tutorials Getting Started with Lasergene . The reason is that all but the restriction cloning projects use the vectors listed in the Vector Catalog. however. Vectors. you can either select inserts before you start cloning or select them during the process. and Clones.

Launch SeqBuilder 2. Hold down the Alt (Windows) or Option (Mac) key and do one of the following: • To create a T-Vector: Click and hold on the CLONING menu. To do this. Highlight the portion of the sequence that you want to use as a vector. if you have custom vectors that you plan to use. Adding custom cloning vectors to a project Objective: To create a custom TA or Directional TOPO vector for use with your cloning project. see the information in the next two tutorials. Or • To create a Directional TOPO vector: Click and hold on the CLONING menu. 5. Getting Started with Lasergene SeqBuilder • 41 . you can either create them during the course of the project or you must import them into the Cloning Vector Catalog before you can use them in a project. then select Create TA Cloning Insert to add the linearized T-vector to the front-most project. Select FILE > Open to display the sequence that you want to create as a new vector. Go to FILE>New Cloning Project. 1. The Untitled Cloning Project window will be displayed.Note: As stated earlier. 3. and then select Create Directional TOPO Insert to add the linearized TOPO vector to the project. 4.

4. Cloning Vectors. and TA Cloning advisors come from both your project and the CloningVectors.sbp is the vectors catalog and is a cloning project. 42 ● SeqBuilder tutorials Getting Started with Lasergene . open that project by selecting FILE > Open. Or • If the vector file is a sequence file. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene 1. change the Files of Type to a SeqBuilder project (. A copy of the sequence file is added to the project file.sbp from the location listed above. To make your custom vector files available to the cloning advisors in all projects. click on one of the folders .sbp catalog.sbp) and select your project. Otherwise.seq to CloningVectors.sbp.Adding vectors to the Cloning Vector catalog The vectors listed in the Gateway® Cloning. Directional TOPO® Cloning. Or • If the vector file is in another project file. Do one of the following: • If the vector file is a sequence file. you will need to add them to this special catalog. select FILE > Import Sequences(s) to Project to navigate to the file you want. and then double-click the sequence to open. 3. Objective: To add a vector to the Cloning Vector catalog. Select FILE > Open to display the Open File dialog. select FILE > Add xxx. Launch SeqBuilder 2. Copy and paste the file from the project to the Cloning Vector catalog. you will only be able to access your vector in the project(s) that contain it. Highlight and double-click the CloningVectors. open the file.

The Untitled Cloning Project window appears. Return to the Untitled Cloning Project window.sbd sequence in the Demo Data folder.sbd insert sequence to open that sequence. Look for the Tn5wPCR. Select EDIT > Copy then close the CloningVectors catalog. Add a sequence to the project by first highlighting the Inserts folder. 9. highlight the Vectors folder and use EDIT > Paste to copy the vector into your project. Select FILE>New Cloning Project. Launch SeqBuilder. and double-click the file to add the file to the Inserts folder.Creating a restriction clone Restriction cloning is a general cloning method that utilizes restriction enzymes to cut insert and vector sequences at specific locations. 2. Objective: To create a virtual restriction clone. Click on Acc651.sbp. 3. Find and select the pcDNA5/FRT/TO/CAT© vector in the Restriction Vectors folder. the standard DNASTAR vector catalog. Once vector and insert sequences are properly prepared. 8. The enzymes and the sequence between them are selected. 6. Then. 4. shift-click on Apal located on the far right of the sequence. 10. Getting Started with Lasergene SeqBuilder • 43 . located at the far left end of the sequence. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene 1. 7. Open the Inserts folder and double click the TN5wPCR. Select FILE > Open and select the CloningVectors. Select FILE > Import Sequence(s) to project to display the Open File window. 5. a ligation reaction is typically used to create a recombinant clone.

located around position 1000. click on Acc651. For this tutorial. The Clone Insert dialog opens displaying the ends of the vector and insert sequence for compatibility. Select CLONING>Clone Restriction Insert. Return to the Untitled Cloning Project window by going to WINDOW>Untitled Cloning Project 1. and the Clone button will be inactive. Double click the pcDNA5/FRT/TO/CAT© vector sequence to open it. 12. Select the restriction enzyme(s) site(s) to clone into. The insert is copied to the clipboard.11. Select CLONING > Copy Restriction Insert. shift-click on Apal located around position 1800. left end and right end checkmarks at the top will be unchecked (Windows only). 15. If ends are not compatible. Then. 13. The enzymes and the sequence between them are selected. 44 ● SeqBuilder tutorials Getting Started with Lasergene . 14.

Modify the ends. When both ends are compatible. and it is stored in the Clones folder of that Project. The new clone is displayed in a new window. enter a name for the new clone in the Save As Copy titled box. 17. Click Clone. by sliding the positional arrows or by clicking Fill in or Trim Blunt. both right and left end checkboxes will be checked (Windows only).16. Getting Started with Lasergene SeqBuilder • 45 . and save to the Untitled Cloning Project listed in the in project box. if needed. When both ends are compatible. 18. and the Clone button will be active.

Choose a location and name the project in the File Name field. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo Data 46 ● SeqBuilder tutorials Getting Started with Lasergene . Creating a TA clone TA Cloning is a cloning method that takes advantage of the terminal transferase activity of some DNA Polymerases that add a 3’dA overhang to each end of a PCR product. Go to FILE>Exit to close your project and exit SeqBuilder. A linearized vector with prepared 3’-T overhangs facilitates ligation of the PCR product. then click Save.19. 20. Objective: To create a virtual TA clone. Save the project and the information in its subfolders by selecting FILE > Save Project As.

Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo Data 1. The Untitled Cloning Project window appears. Select CLONING > Create TA Cloning Insert. 6. Select FILE > Import Sequence(s) to Project to display the Open File window. Look for the Tn5wPCR. Select FILE>New Cloning Project. 4. and the TA Cloning Advisor dialog is displayed. For this tutorial. and double-click the file to add the file to the Inserts folder. Add a sequence to the Inserts folder by first highlighting the folder.sbd insert sequence to open that sequence.sbd sequence in the Demo Data folder. then select the sequence range that has been PCR amplified. 3. 5. click on the neomycin/kanamycin resistance feature to select that range of sequence. the Taq Amplified insert is placed in the Inserts folder. Launch SeqBuilder 2. Open the Inserts folder and double click the TN5wPCR. Getting Started with Lasergene SeqBuilder • 47 .

forward and reverse orientations. then click the Features Displayed check box within the curtain for results. or select a new insert. 8. Verify your selected insert. then select a Linearized T-vector such as pGEM®-T Linearized. are displayed in the Clones folder in the Untitled Cloning Project window. Click Clone. 48 ● SeqBuilder tutorials Getting Started with Lasergene . Note: The T-vector sequence must be linearized at the appropriate base pair and given the T overhangs prior to adding it to the cloning project. 9. Click Circular Map in the View pane.7. The clones. Double click the Untitled TA Clone (forward orientation) in the Untitled Cloning Project window to view the results.

Objective: To create a virtual Gateway clone. This technology eliminates the need for restriction enzyme digestion. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo Data Getting Started with Lasergene SeqBuilder • 49 . Go to FILE>Close. 11. Creating a Gateway® clone Gateway® Cloning is a widely used molecular technique that allows one to transfer DNA inserts between different cloning vectors while maintaining the reading frame and orientation of the insert. 12.Note: Features in the Circular Map view can be moved by double-clicking a feature and then dragging it either up or down. Save the project and the information in its subfolders by selecting FILE>Save Project As. Enter a new name in the File Name field and choose a file location. then click Save. 10. ligation and colony screening for recombinants and is based on the site-specific recombination system of phage I. You may need to move your features to match the picture above.

The Create Gateway® Entry Clone dialog appears. and double-click the file to add the file to the Inserts folder. 3. Open and select the insert sequence of interest. Add a sequence to the Inserts folder by first highlighting the folder. click on the neomycin/kanamycin resistance feature to select that range of sequence. 50 ● SeqBuilder tutorials Getting Started with Lasergene .Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo Data 1. 4. Launch SeqBuilder 2. Select FILE > Import Sequence(s) to Project to display the Open File window. Select CLONING > Create Gateway® Insert/Entry clone. Look for the Tn5wPCR. Select FILE>New Cloning Project. For this tutorial. 5.sbd sequence in the Demo Data folder. 6. The Untitled Cloning Project window appears.

Getting Started with Lasergene SeqBuilder • 51 . The advisor dialog appears. select Untitled Entry Clone and go to EDIT>Rename. 8. 9. Choose your donor vector from pull down list (use PDONR™201). 10. Go to WINDOW>Untitled Cloning Project 1 to return to the project window.7. Go to the Clones folder. then click Create to add an Entry clone to your cloning project. (LR Reaction) Select CLONING > Gateway® Cloning Advisor. Rename the sequence GA clone1. Note: Features in the Circular Map view can be moved by double-clicking a feature and then dragging it either up or down. You may need to move your features to match the picture above.

(use GA clone1). Save the project and the information in its subfolders by selecting FILE>Save Project As. Enter a new name in the File Name field and choose a file location. and destination vector (use pAd/BLOCK-iT™DEST) for recombination. then click Save. 13. 52 ● SeqBuilder tutorials Getting Started with Lasergene .11. Select a suitable entry clone. then click Recombine. 12.

Getting Started with Lasergene SeqBuilder • 53 . and select the sequence range of interest.14. The Untitled Cloning Project window appears. and double-click the file to add the file to the Inserts folder. Objective: To create a TOPO clone. 3. The covalent attachment of DNA topoisomerase I to the vector allows researchers the ability to directionally clone targets with a high degree of specificity. The enzyme functions as both a restriction enzyme and ligase.sbd sequence in the Demo Data folder. which allows researchers the ability to directionally clone DNA targets. click on the neomycin/kanamycin resistance feature to select that range of sequence. Launch SeqBuilder 2. Look for the Tn5wPCR. 5. Select FILE>New Cloning Project. Open the insert sequence. For this tutorial. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo Data Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo Data 1. Add a sequence to the Inserts folder by first highlighting the folder. Creating a Directional TOPO® clone Directional TOPO Cloning utilizes DNA topoisomerase I. Select FILE > Import Sequence(s) to Project to display the Open File window. 4. Go to FILE>Close.

Select CLONING > Create Directional TOPO Insert. Note: The TOPO vector sequence should be linearized at the appropriate base pair prior to adding to the cloning project. The clone is displayed in a new window and it is stored in the Clone folder of the project window. Verify the selected insert.6. 54 ● SeqBuilder tutorials Getting Started with Lasergene . 8. and choose the appropriate cloning vector (use pBAD102/DTOPO® for this tutorial). 7. The new insert is placed in the Inserts folder. Click Circular Map in the View pane then click the Features Displayed checkbox within the curtain. and the Directional TOPO Cloning Advisor dialog is displayed. Click Clone.

Save the project and the information in its subfolders by selecting FILE>Save Project As. as modified by the options selected in a curtain. 9. By default. Further exploration Create and remove panes and curtains The default SeqBuilder document window contains two panes (one above the other). Each pane in turn contains a curtain on the left and a view on the right. Curtains contain options that provide rapid access to the most common elements that you may want to display in a given view. The SeqBuilder document window can be divided into any number of panes. then click Save. the upper pane displays the Sequence view and the lower pane displays the Feature List view for the same file. Enter a new name in the File Name field and choose a file location. You may need to move your features to match the picture above. 10. Create a new pane by clicking and dragging the small box Getting Started with Lasergene SeqBuilder • 55 . Views display data.Note: Features in the Circular Map view can be moved by double-clicking a feature and then dragging it either up or down.

a particular portion of sequence. but you can 56 ● Further exploration Getting Started with Lasergene . this view also shows translations and ORFs. Linear Map: Click to display a linear map of the current DNA sequence. This view shows selected restriction sites. or a specific restriction site. This is where the text of a Genbank entry appears. By default. click Find Next or select EDIT > Find Again. or they may be part of the input file from GenBank or from other Lasergene applications. This view is in the upper pane in the factory default setting. For protein sequences. Remove a pane by double-clicking the divider between two panes or by dragging the divider between any two panes all the way to the divider of another pane. Using the seven SeqBuilder views The Views folder in a curtain permits you to choose from the following views by clicking the icon next to the view name or by clicking on the name. This view displays on one page by default. Features may be user-defined. select EDIT > Find. translations. To find the next instance. Sequence: Click to display the base level DNA sequence. this view displays the ruler and protein sequence. Comment: Click to display a comment that is associated with this DNA or protein sequence. or Sequence view in another pane will likewise display the same highlighting. You may enter any free-form text that you wish in this view. ORFs. and features along the linear map. The options available in a curtain vary with the view selected. This view shows selected restriction enzymes and features. Features are annotations that provide additional information about a particular section of DNA or protein. By default. but you can change the number of pages by going to FILE>Page Layout. the Linear Map. the Circular Map. this view is in the lower pane. Searching the sequence To locate an ORF. by clicking and dragging the small box to the left of a pane’s horizontal scrollbar. Minimap. or by using the Vertical Split or Horizontal Split options from the WINDOW menu. or Sequence views in another pane will likewise display the same highlighting. Circular Map: Click to display a circular map of the current DNA sequence. Comments are optional and not all sequences have them. If a portion of a sequence is selected (highlighted) in one pane. Minimap. The Linear Map view is only available with nucleotide sequences. The Circular Map view is scaled to fit onto one printed page by default.directly above a pane’s vertical scroll bar on the far right. You can also display restriction sites. and features in this view by using options from the curtain. If a portion of a sequence is selected (highlighted) in one pane. Feature List: Click to display features that have previously been identified in the DNA or protein sequence.

the Linear Map. Searching BLAST and text databases SeqBuilder can search BLAST databases for sequences similar to your own.change the number of pages by going to FILE>Page Layout. Viewing & editing restriction site information • To view and edit restriction sites in the Restriction Enzyme Library. The Minimap is scaled to show the whole sequence in a single line. Matches from either search type may be opened in any Lasergene application. or saved. or Sequence view in another pane will likewise display the same highlighting. Minimap: Click to display a smaller version of the current DNA sequence. If a portion of a sequence is selected (highlighted) in one pane. select ENZYMES > Enzyme Manager. The Circular Map view is only available with nucleotide sequences. Additional commands in the NET SEARCH menu allow you to perform a text search of databases such as NCBI’s Entrez database. • To add a new restriction enzyme to the library. Each row in this view shows where a particular restriction site falls along the sequence. Site Summary: Click to display a table of restriction sites and their locations on a DNA sequence. Getting Started with Lasergene SeqBuilder • 57 . The Site Summary view is only available with nucleotide sequences. viewed with the browser. select ENZYMES > New Enzyme. Circular Map. printed. The Minimap view is only available with nucleotide sequences. This view displays restriction sites that are selected in this pane or in another pane.

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If coverage seems unsatisfactory. Getting Started with Lasergene SeqMan Pro • 59 . saving countless hours of analysis and editing. You may also switch between the Pro and Classic assemblers with each SeqMan project. and the Pro assembler. As with all SeqMan parameters. you can add more sequences and reassemble. for projects that utilize data from automated sequencers. or 3) you want to reproduce an assembly made from a previous version of SeqMan. You may also adjust a wide variety of parameters for the assembly process. and allows you to edit and trim constituent and consensus sequences. assembly and consensus calling options you selected. Most importantly. as mentioned above. vector sequence. 2) contains repeated sequences. and identify repetitive sequences so that they will be added last during assembly. and contaminating data from your projects. SeqMan can eliminate poor quality data. SeqMan’s preassembly options let you trim poor-quality data manually or automatically. 2) you do not use vector trimming. In general. you may review tabular reports and graphic views that summarize the results. Prior to assembly. for example. SeqMan provides two different assembly methods: the Classic assembler. After assembly. whether additional coverage may be needed. including which assembly method is used. The Alignment View gives you a more detailed picture of the assembly. 3) has noisy ends. or 4) is being used for SNP analysis. The project begins when you add sequences from any of a wide variety of file types or from NCBI’s BLAST and Entrez databases.SeqMan Pro Overview SeqMan Pro is a sequence assembly tool that lets you assemble anything from two sequences to tens of thousands of sequences into contigs. and then generates the most accurate consensus sequence possible. Once you have selected the options you want. or you can use the Primer Walking feature to drive the closure of gaps or to fill in areas of low coverage. When automated sequencer trace files are available. This system evaluates the quality of the underlying trace data. This will help determine. remove specific vector or contaminant sequences. You may configure SeqMan to apply all of these tools automatically. clicking the Assemble button activates all of the trimming. The Classic assembler should be used when: 1) your data does not contain repeated sequences. you may select one of these assemblers as your default assembly method. we recommend employing the Trace Quality Evaluation system for both trimming and consensus calling. SeqMan includes DNASTAR’s unique Trace Quality Evaluation system. the Pro assembler should be used when your data is: 1) large. Once you have added sequences.

automated 5’ and 3’ end trimming. or split a contig into two or more segments. Use the buttons at the top of the Unassembled Sequences window to prepare sequences for assembly. You may also locate SNPs. We also recommend that you accept the default preassembly option allowing SeqMan to optimize the order in which it assembles sequences. and manual end setting. force contigs to join. 5. Select CONTIG > Strategy View/Scaffold Strategy View to obtain an overview of coverage. 4. 7. When you have selected all the preassembly options you want. 10 Quick-Start Steps for Using SeqMan Here are ten basic steps for using SeqMan: 1. and adjust alignments.override the called consensus. traces and individual consensus conflicts. and then click Scan All. click Assemble. or FILE > Import to open an existing Phred/Phrap assembly project. restore previously trimmed data. Review the PROJECT > Parameters and edit them. Removal of vector sequence(s) is strongly recommended when using Sanger data. conflicts and dual-end data consistency for each contig in your assembly. 2. 3. if desired. Select CONTIG > Alignment View to examine detailed sequence alignments. you may export data or merge contigs with those imported from previous assembly projects. identification of repetitive sequence(s). 60 ● 10 Quick-Start Steps for Using SeqMan Getting Started with Lasergene . If you did not click Scan All in Step 4 (above). vector scanning and contaminant screening. To perform the preassembly processes without assembling the data. You may also view PROJECT > Statistics. Otherwise proceed to step 6. The Project Summary window shows constituent sequences in the bottom pane and contigs in the upper pane. then preassembly and assembly options will run consecutively. Select FILE > New to open the Unassembled Sequences window and an untitled Project Summary window. removal of contaminant sequence(s). or select FILE > Open to open an existing SeqMan project. Once you are satisfied with your assembly. Results will be displayed in both the untitled Project Summary window and in a new Report window. 6. you may wish to save them as a File of Filenames before or after trimming. PROJECT > Trim Reports and CONTIG > Contig Info reports. click Options. Add sequences to the project by selecting SEQUENCE>Add or clicking on the Add Sequences button in the Unassembled Sequences window. If you plan to assemble the same sequences again in the future and do not want to perform preassembly steps over again. Projects may utilize any combination of eligible formats. Preassembly options include the removal of vector sequence(s).

please contact a DNASTAR representative via telephone (608) 258-7420 or e-mail (support@dnastar. then select FILE > Print.com). add new sequences. The previously untitled Project Summary window will now display the name you chose for the assembly project. primer walk. If you have any difficulties or questions. To quit SeqMan and close the current project. separate and recombine projects. make sure the window you want is the active one. adjust the sequence alignment. reassemble data. override the called consensus. SeqMan tutorials This chapter contains four tutorials.8. You may also save various reports as separate files. Getting Started with Lasergene SeqMan Pro • 61 . as each is designed to walk you through a project from start to finish: Assembling Trace Data in SeqMan Pro Assembling 454® Data in SeqMan Pro Designing Sequencing Primers and Improving the Coverage Discovering SNPs The data for each of these tutorials can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo SeqMan The Further Exploration section at the end of the chapter highlights optional features you may wish to use as you gain experience with the software. save consensus. force contigs to join or find locations where they may have been joined erroneously. 10. you may edit conflicts and gaps. select FILE > Exit. FILE > Save the results of your assembly project. 9. restore previously trimmed data. After assembly. The sections in each tutorial should be followed in order. and perform BLAST searches using all or part of a consensus or sequence as the query. search for SNPs. To print the contents of any window.

Each of the fragment sequences is a PE Applied Biosystems. Launch SeqMan. 62 ● SeqMan tutorials Getting Started with Lasergene . Note: The Project Summary window will remain empty until after you assemble your sequences.. you should see a list of sequences with the names Sample 1.. automated sequencer trace file. Inc.Sample 14.Assembling Trace Data in SeqMan Pro Adding multiple sequences to a project Objective: To create a new project and enter sequences for later assembly. Click Add Sequences at the top of the Unassembled Sequences window. Locate and double-click the folder called Demo SeqMan. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo SeqMan 1. 3. shown below) or left of the dialog (Win). The Unassembled Sequences window and an empty Project Summary window should appear. In the pane at the top of the dialog (Mac. 2. The Add to Project/Enter Sequences window (Mac/Win) appears.

click Done. When all fourteen sequence names appear in Chosen Files/Selected Sequences (Mac/Win).4. Add all fourteen fragment sequences to the Chosen Files/Selected Sequences pane (Mac/Win) by using Shift+click to highlight each sequence. 6. then clicking Add File (Mac) or by clicking Add All (Win). 5. Getting Started with Lasergene SeqMan Pro • 63 . The Unassembled Sequences window now contains a list of the fourteen sequences. Expand the window: drag its lower right corner until you can view all the sample names simultaneously.

you can add it to the vector catalog using PROJECT > Vector Catalog. 3. 2. Hint: If the desired vector does not appear on the list. Objective: To remove the Janus vector sequence from the samples and to trim sequence ends based on SeqMan’s trace quality evaluation. Make sure all fourteen of the sequences in the Unassembled Sequences window are highlighted by selecting EDIT > Select All. Specify the vectors to search for by choosing Janus from one of the vector lists and InvJanus from the other.Proceed to the next section (Preparing sequences for assembly). 1. Click Options to open the following dialog. Preparing sequences for assembly Before assembly. 64 ● SeqMan tutorials Getting Started with Lasergene . it is recommended to implement pre-assembly options such as removal of vector and contaminant sequences.

Upside down question marks indicate that the vector was not found in that sequence. then to examine the Trace Data view for one of the trimmed sequences. • The Limits column displays the new sequence endpoints. 4. Getting Started with Lasergene SeqMan Pro • 65 . Each check indicates that the vector was detected in that sequence. perform quality trimming on sequence ends and establish the optimal sequence assembly order. • Note that checkmarks now appear next to all of the Janus vector names in the Vector column and upside down question marks appears next to the InvJanus names. Proceed to the next section (Viewing trace data). A Report window appears. Leave the settings as they are and click Scan All. SeqMan will scan for the vector you selected. Viewing trace data Objectives: To view the Trim Report. reflecting both quality trimming and vector removal. Close it.Hint: Use this dialog to choose whether to trim poor quality and vector sequence and whether to remove reads containing contaminating sequence.

to mask data that should not. Note the good quality of most of the trimmed portion of the sequence. and both trimmed and pre-trimmed lengths. to unmask data that you feel should be included in the assembly. 3. and that beyond base 507. double-click Sample 8 to open its chromatogram in a separate window. amount trimmed. It was trimmed here because the sequence originates from the Janus vector. Select EDIT > Go To Position. by simply dragging the bar at the end of the sequence. its average quality. A Report window appears. followed by the name of each sequence. A vertical black bar appears at the intersection between trimmed and untrimmed sequence at base 45. Close the Trace Data window. SeqMan determined that average peak quality falls below the medium stringency threshold. 66 ● SeqMan tutorials Getting Started with Lasergene . as shown below: Data trimmed from the 5’ end appears dimmed. It is possible to drag this bar left. 1. Proceed to the next section (Assembling & checking coverage). Close the report when you are finished viewing it. 4. Note that the peak quality falls in this region. Select PROJECT > Trim Report to open a report showing current end-trimming parameters. 5. Note that this data can be recovered.1. Assembling & checking coverage Objectives: To assemble the fourteen fragments. or right. 2. type 500 into the Position text box and click OK. From the Unassembled Sequences window. if desired. Click Assemble to assemble the 14 sequences into a single contig. then to use the Strategy View to examine the number of conflicts and degree of coverage for the resulting contig. denoting that it will not be included during sequence assembly.

The color and thickness of the band represent the amount of coverage.2. indicating little or no conflict in most areas. 4. • The thick green band that appears between 200 and 900 denotes a region sequenced on both strands and above the minimum coverage threshold. Enlarge the Strategy View by dragging its lower right corner down to view all of the samples. In the Project Summary window. click on “Contig 1” to select it and select CONTIG > Strategy View to open the following window. Getting Started with Lasergene SeqMan Pro • 67 . Review the data in the Report window and then close that window. • The thin red line appearing at each end of the contig means the region was sequenced only once. 5. (This threshold may be adjusted using PROJECT > Parameters > Strategy Viewing). 3. Several dark blue bands indicate regions where conflicts occur in 10%20% of the bases. you may enlarge the Strategy View by clicking . • The medium blue line to its right indicates a region sequenced in one direction only. Check the Conflicts box at the top left of the window to view a histogram showing agreement between fragment sequences. This histogram appears mostly black. If you can’t see these blue bands. • Note the colorful band summarizing sequencing coverage that appears just below the ruler.

Click on the triangle to the left of the word “Consensus” to compare two consensus calculated using different methods. which appears as a black vertical bar on the left end of the sequence. Restore data to the Sample 9 sequence by dragging the left trim bar. Drag the trim bar to approximately 0. Objectives: To use the Alignment View to restore trimmed ends manually and to compare the results of two consensus calculation methods. indicated in red. with constituent sequences listed in the lower part. SeqMan trimmed ends for the constituent sequences based on trace data quality and presence of vector.Proceed to the next section (Comparing consensus & restoring sequence ends). In the list of constituent sequences. This opens the Alignment View for that region The Alignment View displays the consensus sequence at the top of the window. Any differences between the primary method (Trace) and comparison method (Majority) appear in red. zoom in using until you can see and double-click on the first blue stripe on the left of the Conflicts histogram (near base 50). 68 ● SeqMan tutorials Getting Started with Lasergene . 1. visible as shading in the graphic below. View the underlying trace data for Sample 9 by clicking on the triangle to the left of the sequence name. A line opens under the original consensus. 4. From the Strategy View. You may also wish to restore data in order to verify the consensus in a low-coverage area. Although sufficient data remained to assemble the sequences into a single contig. Note that previously trimmed sequence is easily recognized by its bright yellow background. note that Sample 9 has a number of conflicts. there are cases when restoring some of the trimmed data may allow SeqMan to join multiple contigs into a single contig. Comparing consensus & restoring sequence ends Prior to assembling the contig. 2. 3. and the names of the calculation methods are displayed.

6. Getting Started with Lasergene SeqMan Pro • 69 . That’s because the Trace method judges the peak quality. Open the trace data for Sample 9 again and slide the trim bars toward the central sequence again until the trimmed (highlighted) data are no longer visible. To view conflicts in the newly-restored sequence more easily. Regardless of its deceptively high peak quality. 5. it is not recommended that you restore vector data. In this case. • The red letters appearing in the leftmost portion of the Sample 9 sequence signify conflicts between the restored data and the consensus. the Trace consensus call remains G even though the sample 9 call is C. sequence had originally been trimmed due to inferior peak quality. you may wish to view an example of why the Trace consensus call is often superior to the simple Majority consensus. 7.Note: Restoring trimmed sequence often reveals conflicts between the restored data and the consensus sequence. 8. On the other hand. data removed due to vector contamination would have been characterized by normal peak quality in combination with a high number of conflicts. while the Majority method (which calls S) does not get the correct consensus here. Click on the Project Summary window and select FILE>Close to close the current project. It is sometimes beneficial to restore such data. • Before continuing. At position 46. Click No (Win) or Don’t Save (Mac) when prompted to save the changes. Proceed to the next section (Joining multiple contigs into a single contig). close the trace data by once again clicking on the dark triangle to the left of Sample 9.

Joining multiple contigs into a single contig Objective: To join multiple contigs into a single contig. Proceed to the next section (Assembling dual-end data). 12. and select FILE > Close. respectively. 7. The Trim Ends dialog appears. Assembling dual-end data A dual-end sequence pair is a pair of reads that are known to be related with respect to orientation and distance. Enter 200 and 500 into the 5’ End and 3’ End boxes. 5. In the Project Summary window. highlight both contigs by holding down the Shift key while clicking them. Remove the checkmark next to “Don’t add single sequence contigs. 10. With both contigs highlighted in the Project Summary window. A dialog requests confirmation. Click Scan Later. 3. 13. 4. 1. SeqMan assumes the pair will be from opposite ends of the same DNA fragment. Click Options. 14. 11. 9. Click Scan Later. 6. Select Fixed End Points. select CONTIG > Align Contigs to combine the two contigs into a single contig. Select CONTIG > Extend Contig Ends. Two contigs are produced.” 8. and 70 ● SeqMan tutorials Getting Started with Lasergene . Click Trim Ends. Click on the Project Summary window. Discard any changes made to the project. Click Extend. 2. Click Assemble. Repeat all of the steps in Adding multiple sequences to a project to enter the fourteen sequences as in the Demo SeqMan folder to the project.

zip from the Demo SeqMan folder. Select SEQUENCE > Add and select the file Dual-End ABI Files. Click Add File (Mac) or Add All (Win). 4. After assembly is finished. Click Assemble.sequenced from the end of the fragment inwards. 3. 7. Make sure that all of the sequences in the Unassembled Sequences window are highlighted by selecting EDIT > Select All. 5. as shown below: Getting Started with Lasergene SeqMan Pro • 71 . 2. highlight Unlocated Contigs and select CONTIG > Scaffold Strategy View. Create a new project using FILE > New. Select Janus vector from one of the Set Vector pull-down menus. Objective: To assemble sample data using dual-end sequence information. (See the beginning of this chapter for the folder location). 8. Note that each sequence is represented with a single black arrow. Click Done. go to the Untitled Project Summary window. 6. 1. The sequence naming convention is codified in the PROJECT > Parameters > Pair Specifier dialog.

Click on the Project Summary window and select FILE > Close to close the current project. 1. From the Project Summary window. 72 ● SeqMan tutorials Getting Started with Lasergene . Click the Project Summary window and select FILE > Close to exit from the project. The colorful appearance of this Strategy View signifies that its dual-end specifier parameters have enabled SeqMan to utilize the data more effectively. highlight Scaffold 1 and select CONTIG > Scaffold Strategy View. Select FILE > Open to open Practice.sqd. 12.sqd. The next section (Using dual-end specifier parameters) will show you how to use these same dual-end specifier parameters in the Practice. Select FILE > Open and open Dual-End Sample. 13. 10.sqd project.9. 11. Select FILE > Save to save the current project under the name Practice. enabling SeqMan to use data more effectively. (See the beginning of this chapter for the folder location). This is a demo project showing how the Scaffold Strategy View should look when dual-end data are properly handled.sqd from Demo SeqMan/Dual End Demo folder. Using dual-end specifier parameters Objective: To use dual-end specifier parameters.

abi <> sample_r. 8. Select PROJECT > Parameters to open the Parameters dialog. Select Pair Specifier from the list at left on the dialog.sqd. click Unlocated Contigs and select CONTIG > Scaffold Strategy View. Getting Started with Lasergene SeqMan Pro • 73 . and light blue shows reads that would be consistent if contigs were re-ordered or complemented. respectively. SeqMan requests confirmation of this decision. • Green denotes consistent pairs within the same contig. Select sample_f.abi from the drop down list at the top of the dialog.abi in the “Forward” and “Reverse” boxes. 4. 3. but now its arrows appear in green. • Practice.sqd isn’t identical to Dual End Example. 7. Click OK to apply these parameters to the project and close the Parameters dialog.abi and 01r. Type in 01f. Close the Scaffold Strategy View and select PROJECT > Order Contigs. black means that no pair was found. From the Project Summary window. black and light blue. Your parameters dialog should look like this: 6. 5.2.

9. The Unassembled Sequences window and an Untitled Project Summary window will appear. • The Scaffold Strategy View for Practice. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan\SFF Demo Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan\SFF Demo Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo SeqMan\SFF Demo 1. When prompted to save the document. 11. 10. Close all open SeqMan windows using FILE > Close.sff. 3. click Scaffold 1 and select CONTIG > Scaffold Strategy View. The 1. Click the Add Sequences button located at the top of the Unassembled Sequences window.sff file will be added and displayed in the Unassembled Sequences window. Click Add File (Mac) or Add (Win) and then click Done. 74 ● SeqMan tutorials Getting Started with Lasergene .154 sequences from the .sff files from 454® using the Pro Assembler.sqd. • You can use this information to get a better determination of what the final consensus should be. distance and naming) but are in two different contigs. From the Project Summary window. Launch SeqMan. The light blue arrows have changed to dark blue indicating that the pairs are consistent with the parameters (i. Confirm by clicking Order. 2. The Enter Sequences/Add to Project window (Win/Mac) is displayed.. Open the SFF Demo folder and select ecolireads. 4. click No (Win) or Don’t Save (Mac). given additional data to help fill in the gaps between the contigs and create deeper coverage.sqd is now identical to that of Dual End Sample.e. Please proceed to the next tutorial (Assembling 454® Data in SeqMan Pro). Assembling 454® Data in SeqMan Pro Objective: To assemble .

5. This value represents the depth of coverage expected in this assembly. This setting is typically recommended for 454® data. choose Strategy Viewing from the list on the left.sff file contains some short sequences. 6. Your SeqMan Parameters window should appear as shown on the following page: 9. Go to Project>Parameters. 7. Select Assembling from the list on the left. and make sure that “Use Pro Assembler” is selected as the Assembly Method. Next. Since our . The SeqMan Parameters window is displayed. 8. as it allows each entire read to be used when computing pairwise similarity. we need to change the Minimum Sequence Length parameter so that the shorter sequences will be included in the assembly. Change the Maximum Expected Coverage value to 40. Change the Minimum Sequence Length value to 40. Also change the Maximum Mismatch End Bases to 0. Getting Started with Lasergene SeqMan Pro • 75 .

148 of the 1. information about sequences not added is found at the beginning and the end of the report. look at the Report window that is open on your screen. 10. To allow these 6 sequences to be assembled into the existing contig. Notice that 1. Typically. Next. red areas in the Coverage bar found in the Strategy View. 14.Once the sequences are assembled. but notice at the bottom of the report.154 reads are assembled into one contig. Reduce the Match Size to 15 and the Minimum Match Percentage to 55. 11. the following notification is given: “Single sequence contigs were not added to your assembly. areas exceeding this maximum value will be indicated by thick. 12. so that your parameters appear as shown below: 76 ● SeqMan tutorials Getting Started with Lasergene .” This lets us know that the 6 sequences not included in the assembly were likely all single sequence contigs. Click OK to close the SeqMan Parameters dialog and return to the Unassembled Sequences window. we will need to relax our assembling parameters. Go to Sequence>Add to view the remaining 6 sequences. To find out why some sequences were not added. there is no information at the beginning. Go back to Project>Parameters and select Assembling from the list on the left. 13. as shown in the Untitled Project Summary window. 15. In this case. Click the Assemble button.

Getting Started with Lasergene SeqMan Pro • 77 . 18. Double-click on Contig 2 to view your assembly in the Alignment View. Click the Assemble button. The remaining 6 sequences are added to the existing contig.16. Click OK to return to the Unassembled Sequences window. as shown in the Untitled Project Summary window. 17.

Grayed out sequence may appear. 78 ● SeqMan tutorials Getting Started with Lasergene . Scroll to either end of the sequence you selected in the flowgram window. The Quality scores for each called base will be displayed. Click and hold the button located in the top left corner of the flowgram window and select from the list provided. the quality score for each consecutive base will be listed in order of orientation from bottom to top. Click anywhere within a sequence to select that sequence. For bars that represent more than one base. 21.19. and then go to Sequence>Show Original Trace/Flowgram Data. The flowgram for the read you selected is displayed. 20. representing trimmed sequence.

Then. You may grab the trim bar located on either end of your sequence and drag it to the left or right to either restore previously trimmed sequence. These sections of data cannot be restored. These red sections indicate areas of your assembly that exceed the Maximum Expected Coverage value that we defined as 40 prior to assembly.22. Click the button several times to zoom-in on the Strategy View until you can clearly see the thick.sff files may already contain trimmed portions of sequence. 23. Getting Started with Lasergene SeqMan Pro • 79 . go to Contig>Strategy View to view a graphical summary of each constituent sequence in your contig. 24. red areas in the Coverage bar around position 5500. representing removed adapter sequence. Close the flowgram window. Note: Some . or to manually trim the ends.

If the coverage appears too deep. From the Alignment View. Designing sequencing primers Objective: To design sequencing primers that extend upstream and downstream from an existing contig that “walks” into a region lacking in trace data. In this example. you can visually assess the coverage for the indicated area. the coverage is only slightly above the threshold. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan 80 ● SeqMan tutorials Getting Started with Lasergene . Close all open SeqMan windows using FILE > Close. Double-click on one of the red bars to launch the Alignment View for that area of the assembly. you may want to turn on the Use Repeat Handling Assembling parameter (located under Project>Parameters) and then reassemble your project. click No (Win) or Don’t Save (Mac).25. When prompted to save the document. Designing sequencing primers and improving the coverage The Primer Walking feature in SeqMan identifies weak coverage and gaps and helps you design sequencing primers. 26. so we do not need to take further action. Please proceed to the next tutorial (Designing sequencing primers and improving the coverage).

abi by holding Control while clicking them and click Add. 6. 2. When the assembly is complete. the Report window appears along with the Untitled window listing the contig. if it is not already launched.abi. the length. with a ruler at the top. Click Assemble to create a contig. Do one of the following: • Windows: Select Sample 1. number of sequences and position.abi and Sample 6. • Macintosh: Double-click on Sample 1.Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo SeqMan 1. select CONTIG > Strategy View • The coverage window appears. 3. Getting Started with Lasergene SeqMan Pro • 81 . Close the Report window. Select SEQUENCE > Add. 9. To display the Strategy View. 4. click Done. a summary of conflicts (if checked). 5. and a list of constituent sequences at the bottom. When both sequence names appear in the Selected Sequences pane (Win) or Chosen files pane (Mac).abi and Sample 6. a coverage graphic just below the ruler. Locate and open the Demo SeqMan folder. 8. 7. Launch SeqMan. Click on “Contig 1” to select it.

From this window. Improving the coverage Objective: To use SeqMan to improve coverage by either extending contig ends into unsequenced areas (primer walking) or by filling in areas that need further confirmation. beneath the conflicts summary. The overlaps are green. 82 ● SeqMan tutorials Getting Started with Lasergene . Note: The two trace files that make up this contig are from upper and lower strands and overlap at all but the ends of the contig.• If dual-end sequence data are present and correctly specified. Proceed to the next tutorial. 1. Select CONTIG > Primer Walk… to view the Primer Walking setup parameters. • Click the bottom right corner of the Strategy View window and drag it to create a larger viewing window. • Use the zoom in tool to increase scale of strategy view. then a graphical summary of pairs information will also be present. you can do the following to change the look of the window.

Getting Started with Lasergene SeqMan Pro • 83 . Click OK to locate sequencing primers. Click the Require Clone Coverage checkbox to eliminate primers selected in areas that have no sequence coverage on the same strand as the primer. The Primer Walking Report appears. 4. Click the Improve Coverage radio button to select a search for coverage primers.2. 3.

The Strategy view window shows a thin red horizontal line around base pair 100 corresponding to the bottom strand sequencing primer. Note: The report is connected to the Alignment View window. 84 ● SeqMan tutorials Getting Started with Lasergene . 6. Double-click the top-scoring primer from the report to open the Alignment view. Select CONTIG > Strategy View .5.

Click Save. Double click on the primer shown around base pair 100 to open the Alignment view displaying contig 1. Select FILE > Save Primer Info. Click on the Primer Walking Report to bring it into view. Change the Save As Type (Win) or Format (Mac) pull down menu to Tab-Separated. 1. Choose a location and file name. Getting Started with Lasergene SeqMan Pro • 85 . 3. Saving the Primer Walking Report Objective: To save the Primer Walking report as tab delimited text. 4. 2.7.

Click Don’t Save (Mac) or No (Win) when prompted to save changes. 86 ● SeqMan tutorials Getting Started with Lasergene . When all sequence names appear in the Selected Sequences pane (Win) or the folder appears in the Chosen Files pane (Mac). Select SEQUENCE > Add in the Unassembled Sequences window.seq. 3. The sequence is selected as the reference sequence and its name is shown in italics. 2. 6.5. 6. Please proceed to the next tutorial (Discovering SNPs). Launch SeqMan. Locate and open the Demo SeqMan folder. 5. Discovering SNPs Aligning sample sequences Objective: To align sample sequences to a reference sequence. and click Mark Ref. 4. click Done. • Macintosh: Select SNP Demo and click Add Folder. select the sequence named Reference. Click on the Project Summary window and select FILE>Close to close the current project. In the Unassembled Sequences window. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo SeqMan Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo SeqMan 1. Do one of the following: • Windows: Double-click on the folder SNP Demo and then click Add All.

abi <> sample_r. In the Verify Pattern section.scf in the Forward field and Sample1_r.scf in the Reverse field. type in Sample1_f. In SNP discovery.Note: A reference sequence is a known sequence used for comparison with your sample sequences. Select PROJECT > Parameters > Pair Specifier. Select sample_f. SeqMan will determine where your sample sequences differ from the reference sequence. 8. Getting Started with Lasergene SeqMan Pro • 87 . The Success message indicates that the example names match the pair pattern you selected.abi from the Pair Pattern drop-down menu. 9. 7. The Pair Specifier dialog appears.

From the top pane of the Untitled window. 11. Proceed to the next tutorial. 88 ● SeqMan tutorials Getting Started with Lasergene . Alignment view. then select CONTIG > Strategy View to display an overview of the contig. Click Assemble. The sample sequences align into a single contig with the reference sequence. Click Zoom In twice and drag the lower right corner of the window to enlarge the view. 1.10. it appears at the top of the view with its name in italics. Select SNP > Show SNPs to change the display of the Strategy View as follows. 2. click Contig 1. Click OK to save the changes and close the SeqMan Parameters dialog. Reviewing SNPs Objective: To review SNPs using the Strategy view. and SNP Report. Note: After assembly. whenever the reference sequence is displayed. and close the Report window.

When you look at this view. • Locations of bases in the contig that correspond to putative SNP base locations in the aligned sample sequences are indicated with blue hash marks on the contig goalpost. Select CONTIG > Alignment View. 3. so that it looks like the following window. • Locations of bases in sample sequences identified as putative SNPs by SeqMan are indicated with blue hash marks on the sequence arrows.When you look at this view.seq file are indicated with green hash marks on the reference sequence arrow. notice the following: Getting Started with Lasergene SeqMan Pro • 89 . A light arrow overlapping a sample sequence arrow represents the sequence that is paired with the sample. • Locations of bases annotated as features of type “variation” in the Reference. notice the following: • Bold arrows represent sample sequences. and drag the lower right corner of the window to enlarge the view.

The ‘T’ in position 58 is an example.4. select a row and then press the up and down arrow keys to select other rows. notice that the corresponding SNPs are selected in the Alignment View. 6. • Bases in sample sequences identified as putative SNPs by SeqMan are displayed in blue throughout the view. information in the SNP Report window can be copied and pasted into other documents such as MS Excel spreadsheets. • A row in the All Found SNPs report lists information for a single SNP base in a sample sequence. The SNP report can also be saved in tab-separated file format by selecting FILE > Save SNP ‘Contig 1’ Info…. • A base in a sample sequence that SeqMan has determined is a heterozygous putative SNP is displayed using the appropriate ambiguity code. Select SNP > Sort by SNP. See the ‘y’ and ‘c’ bases in position 58 as examples. Note: For further analysis. 90 ● SeqMan tutorials Getting Started with Lasergene . allowing you to quickly scan through the SNP bases. • A row in the SNPs Summary report summarizes information for all of the SNP bases in an aligned column. When you look at the window. Select SNP > SNP Report. notice the following: • Tabs on the SNP Report allow you to change the display between two different SNP reports: All Found SNPs and SNPs Summary. 5. • Bases annotated as features of type “variation” in the reference sequence file are displayed in green. As you do so. In either the All Found SNPs or the SNPs Summary report.

Sample sequences with SNP bases in the aligned column are sorted to the top and samples with non-SNP bases are sorted to the bottom. sample sequences in the Alignment View are reordered automatically according to the SNP bases in the selected column. Note: A sample sequence in the Alignment View can be manually reordered by clicking on its name and dragging it up or down. Selecting CONTIG > Sort > by Offset or CONTIG > Sort > by Name automatically reorders sample sequences by offset or name. Getting Started with Lasergene SeqMan Pro • 91 . Proceed to the next tutorial.• Because you selected SNP > Sort by SNPs.

Double click on the sequence name in the row. any base that exhibits a secondary trace peak that is at least 80% of the intensity of the primary peak will be identified as heterozygous. The SNP column entry in the report changes to an x to indicate that you have rejected that the base is an actual SNP. 3.Analyzing putative SNP bases Objective: To analyze putative bases and determine if you want to confirm or reject them as actual SNPs.scf at contig position 37. The question marks in the column indicate that each base listed is a putative SNP. The default is 80% – at that setting. Select SNP > Reject SNP. Select PROJECT > Parameters > SNP Discovery to open the dialog for setting the threshold for discovering heterozygous SNP bases. 6. 5. which is the default color for a rejected SNP base.scf sequence name to reveal the underlying trace data. Locate the row for the putative SNP in Sample1_f. click on the All Found SNPs tab and look at the first column. 8. 7. Examine the trace corresponding to the putative SNP base to determine that the base is not an actual SNP.scf selected. The color of the SNP base changes to red in the Alignment View. You can see that the A trace peak preceding the putative SNP base has slipped and is overlapping the T trace peak. which is labeled SNP. In the SNP Report window. The Alignment View displays with the putative SNP base at contig position 37 in sequence Sample1_f. 4. Change the threshold to 50% to lower the stringency and find more putative SNPs. 92 ● SeqMan tutorials Getting Started with Lasergene . 1. Click on the triangle to the left of the Sample1_f. 9. Click OK to save the change and close the SeqMan Parameters dialog. 2.

The x indicates that the base in that row is not an actual SNP.scf at contig position 37 reveals that this base is also not an actual SNP. click the tab that selects the SNPs Summary report. by using Shift+click or a similar method. Getting Started with Lasergene SeqMan Pro • 93 . click once on the question mark in the SNP column to change the symbol to a checkmark. select the two rows with an x in the SNP column that correspond to contig position 37. 11. 12. and click the checkmark a second time to change it to an x. In the corresponding row in the All Found SNPs report. In the SNP Report window. Select EDIT > Clear to remove the SNPs from the report.10. In the All Found SNPs report. Examining the trace data for the base in Sample3_f.

13. Look at the SNP column in the SNPs Summary report. The symbol in the column indicates that
the SNP bases in the column are all putative (question mark), all confirmed (checkmark), all
rejected (x), or a combination of the three (–).
14. Locate the row in the SNP Summary report for contig position 58.
15. Double click on the contig position in the row. The Alignment View is displayed with the
column at position 58 selected.
16. Select SEQUENCE > Show Original Trace/Flowgram Data. As shown next, one window of
trace data is opened for each of the sample sequences in the aligned column.

17. Examine the trace data for the bases aligned in that position to find that the putative SNP bases
in that column can be confirmed as actual SNPs.
18. In the SNPs Summary report, click on the row for contig position 58 to highlight it, then select
SNP>Confirm SNP. The mark changes to a checkmark to indicate that you are confirming
that the putative SNP bases in the column are actual SNPs. In the Alignment View, the color of
the SNP bases in the column changes to green, which is the default color for confirmed SNP
bases.
19. Click on the Project Summary window and select FILE>Close. When prompted to save the
document, click Don’t Save (Mac) or No (Win).
Note: The default colors for display of putative, confirmed, and rejected SNP bases, as well as non-SNP
bases, can be changed in the dialog opened by selecting PROJECT > Parameters > SNP Discovery.
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Further exploration
Importing Phrap data
Phrap assembly documents (*.ace) may be opened in SeqMan using FILE > Import.
The Alignment View for an imported Phrap project displays Phred base calls and Phrap consensus
calls. By default, the top consensus (next to the word Translate) is the Imported Phrap consensus. Just
below this is the SeqMan Trace or Majority consensus. These consensus only show differences from
the Phrap consensus based on the original Phrap assembly.

Using the vector catalog
SeqMan contains a catalog for a number of common vectors. To add, delete, or modify entries in the
catalog, select PROJECT > Vector Catalog.

Handling repetitive & contaminant sequences
To remove contaminant sequences, use the following procedure.
1.

Store sequence files of known contaminants in the Contaminant Seqs folder located:

Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\
Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7
Data
Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene
2.

Prior to sequence assembly, select PROJECT > Contaminant Seqs and select which to
screen for from the list of available files.

3.

From the Unassembled Sequences window, click Options and check Remove Contaminant
Sequences.

To add known repetitive sequences last to an assembly, use the following method.
1.

Store sequence files of known repetitive sequences in the Repetitive Seqs folder located:

Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\

Getting Started with Lasergene

SeqMan Pro • 95

Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7
Data
Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene
2.

Prior to sequence assembly, select PROJECT > Repetitive Sequences and select which files
you wish to screen for in the current project.

3.

From the Unassembled Sequences window, click Options and check Optimize Sequence
Assembly Order.

Note: Optimizing assembly order is a parameter under the Classic assembler only, and is recommended
for most assemblies using the Classic assembler whether or not repetitive sequences are specified.

Negating weights
To cancel the internal weight for a selected portion of a sequence so that it does not contribute to the
consensus, highlight the region of sequence in the Alignment View and select EDIT > Negate
Weights. This command is particularly useful when building the assembly around a “backbone”
sequence from a closely related species. Use EDIT > Restore Weights to restore.

Searching BLAST and text databases
In SeqMan, NET SEARCH menu commands may be used to search for similar sequences in the
BLAST database. Additional commands in the NET SEARCH menu allow you to perform a text
search of databases such as NCBI’s Entrez database. Matches from either search type may be opened in
any Lasergene application, viewed with the browser, printed, or saved. See Performing a text search for
an example of a text search. If you already know the locus name or accession number of a sequence in
NCBI’s Entrez database, you may also open it directly using SEQUENCE>Add Entrez Sequence.

Exporting data

To save an assembly project, select FILE > Save.

To export all the constituent sequences for one or more contigs, select CONTIG > Export
Sequences.

To export the consensus sequence for one or more contigs, select CONTIG > Save Consensus.

To export assembly projects to Phrap or DOS SeqMan formats, select FILE > Export PROJECT.

96 ● Further exploration

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Protein Finder—Identifies regions whose translation matches a user-specified protein sequence file. • Patterns .Signal—Uses a text-based pattern database of known DNA transcription factor sites.Matrix—Uses files containing log odds or positional frequency matrix patterns.Dyad Repeats—Locates dyad repeats and palindromes. The next step is to apply some of GeneQuest’s many analysis methods to the sequence. The display combines results for all six reading frames. These methods include: • Title—Adds a title to the document. The project begins when you enter a DNA sequence.Type-In Pattern—Uses patterns that have been typed in using the keyboard.DNA Finder—Identifies regions that match those of a user-specified DNA sequence file. • Patterns . • Ruler—Adds a ruler to the document. • Gene Finding . • Sequence—Displays the sequence on the document.Inverted Repeats—Locates inverted repeats. • Repeats . • Patterns . • Repeats . Getting Started with Lasergene GeneQuest • 97 .GeneQuest Overview GeneQuest aids you in identifying and annotating coding regions and other features of interest in your DNA sequence. • Repeats . Displays results separately for the top and bottom strands. • Gene Finding .Direct Repeats—Locates direct repeats.

In addition. Create a new project or open an existing GeneQuest project. GeneQuest also reads and interprets features from the comment or feature panes of GenBank-formatted sequence files. the results are displayed graphically on a common horizontal scale. 3. menu commands let you display tabular data summaries (e. if they are not already there. It also plots the local strand skew of the complements AT and GC. 4.Starts Stops ORFs—Locates and summarizes open reading frames longer than a user-specified minimum length.. • Bent DNA . 98 ● 10 Quick-Start Steps for Using GeneQuest Getting Started with Lasergene . Starts and stops are displayed independently for all frames. or view the predicted electrophoretic separation of restriction fragments through an agarose gel. Apply methods to the sequence by using the mouse to drag them from the Method Curtain to the assay surface.Borodovsky—Uses Borodovsky’s Markov method to identify potential coding regions.• Enzymes .Local Compositional Complexity—Uses the Shannon information theory to find regions rich with information. 2. you may create your own annotations and link several features together under a common title and description. • Coding Prediction . • Base Contents . You can designate whether or not a start codon is required.Bending Index—Predicts the bending of free DNA by showing localized angles of helix trajectory computed for a specified window size. • Coding Prediction . codon usage). 10 Quick-Start Steps for Using GeneQuest Here are ten basic steps for using GeneQuest: 1. and choose which of these you would like to apply to the sequence. fold a portion of the sequence as RNA. Move the methods you want from the More Methods menu to the Method Curtain. Select ANALYSIS > Show Available Methods to display a list of methods.g.Base Distribution—Plots the local frequency of the four bases and A+T and G+C pairings. • Coding Prediction . You may wish to follow the formal strategy for locating potential genes outlined later in this chapter. Once you have discovered areas of interest in the sequence.Restriction Map—Creates restriction maps using any of the enzymes from the DNASTAR enzymes catalog. View the results in graphical or tabular form. base content. Once the methods have been applied.

rearrange and “decorate” methods until you are satisfied with the look of the document. Then add. 10.com). or other sequences of interest. delete. If you have any difficulties or questions. Getting Started with Lasergene GeneQuest • 99 . Select FILE > Open. 2. Search BLAST or Entrez databases for matches to your sequence. 7. Opening an Existing Project and Organizing Methods All folders referenced in this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\ Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene: 1. 9. Select FILE > Print to print your results. Use the graphical and tabular results to form hypotheses about your sequence. GeneQuest tutorials The tutorials in this chapter should be followed in order. as they are designed to walk you through an example GeneQuest project from start to finish. Select FILE > Save to save your results.5. please contact a DNASTAR representative via telephone (608) 258-7420 or e-mail (support@dnastar.dad in the Demo Data folder and double-click it to open. Edit method parameters at any time. if desired. Select either FILE > Exit (Win) or GENEQUEST > Quit (Mac) to exit GeneQuest. Locate the file WormProblem_FinalResult. 6. Launch GeneQuest. The Further Exploration section at the end of the chapter highlights optional features you may wish to use as you gain experience with the software. and then annotate any interesting features. 8.

Ensure that the Method Curtain is displayed at left.dad project. Note: At this point. 2. Select FILE > New. and make sure the Files of Type (Win) or Enable (Mac) drop-down menu shows Lasergene DNA files (*. Proceed to the next section. only the scale and sequence have been applied to the assay surface. 100 ● GeneQuest tutorials Getting Started with Lasergene . then click Open. 3.seq file in the Demo Data folder. This curtain displays the names of the methods that have been applied to the assay surface. The Method Curtain at left displays a list of available analysis methods.The main portion of the window is the assay surface.seq). In the Method Curtain. Applying Methods to the Sequence 1. The Legend Curtain is displayed to the right of the assay surface. click on the plus sign (Win) or the triangle (Mac) to the left of the method Coding Prediction – Starts Stops ORFs to reveal a list of available displays. 4. Select the WormProblem. Select ANALYSIS > Show Legend. Select FILE > Close to close the WormProblem_FinalResult. It contains graphic displays of analysis methods that have already been used to analyze the sequence. 5.

4. Shift+click each of the region plot symbols for ORFs 1. Click the white space to the left of the displays to ungroup them. then click OK to close the dialog. 9. Click Open. 7. 6. ORFs are calculated and displayed as boxes on the assay surface. Drag the selected displays onto the assay surface and drop them under the existing displays.mat file from theBorodovsky Matrices folder. In the Method Curtain.3. 5. 8. Getting Started with Lasergene GeneQuest • 101 . 2. Click Set Data File and select the ce_lo_4. and 3 to select them.mat so that the method parameters dialog is displayed. as shown. double-click on the method Coding Prediction-Borodovskyvolvox_0. Click on the plus sign (Win) or the triangle (Mac) to the left of the method Coding Prediction – Borodovsky – ce_lo_4. and 3 to select these three displays.mat to reveal the list of possible displays. Shift+click each of the graph symbols next to Frames 1. 2. Drag the selected displays onto the assay surface and drop them under the existing displays.

Matrix. Repeat steps 12-13 to add two matrix patterns celegans_as_2. A new entry called Patterns . Shift+click the two frame 1 displays to select them. 102 ● GeneQuest tutorials Getting Started with Lasergene . 15. Note: GeneQuest calculates potential translation start. Select the Top Strand of this method and drag it to the bottom of your assay surface.Note: GeneQuest calculates potential coding regions and displays them as peaks. 12. The higher the Borodovsky peak. • • . To view the scores only. select the Range Selector tool hold on a site until an information box is displayed. At the top of the Method Curtain. click on the plus sign (Win) or the triangle (Mac) to the left of the method Patterns . select MORE METHODS > Patterns > Matrix. then click and The cursor snaps to a particular point on the site.mat and celegans_atg_2 to this new method. the higher the probability that the area is a coding region. and 3 of the Borodovsky methods. • • • • Click the Object Selector tool . donor and acceptor sites and displays them as vertical lines. Shift+click frames 1. 14. The exact base to which the cursor snaps is shown on the header at the top left of the window next to the word “Position. 2. but select Green for the two frame 2 displays and Blue for the two frame 3 displays. select the Zoom In tool and then click repeatedly on the assay surface until you can read the numbers inside the boxes. In the Method Curtain.mat from the Matrix Patterns folder and click Open. 11. To view the scores and other information. Each apparent line is really a narrow box containing a numeric score denoting the likelihood of that area being a site. Locate and select the file celegan_ds_2. 10.” The pop-up information box displays the bases spanned by the pattern and a candidate ranking score.Matrix will appear in the Method Curtain. Repeat the above steps for the remaining frames. 13. drag the Top Strand to the bottom of your assay surface. You can use colors to differentiate each frame of the newly applied Borodovsky and ORFs displays from each other. then select OPTIONS > Superimpose Objects. Select OPTIONS > Line Color > Red. For each method. This method will now be listed in the Method Curtain. 16.

000 bp. Close the method curtain by selecting ANALYSIS > Hide Available Methods.000or to enlarge or reduce the view until the sequence and 12. Use the Zoom tools Legend Curtain combine to fill the entire window. Proceed to the next section.000. To make it easier to locate potential genes. click the Object Selector tool . For example. 3. choose green for the donor sites. blue for the acceptor sites. Use the scroll bar at the bottom of the window to locate the area of the sequence from 9. Drag and drop the method names so they appear in the in the Legend Curtain in the order shown: 2. then use the OPTIONS menu to change the line color. Predicting Coding Regions and Creating Features 1. Click the Range Selector tool Getting Started with Lasergene and then click on the frame 3 ORF centered on base 10. and black for the start sites.17. 4. Use the Object Selector tool to select each matrix pattern result in turn. GeneQuest • 103 .

it must end with a donor splice site that precedes the stop codon for the ORF in the same frame. It is likely that this area represents an exon.0 match from 9935 to 9943 on top strand 104 ● GeneQuest tutorials Getting Started with Lasergene . • • The header shows the position of the candidate. In the Top Strand…atg_2.• • 5. In the Top Strand…ds_2. If this is the first exon of a multi-exon gene. Pattern: caenorhabditis & elegans start 9. You are now ready to find the beginning of the potential gene. while the pop-up information box shows the score.mat display. observe that there are two candidate donor splice sites (shown circled in the following figure) beyond the Borodovsky peak and still within the corresponding frame 3 ORF.mat display. Note that a large peak in the frame 3 Borodovsky graph lies entirely within this highlighted ORF. This site is shown enlarged below. click and hold on the candidate start site at the far left end of the ORF.

• Exon 1 Donor: Base 10028; score = 4.6
• Exon 1 Donor: Base 10056; score = 9.6

6.

Select EDIT > Go To Location. Enter the range 9939,10055 and then click OK.

7.

You are now ready to annotate the first exon.

The position 10056 indicates the beginning of the acceptor splice site; therefore, the actual
range of the exon extends from 9939 to 10,055.

Click the Annotation tool
appears in the Segment box.

Getting Started with Lasergene

to open the Feature Editor. The location of the gene segment

GeneQuest • 105

8.

9.

Enter genE into the Title box.

Enter exon1 into the Segment Name box, then click OK.

Now that the first exon has been annotated, you may continue searching for additional exons.

Any further exons must begin with an acceptor splice site, and that site should be in an ORF
for which there is Borodovsky evidence for coding potential.

Observe that the next significant Borodovsky peak is in frame 2, centered near base 10,600.

There is one very likely acceptor candidate in the Top strand…as_2.mat display, between
bases 10,207 and 10,219: Exon 2 Acceptor: Base 10,217; score = 12.0

If there are more than two exons to this gene, Exon 2 must terminate with a donor splice site.
There is a strong candidate in the Top strand...ds_2.mat display at position 10,778, some
distance to the right of where the frame-2 Borodovsky peaks fall to the baseline: Exon 2
Donor: Base 10,778; score = 12.6.

Now you may annotate this exon and join it to the first gene segment.

Select EDIT > Go To Location.

10. Enter the range 10217,10777 and then click OK.

106 ● GeneQuest tutorials

Getting Started with Lasergene

11. Click the Joining Tool

.

12. Select genE - misc_feature from the list and then click Join.
13. The next Borodovsky peak is not only in the same frame as exon 2, but also in the same ORF. The
best candidate acceptor splice site to start exon 3 has a likelihood value of 14.2 out of a maximum
16.7—an excellent candidate. Two candidate donor splice sites are available to define the 3′ end of
this exon, at 10,973 and 10,993. The first site scores higher at 6.6.

Exon 3 Acceptor: Base 10,826; score = 14.1

Exon 3 Donor: Base 10,973; score = 6.4

14. The next Borodovsky peak is once more in frame 2, but there is no candidate acceptor site (at the
default threshold) under this peak. However, there is a site to the left of the peak, at position
11,021, that scores an impressive 15.1; this is the best candidate start for exon 4. You will note that
there is no candidate donor splice site within the corresponding frame 2 ORF.
15. This indicates that Exon 4 is probably the final exon in the gene. Therefore use the splice site at
base 11,021 to start this exon and the stop codon at the end of this ORF (base 11,155) to terminate
this exon.

Exon 4 Acceptor: Base 11,021; score = 15.1

Exon 4 Donor: No likely candidates

Stop Codon: Base 11,155

16. After locating the endpoints for Exons 3 and 4, annotate them by repeating steps 9-12 for each
exon.
17. Now that all four gene segments have been annotated, click on either the Object Selector tool
or the Range Selector tool
Editor.

and then double-click on the feature to reopen the Feature

18. In turn, click on the coordinates for the three unnamed segments and type their names into the
empty Segment Name boxes (exon2, exon3, exon4).

Getting Started with Lasergene

GeneQuest • 107

Leave nr selected in the Database drop-down menu. protein). Corroborating Predictions by BLAST Search 1. The feature should resemble the figure below: Proceed to the next section. • • 3.19. GeneQuest displays search results when they are returned from the server—this may take a few minutes: Highlight the match with Identifier 17553974 (this will likely be the first entry) and click the Put In Document icon. 2. then click OK. However. Click the Range Selector tool and then click anywhere in the genE feature. This sends the translation of the selected DNA sequence to NCBI’s BLAST server as a blastp query (protein vs. Click OK to finish. Select NET SEARCH > Blast ORF. 108 ● GeneQuest tutorials Getting Started with Lasergene . the Selection line in the header above the assay surface indicates that only the annotated segments of the feature are selected. Note: The entire sequence spanning the feature is highlighted.

Getting Started with Lasergene GeneQuest • 109 . go to the next step. search the nucleotide database at NCBI and click OK. click Save to save a local copy of the sequence and open it in GeneQuest.to display the features. 3. In the ensuing dialog. It also appears in the Method Curtain as the Gene Finding . 2. Drag the K01G5. Select FILE > Open Entrez Sequence. You can use the Zoom In tool sequence. applying the default method set. Note: The Z92803 sequence is exactly the same length as your problem sequence—19. How does it match up to yours? 4. as pictured below. It now appears on the assay surface under the genE feature. keep the defaults and click OK to save copy of the highlighted sequence. You can apply it to the assay surface as you would any other method. In the Method Curtain for Z92803.Protein Finder method.442 bases. When the Entrez result is returned.4. • • 5. Enter Z92803 in the dialog box. 1.3 gene to the Assay Surface. Select FILE > Save to save the project if you wish and close GeneQuest. expand the Features method. we will be comparing our results with the results the authors of this sequence obtained. Examine the annotated gene in the region 9-12 kb. To see all the annotations made by the authors. and click repeatedly on the assay surface to read the Proceed to the next section. Checking Predictions against External Data In this section. This method aligns the protein sequence with the corresponding DNA region(s) in your sequence where the protein matches the sequence translation.

apply one or more Enzymes . nucleotide composition or codon distribution. showing the predicted electrophoretic separation of the restriction fragments: Note: Select a portion of sequence prior to using the following commands. 110 ● Further exploration Getting Started with Lasergene .Restriction Map methods. respectively. then select SITES & FEATURES > Agarose Gel Simulation. • To simulate multiple digestion experiments and examine fragment sizes in predicted agarose gel migration patterns. apply one or more Enzymes . ANALYSIS > Base Composition or ANALYSIS > Codon Usage. select ANALYSIS > Tabular Data.Further exploration Additional analyses • To view a tabular summary of restriction fragments. • To view tabular summaries showing method results.Restriction Map methods. The following window opens. then select SITES & FEATURES > Restriction Fragment Summary.

then change method parameters separately for the added copy of the method. • To predict how a nucleotide sequence might fold as RNA. click move the pointer to a location. Getting Started with Lasergene GeneQuest • 111 . Similarly. add another copy of the method to the method curtain. Changing method parameters Access method parameters by double-clicking on a method name or display anywhere in the GeneQuest window. but still read the sequence. select a <1500 bp portion of sequence and select ANALYSIS > Fold as RNA: Using the microscope view To view the sequence for any region under the mouse pointer. To view multiple displays of a method using different parameters. apply one or more Repeats methods and select ANALYSIS > Repeat Summary. parameter changes made to a display on the assay surface are applied to all displays originating from the same method in the method curtain.• To view repetitive portions of a sequence. Note that parameter changes made to a method in the method curtain affect all current and future displays created from that copy of the method. The microscope tool lets you stay zoomed out for the big picture.

select ANALYSIS > Save Method Outline. or Fill Color. • To apply an outline from another document or saved as a separate file. GeneQuest uses a file called the default method outline (“default. select ANALYSIS > Apply Method Outline. • To optimize the look of a selected display(s). and optimized method parameters and display options. you may also open it directly using FILE > Open Entrez Seq. you may have deleted some of the default methods. Searching BLAST and text databases As discussed earlier in this chapter. GeneQuest can search BLAST databases for sequences similar to your own. • To save the current method outline as a file without saving it as the default. GeneQuest allows you to save the method outline from the current assay document and apply it to selected assay documents or to all new assay documents. Matches from either search type may be opened in any Lasergene application. select OPTIONS > Font or Size. If you already know the locus name or accession number of a sequence in NCBI’s Entrez database. • To save the current outline as the default method outline. You may wish to use a similar setup in other assay documents. Using method outlines When you create a new assay document. “Default DNA Outline” on Macintosh) to determine which methods to place in the method curtain and which of those to apply to the assay surface. select OPTIONS > Line Color. printed.dao” on Windows. or saved. viewed with the browser. Line Weight. In the course of revising your assay document. select ANALYSIS > Save As Default Method Outline.Changing formatting options Note: Click and select one or more displays prior to using the following commands. 112 ● Further exploration Getting Started with Lasergene . Fill Pattern. added new methods of your own. Additional commands in the NET SEARCH menu allow you to perform a text search of databases such as NCBI’s Entrez database. • To change the font and point size of text other than feature annotations.

• Secondary Structure .Garnier-Robson—Examines the propensity of a given residue to exist in a certain structure. • Patterns . • Proteases . • Secondary Structure .Protease Map—Identifies proteolytic sites and displays them as a mini-map. • Secondary Structure . The next step is to evaluate secondary structural characteristics of the polypeptide sequence by applying some of Protean’s many analysis methods to the sequence. • Ruler—Adds a ruler to the document. The project begins when you enter a protein sequence.Prosite Database—Searches the Prosite database to find matches to your sequence. These methods include: • Title—Adds a title to the document.Coiled Coil—Predicts transmembrane alpha helices. • Sequence—Displays the sequence on the document.Protean Overview Protean aids you in predicting and annotating the structural character of your protein sequence and provides a graphical display and tabular summaries of sequence composition and other data. Getting Started with Lasergene Protean • 113 . • Charge Density – Charge—Predicts regions of positive and negative charge by summing charge over a specific range of residues.Ariadne File—Locates matches between a user-specified pattern descriptor and the sequence.Deléage-Roux—Uses an independent prediction of the protein’s structural class to bias the prediction of its secondary structure. • Patterns .

• Antigenicity .Rothbard-Taylor—Locates potential T-lymphocyte antigenic determinants which contain a common sequence motif. • Hydropathy .• Secondary Structure . You may also wish to take advantage of Protean’s specialized protein fonts. menu commands let you display tabular data summaries. Once the methods have been applied. Furthermore. • Antigenicity . • Hydropathy .Hopp-Woods—Finds protein antigenic determinants by searching protein sequences for the area of greatest local hydrophilicity. create protease digestion maps. • Amphiphilicity – Eisenberg—Predicts the Eisenberg Moment. • Flexibility .Sette MHC Motifs—Predicts peptide antigenic sites which interact with mouse MHC II haplotype d proteins. the results are displayed graphically on a common horizontal scale. calculate titration curves. you may create your own annotations for features of interest and even link several features together under a common title and description. You can also use Protean to create images for publication.Chou-Fasman—Predicts secondary structure of proteins from the crystallographic structures of their amino acid sequences. • Hydropathy . • Surface Probability – Emini—Predicts the probability that a given region lies on the surface of a protein. • Antigenicity . • Antigenicity – AMPHI—Predicts immunodominant helper T-lymphocyte antigenic sites from primary sequence data. which allow you to view sequences as chemical formulas or space-filling models. and perform SDS PAGE simulations.Goldman-Engleman-Steitz—Predicts non-polar alpha helices which may span a cell membrane.Kyte-Doolittle—Predicts regional hydropathy of proteins from their amino acid sequences. In addition. You can juxtapose and superimpose method results graphically or utilize one of Protean’s tabular data summaries. 114 ● Overview Getting Started with Lasergene . Protean reads and interprets GenBank-formatted features from the comment or features pane of sequence documents.Jameson-Wolf—Predicts potential antigenic determinants by combining existing methods for protein structural predictions.Karplus-Schulz—Predicts backbone chain flexibility.

2. The method display is updated after each edit. 5. if desired. If you have any difficulties or questions. 9. Select ANALYSIS > Show/Hide Available Methods to display a list of methods. please contact a DNASTAR representative via telephone (608) 258-7420 or e-mail (support@dnastar. FILE > Save your results. Apply methods to the sequence by using the mouse to drag them from the Method Curtain to the assay surface. Use the graphical and tabular results to form hypotheses about your sequence. and select which of these you would like to apply to the sequence. If you have applied proteolytic digestion methods to the sequence. or calculate its titration curve. Search BLAST or Entrez databases for matches to your sequence. as they are designed to walk you through an example Protean project from start to finish. 8. you may run a SITES & FEATURES > SDS Page Simulation. Protean tutorials The tutorials in this chapter should be followed in order. If you wish. and then annotate any interesting features. 4.com). Move the methods you want from the More Methods menu to the Method Curtain. Getting Started with Lasergene Protean • 115 . 6. FILE > Print them. 10. either by double-clicking on the method display or on the method name in the Method Curtain. and quit using FILE > Exit (Win) or PROTEAN > Quit Protean (Mac). create a structural model of your protein. delete or rearrange method displays until you are satisfied with the look of the document. 7. or other sequences of interest. Edit method parameters at any time.10 Quick-Start Steps for Using Protean Here are ten basic steps for using Protean: 1. 3. The Further Exploration section at the end of the chapter highlights optional features you may wish to use as you gain experience with the software. Create a new project (FILE>New) or open an existing project (FILE>Open) and review the results of the current method outline. View the results in tabular form. if desired. Continue to add.

Protean opens the project in a new window. The Assay Surface contains the ruler. • Several analytical method displays are already present on the Assay Surface. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo Data Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo Data 1.Correlating annotations with predicted structures Objective: To open an existing protein assay and to view the locations of known calcium-binding sites and correlate them with predicted turns.pad from the Demo Data folder. 116 ● Protean tutorials Getting Started with Lasergene . the main area of the Protean window. sequence. Launch Protean. 2. as displayed on right side of the figure above. and methods. Select FILE > Open to open Human Calmodulin.

• 3. The Features method appears at the bottom of the method curtain. the first option under ANALYSIS will be Hide Available Methods. widen it by dragging the curtain pull ( ) to the right. Using Shift+click. highlight each of the four features named Calcium binding region: Region. click in the white space to their left to ungroup them.” You will learn later how to access the complete list of methods. proceed without taking any action. • • If the Method Curtain is open. This figure displays the Method Curtain. Select OPTIONS > Superimpose Objects to place all four calcium binding site locations (represented by cylinders) on the same line. Methods listed in the Method Curtain are ready to be applied to the sequence. 4. Open the Method Curtain (if it is not already open) using ANALYSIS > Show Available Methods. Drag the highlighted features to the right and drop them on the assay surface. If the items in the list appear highlighted. Getting Started with Lasergene Protean • 117 . In this case. 7. • If you cannot see the complete names of the features in the Method Curtain. 5. The four features now appear on the assay surface and remain selected. 6. and were specified by the “default method outline. In the Method Curtain. click on the triangle (Mac) or the plus sign (Win) to the left of the method Features to reveal a list of annotations that were imported with the sequence. open at the left. The Method Curtain contains methods that can be applied to the assay surface using a drag and drop method.

• Note that the calcium binding sites are all located in predicted turn regions. This is consistent with the known structure of these binding sites. When you have predicted structures and features displayed in this way. To make it easy to compare the calcium-binding site locations with predicted turns. 118 ● Protean tutorials Getting Started with Lasergene . 1. A new copy of the method now appears at the top of the method curtain list. Regions . and to compare it to the already applied Hydropathy – Kyte-Doolittle method. Similarly. and drag it down to be just above the calcium binding site display name. From this menu. to change its parameters. Note that parameter changes made to a method in the Method Curtain affect all current and future method displays created from that copy of the method. which are classical “EF Hand” calcium-binding sites wherein calcium is bound at the “elbow” between two adjacent alpha-helical regions.) copy of the method to the Method Curtain. To view multiple displays of a method using different parameters. you can select methods that aren’t listed in the lower part of the method curtain or make additional copies of methods that are. Click More Methods at the top of the Method Curtain to open a menu containing methods. Calculating methods with different parameters You can access method parameters by double-clicking on a method name or display anywhere in the Protean window. etc. 2. Select Hydropathy – Kyte-Doolittle. you can add a second (or third. Proceed to the next tutorial. then change method parameters separately for the added copy of the method. select the name “Turn.• 8. parameter changes made to a display on the assay surface are applied to all displays originating from the same parent method in the method curtain. you can begin to evaluate the structural characteristics of the protein and its corresponding features.Garnier-Robson” from the list. therefore. Objective: To apply a new Hydropathy – Kyte-Doolittle method to the sequence. scroll to the far right of the assay surface.

shown below. • Predicted hydrophilic regions of your protein are shown as a black and white filled graph in the new method. From More Methods at the top of the Method Curtain. Predicting proteolytic cleavage sites & performing SDS PAGE simulations Objectives: To identify and display proteolytic sites. 5. it may be applied to the sequence. then to simulate an SDS PAGE run. If the items in the list appear highlighted. Now that the method is in the main body of the method curtain. Click on Hydrophilicity Plot. drag it to the assay surface and drop it below the existing Hydrophilicity Plot – Kyte-Doolittle display. click in the white space to their left to ungroup them. 4. Getting Started with Lasergene Protean • 119 . Double-click on the newly added display to open its method parameter dialog. Click on the triangle (Mac) or the plus sign (Win) to the left of one of the Hydropathy – Kyte-Doolittle methods to reveal the four possible display types. Change the Residues to Average to 3 and click OK.3. select Proteases – Protease Map. • The original display used the default value of 9. 1. 6. • The two displays now show the effects of applying the different parameter values: Proceed to the next tutorial.

4. select SITES & FEATURES > SDS PAGE Gel Simulation. An image of the simulated separation will appear in a new window. 120 ● Protean tutorials Getting Started with Lasergene .2. as below. If the items on the list appear highlighted. Leaving the displays highlighted. 6. Click on the triangle (Mac) or the plus sign (Win) to the left of Proteases . The resulting method displays show recognition sites as vertical bars. click in the white space to their left to ungroup them. When you are finished. View the following information about the available proteases by choosing SITES & FEATURES > Show Protease List. select FILE > Close to close the Protease List. and then drag them onto the assay surface. 5. Use Shift+click to highlight both Chymotrypsin and CNBr. Above each column in the gel is the name of the corresponding protease or molecular weight standard. 3.Protease Map method name to view the list of proteases.

HPLC retention time and predicted pI.7. 8. This opens a summary of information about the proteases applied to the sequence. Getting Started with Lasergene Protean • 121 . Move your cursor over the surface of the gel and notice how the header at the top of the window changes. When the cursor is over a fragment. sequence range. Select SITES & FEATURES > Protease Fragment Summary. the header displays its molecular weight.

Further exploration Additional analyses • To view tabular summaries of method results and sequence composition. select ANALYSIS > Tabular Data or Composition. Helical Net. Beta Net. A helical wheel structure is shown below: 122 ● Further exploration Getting Started with Lasergene . Linear Space Fill or Chemical Formula. select ANALYSIS > Model Structure and select from Helical Wheel. • To view the secondary structure of the sequence.

• To view a titration curve like the one below. select ANALYSIS > Titration Curve: Getting Started with Lasergene Protean • 123 .

“Default Outline” on Macintosh) to determine which methods to place in the method curtain and which of those to apply to the assay surface. • To apply an outline from another document or saved as a separate file. select ANALYSIS > Save Method Outline. Using the microscope view To view the sequence for any region under the mouse pointer. and optimized method parameters and display options. Additional commands in the NET SEARCH menu allow you to perform a text search of databases such as NCBI’s Entrez database. • To save the current method outline as a file without saving it as the default. If you already know the locus name or accession number of a sequence in NCBI’s Entrez database.Using method outlines When you create a new assay document. viewed with the browser. or saved. You may want to use the same setup or a similar setup in other assay documents. you may also open it directly using FILE > Open Entrez Protein. • To save the current outline as the default method outline.pao” on Windows. printed. The microscope tool lets you stay zoomed out for the “big picture. Matches from either search type may be opened in any Lasergene application. you may have deleted some of the default methods. Protean uses a file called the default method outline (“default.” but still read the sequence. added new methods of your own. as well. In the course of revising your assay document. Protean allows you to save the method outline from the current assay document and apply it to selected assay documents or to all new assay documents. activate (the Microscope palette tool) and move the mouse pointer to the desired location. select ANALYSIS > Apply Method Outline. select ANALYSIS > Save As Default Method Outline. Searching BLAST and text databases Protean can search BLAST databases for sequences similar to your own. 124 ● Further exploration Getting Started with Lasergene .

10 Quick-Start Steps for Using MegAlign Here are ten basic steps for using MegAlign: 1. Four algorithms are available for pairwise alignments: Martinez-Needleman-Wunsch and WilburLipman for DNA alignments. MegAlign can utilize sequence files from a wide variety of file types or from NCBI’s BLAST or Entrez databases. ClustalV and two ClustalW options: Slow-Accurate and Fast-Approximate. Four additional algorithms are available for multiple alignments: Jotun Hein. Once you are satisfied with the alignment. Sequences may be realigned using different alignment parameters. you may specify a portion of the sequence by coordinates. The DotPlot method may be applied to both DNA and protein alignments. the versatile Alignment Report allows you to create a customized display of results. and Lipman-Pearson for protein alignments. as well as reports and tables showing the numerical data underlying the comparisons. and manual adjustments may be made using the Worktable palette tools. If a sequence file is longer than you need. Getting Started with Lasergene MegAlign • 125 .MegAlign Overview MegAlign generates pairwise and multiple sequence alignments of DNA and/or protein. by selecting a feature from the feature table. or by highlighting a sub-region of a previously aligned project. MegAlign can also create phylogenetic trees. Bootstrapping analysis may be applied to the tree when one of the Clustal algorithms is used to align your sequences. Select FILE > New to open a new project or FILE > Open to open a previously saved project. Your alignments may also be displayed in the form of a phylogenetic tree.

Choose a pairwise or multiple alignment method. You can use a sub-range of a sequence. use the Alignment Report. 5. You may want to edit the ALIGN > Method Parameters before performing the alignment. If you do not want to use the Standard Genetic Code. Select FILE > Print your results. If desired. The Further Exploration section at the end of the chapter highlights optional features you may wish to use as you gain experience with the software. For multiple alignments. all sequences in the Worktable are aligned whether or not they are highlighted. View and edit weight tables using ALIGN > Set Residue Weight Table. 126 ● MegAlign tutorials Getting Started with Lasergene . Genetic codes are only important if you will be aligning DNA and protein sequences together. if desired. Codes may be edited using OPTIONS > Edit Genetic Code. and if necessary. Add sequences to the Worktable as directed in the tutorial. 4. To locate a particular position in a sequence. 3. Select FILE > Exit (Win) or MEGALIGN > Quit MegAlign (Mac) to exit MegAlign. but not for pairwise alignments. 6. you may edit gaps in the alignment from the Worktable or realign residues using the Worktable palette tools. select ALIGN > Unalign All. or translating DNA. 10. first highlight two or more sequence names by using Ctrl/Cmd+click (Win/Mac) or Shift+click. You can select OPTIONS > New Consensus and OPTIONS > New Decoration to change the appearance of the report and the definition for the consensus. For pairwise methods. select EDIT > Go To Position or EDIT > Find Disagreement.com). If you have performed a multiple alignment. select OPTIONS > Genetic Codes. as they are designed to walk you through an example MegAlign project from start to finish. please contact a DNASTAR representative via telephone (608) 258-7420 or e-mail (support@dnastar. 9. 8. MegAlign tutorials The tutorials in this chapter should be followed in order. Sequence Distances. 7. customize the OPTIONS > Alignment Report Contents. If you have any difficulties or questions. To undo a multiple alignment. To examine numerical data about completed alignments. Residue Substitutions or Phylogenetic Tree commands from the VIEW menu. Search commands can be used for unaligned or multiply aligned sequences.2. you can select OPTIONS > Font and Size to reformat characters until they are clearly legible.

2. On Macintosh. Highlight both Tethis21. On both platforms. 4. Objective: To add two sequences to the MegAlign Worktable using the drag and drop procedure. MegAlign will launch at this point. • Macintosh: open the Finder window containing the MegAlign program icon. the MegAlign Worktable will appear as follows: Getting Started with Lasergene MegAlign • 127 . The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign\Histone Sequences Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign\Histone Sequences Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo MegAlign:Histone Sequences 1.Entering sequences using drag and drop Drag and drop is a feature available in all Lasergene applications. and is very useful for entering large numbers of sequences. Using Windows Explorer or Macintosh Finder. 5.seq using Shift+click or a similar method. Adjust the windows so you can view the folder contents and the MegAlign window (Win) or program icon (Mac) simultaneously.seq and Tethis22. Use the mouse to drag the selected files onto the MegAlign Worktable (Win) or MegAlign program icon (Mac) and drop them there. 3. Do one of the following: • Windows: launch MegAlign. locate and open the Histone Sequences folder.

Select OPTIONS > Set Sequence Limits > From Feature Table.seq appears first. because the sequences are almost the same length. You can do this by first selecting the sequence you want to move. the final base pairs of both sequences appear in the right pane. 2. In this case. Note: The lengths of these sequences are 906 bp for Tethis21 and 905 bp for Tethis22. 128 ● MegAlign tutorials Getting Started with Lasergene . • You may select OPTIONS > Size to increase or decrease the font size.seq appears before tethis21.seq. • You can use this dialog to see the length of a sequence in the “Segments” section of the dialog. Objective: To set sequence ends using one of the GenBank features imported automatically when the sequences were entered. rearrange the sequences so that tethis21. • The left pane of the worktable displays the first (starting) base pairs of both sequences. Select tethis21 by clicking its name in the left pane of the Worktable.Note: If tethis22. and then dragging it to the desired position in the list. The Feature List appears. you may set sequence ends by typing in the desired coordinates or by selecting a feature. Setting ends using features In MegAlign. 1. The right pane displays the final (ending) base pair of the longest sequence.

3. Click Change the Rest to limit the tethis22 sequence using the same feature. MegAlign always needs to know which pairs you wish to align. Performing a pairwise alignment Since the current sequences are DNA. Objective: Use the Wilbur-Lipman method to align two sequences. The lower pane now shows the feature name and range (232 > 600) of the highlighted feature. Note that the selected range of the tethis21 sequence has limited the range of both original sequences on the worktable. MartinezNeedleman-Wunsch and DotPlot. 4. Because there may be many sequences. Hold down the Shift key while you click each of the two sequence names to highlight them. 5. 1. Select ALIGN > One Pair > By Wilbur-Lipman Method. the pairwise methods available are Wilbur-Lipman. 2. Highlight the histone H2B-1—CDS feature in the upper pane of the dialog. Click OK to return to the MegAlign Worktable. Getting Started with Lasergene MegAlign • 129 . The method parameter dialog opens. Notice that the worktable now reflects that both sequences end at bp 369.

the aligned pair of sequences flanks a central consensus that shows mismatches as spaces. Click OK to use the default parameters for the alignment. The Alignment View Options dialog Getting Started with Lasergene . Check and uncheck each of the four boxes just under the color selection boxes. choosing Red for Mismatch Color and Green for Consensus Color. From the Match Color list.3. 7. 5. In the lower portion of the Alignment View. gap number. All matching residues immediately turn blue on the Alignment View. The window header displays the similarity index (percent of all residues that are matching). total gap length and consensus length. select Blue. 6. Repeat step 5. Change the color scheme of the window by clicking appears. and note how the Alignment View changes. MegAlign calculates the alignment and displays results in the Alignment View. 130 ● MegAlign tutorials . 4. shown below.

Select ALIGN > One Pair > Dot Plot. Objective: To use the DotPlot method to align two sequences. Leave the parameters set to their defaults.8. green. Click Vertical Bars to show agreement between the two sequences as vertical lines instead of letters. Once you have finished experimenting with the Alignment Color dialog. • Diagonal lines indicate regions of the two sequences that meet the threshold for similarity specified in the Dot Plot parameters. with one sequence on each axis. 3. allowing you to set the parameters of the alignment. 2. and click OK. • The long red diagonal line indicates that these two sequences align well over most of their length. Close the Alignment View to return to the Worktable. The DotPlot opens in a separate window. yellow. close it using the close icon in the title bar. Highlight the two sequence names (if not still highlighted from the previous tutorial). 10. with progressively stronger similarities indicated by pale blue. The Filtered DotPlot dialog opens. 1. Getting Started with Lasergene MegAlign • 131 . • Dark blue diagonals show weaker similarities. Performing a Dot Plot pairwise alignment The Dot Plot method compares sequences by plotting matches between two sequences on a chart. orange and red. 9.

click on the Worktable and close the current MegAlign project using FILE > Close. Once you have finished viewing the window. click Don’t Save (Mac) or No (Win). 132 ● MegAlign tutorials Getting Started with Lasergene .4. 5. When prompted to save the document. Double-click on any diagonal line to open a window that shows the underlying alignment of that diagonal.

• MegAlign’s residue weight tables are used in scoring multiple alignments so that mismatched residues that are chemically similar score higher than chemically different residues. Select ALIGN > Set Residue Weight Table. Since calmodulins are highly conserved. 7. When all fourteen sequence names appear in the Selected Sequences pane (Win) or the folder appears in the Chosen Files pane (Mac). 3. Locate and open the Demo MegAlign folder. 4. • The title bar for the current window reflects MegAlign’s default settings. the PAM100 table should be used for this alignment. click Done. Do one of the following: • Windows: Double-click on the folder “Calmodulin Sequences” and then click Add All. 6. Select all of the sequence names in the Worktable by holding Shift while clicking them. and the PAM250 table should be used for divergent sequences. Getting Started with Lasergene MegAlign • 133 . The Residue Weight’s table appears. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign\ Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo MegAlign 1. • For the ClustalV method.Performing a multiple alignment Objective: To align fourteen calmodulin sequences using the ClustalV method. • Macintosh: Click “Calmodulin Sequences” and click Add Folder. 2. 5. Select FILE > Enter Sequences. Select PAM100 from the pull-down menu. The title will later change to reflect the weight table and alignment method you choose. the PAM100 table should be used when aligning conserved sequences.

To align the sequences. 9. select ALIGN > Method Parameters and select the ClustalV tab. leave the default values. select ALIGN > By Clustal V Method. select VIEW > Sequence Distances: 134 ● MegAlign tutorials Getting Started with Lasergene . To view the current ClustalV settings.8. and then click Later. the Worktable reappears with the aligned sequences. When the alignment is complete. 10. The Alignment Progress window shows what percentage of the project has been aligned. To view a table showing the divergence and percent identity of the sequences. Review the settings. 12. 11. Click OK to return to the Worktable.

15. To view a table showing residue substitutions. select VIEW > Residue Substitutions: 14. select ALIGN>Perform Bootstrapping. Review the settings and then click OK. and the bootstrapping is applied to the phylogenetic tree. Getting Started with Lasergene MegAlign • 135 . select VIEW>Phylogenetic Tree. 1000) are generated. A separate window containing the bootstrap analysis settings appears. The numbers displayed refer to the percentage of times the same branches appear when a number of sample trees (in this case. Bootstrapping analysis begins. To apply bootstrapping analysis to the tree.13. To view the phylogenetic tree.

On the Alignment Report you may also define and display one or more custom consensi built upon your own criteria. 2. shade. as shown below: 136 ● MegAlign tutorials Getting Started with Lasergene . hide and highlight various components and consensi.16. Creating an alignment report Alignments can be modified to show. 1. Close all windows except for the Worktable. select OPTIONS > Alignment Report Contents. To modify the contents of the report. Take a moment to review its contents. Graphics in the Alignment Report that box. Select VIEW > Alignment Report to open a separate window containing the results of the multiple alignment. Check Show Consensus Strength and leave all other settings at their defaults. 3. or hide residues are referred to as “decorations”. Objectives: To modify the contents of the Alignment Report and to create a decoration that shades those amino acids that disagree with the consensus sequence.

4. The dialog should now look like the one below: Getting Started with Lasergene MegAlign • 137 . The New Decoration dialog appears. 8. Select OPTIONS > New Decoration. Choose a color from the first menu and the darkest shading scheme from the second menu. type “Shade disagreements with consensus”. The next row contains three pull-down menus. Leave the third menu. 6. Click OK to update the already open Alignment Report. at the default setting. Note the color-coded histograms showing the strength of the consensus at each base. the one on the right. 5. the Consensus. In the title box. Change the first pull-down menu to Shade and the middle menu to residues differing from. Choosing Shade caused two additional pull-down menus to open below it. 7. Leave the distance units box at “0”.

Click OK. 138 ● MegAlign tutorials Getting Started with Lasergene . In the Alignment Report window.9. shown below. (The sequences may be listed in a different order in your project). any constituent sequence residues that do not match those in the consensus sequence now appear shaded.

Straighten Columns inserts gaps preceding selected residues and aligns residues along their left sides. remove or change decorations and consensi. A new Worktable will open containing only the highlighted portion of sequences. When prompted. Getting Started with Lasergene MegAlign • 139 . you may manually adjust selected portions of the alignment using the palette tools to the left of the Worktable.Further exploration Creating an alignment using a portion of the sequences To create a new alignment from a portion of the sequences. Exporting data • To export sequences as separate Lasergene DNA or protein files. select OPTIONS > Decorations > Decoration Manager or OPTIONS > Consensi > Consensus Manager. Realigning residues After completing a multiple alignment. select OPTIONS > New Consensus. Shuffle Right moves gaps within selected regions so they precede the selected residues. highlight a portion of the consensus sequence in the Worktable and select ALIGN > Create Alignment from Selection. • To add. Shuffle Left moves gaps within selected regions so they follow the selected residues. If desired. Working with the Alignment Report • To add a decoration to the Alignment Report use OPTIONS > New Decoration (discussed in this chapter). choose a name and location for the new project and click Save. you can change the standard amino acid categories from the same dialog by clicking Set Groups then Standards. • To add a consensus to the Alignment Report. highlight the names of the sequences in the Worktable and select FILE > Export Sequences.

Located matches may be opened in any Lasergene application. If you already know the locus name or accession number of a sequence in the Entrez database. printed. select FILE > Export Consensus. you may also open it directly using FILE > Enter Entrez Sequence. NET SEARCH menu commands may be used to search for similar sequences in a BLAST or text database. BLAST and Entrez Searching In MegAlign. 140 ● Further exploration Getting Started with Lasergene .• To export the consensus as a Lasergene DNA or protein file. Gaps in the sequence are represented by Xs. • To export the alignment for further analysis in PAUP. added to the MegAlign Worktable. See Online database searches for additional information. GCG Pileup or as a Phylip tree file. or saved. such as the NCBI BLAST or Entrez databases. viewed. select FILE > Save As and select the format you want.

PrimerSelect identifies and scores primers based on your specified criteria. The window has five views. Once you have selected and optimized your primer. Primer “Workbenches” allow you to modify your primer and see how any edits affect translated reading frames. and amplification products. Add one or more template sequences to the Document Window using any of the four methods. which display templates. 10 Quick-Start Steps for using PrimerSelect Here are ten basic steps for using PrimerSelect: 1. Once you have entered one or more template sequences. select it from the FILE > Recent Documents list. To reopen a document that has recently been opened in PrimerSelect. false priming sites and restriction sites. secondary structure. These primary calculations are all controlled by initial conditions that you set yourself. PrimerSelect processes the template for melting temperatures. PrimerSelect lets you create documents recording the theoretical activity of the templates. the principle working area in PrimerSelect. free energy. The Document Window is used to collect and inspect template sequences and to evaluate search results. you may specify a portion of the sequence by coordinates or by selecting a feature from the feature table. sequencing. Getting Started with Lasergene PrimerSelect • 141 . Select FILE > New to create a new project or FILE > Open to open a previously saved project. 2. If a sequence file is longer than you need. Sequences added to a project are arranged in the Document Window. After template processing. and hybridization experiments. primers and products. primers. The project begins when you enter a template sequence from any of a wide variety of sequence files or from NCBI’s BLAST or Entrez databases. and terminal free energy in pentamer windows.PrimerSelect Overview PrimerSelect helps you design primers and probes for PCR.

Activate a primer pair in the Located Primer Pairs window by double clicking on it. 8. You may save these settings for application to other template sequences using CONDITIONS > Save Conditions. Order your final oligonucleotide choices by using LOG > Oligo Request Form. Primer Pair Dimers. Define your starting conditions and desired primer characteristics. please contact a DNASTAR representative via telephone (608) 258-7420 or e-mail (support@dnastar. or enter your own primers. Search for primer pairs by using LOCATE > PCR Primer Pairs. 9. 4. select LOCATE > Only Catalogued Primers. PrimerSelect tutorials The tutorials in this chapter should be followed in order. Select LOCATE > Adjust Scoring to change the weight of such factors as internal stability and product length. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo Data 142 ● PrimerSelect tutorials Getting Started with Lasergene . To use only those primers in the catalog. by using LOCATE > Sort Primers. as they are designed to walk you through an example PrimerSelect project from start to finish.com). If you have any difficulties or questions. 7. 6. Primer Self Dimers. Sort the resulting primers. View results in tabular form by going to the REPORT menu and choosing Amplification Summary. if desired. 10. Composition Summary. Make any necessary modification to the active primers using the Workbenches. then FILE > Exit (Win) or PRIMERSELECT > Quit PrimerSelect (Mac) to exit PrimerSelect. Let PrimerSelect choose primers for you by using LOCATE > Primers & Probes. or Primer Hairpins. The mutated primers can then be saved in the primer catalog. The Further Exploration section at the end of the chapter highlights optional features you may wish to use as you gain experience with the software. FILE > Save your results.3. if desired. Defining primer design criteria Objective: To specify primer characteristics and their locations using the cloning vector pBR322 as the template sequence. 5.

• The default primer length has been set to 17-24 bp. 4. Click Add (Win) or Add File (Mac) to move the sequence into the Selected Sequences/Chosen Files list. Click Done to open the selected sequence in PrimerSelect’s Document Window.Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo Data Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo Data 1. The window should appear as follows: 5. and that dimer and hairpin duplexing of 1 or 2 bp will be accepted. Select FILE > Enter Sequence and highlight pbr322.seq from the Demo Data folder. Launch PrimerSelect. Select CONDITIONS > Primer Characteristics to open the dialog below. 2. Getting Started with Lasergene PrimerSelect • 143 . 3.

144 ● PrimerSelect tutorials Getting Started with Lasergene . Text boxes appear to permit you to define these numbers. or a combination of both. select Upper and Lower Primer Ranges. 8. From the Restrict Locations By list. a range in the template. Select CONDITIONS > Primer Locations to open a dialog for restricting primer locations based on a given product length.6. 7. Leave the default settings and click Cancel to exit the dialog.

In Lower Primer Locations enter 2250 and 2500 respectively. enter 1700 and 1950 respectively.• 9. The Located Primer Pairs window appears. Getting Started with Lasergene PrimerSelect • 145 . with the best choices shown at the top of the window. This is the best choice when designing PCR primers for a particular region of a sequence. 1. Paired green and red triangles mark the allowable ranges for the upper and lower primers. displaying a list of primer pair candidates. which now appears as follows. 11. In Upper Primer Locations. respectively. 10. Proceed to the next tutorial. Click OK to return to the Document Window. Searching for primer pairs Objective: To search automatically for the best primer pairs. Select LOCATE > PCR Primer Pairs.

• The upper pane of the Located Primer Pairs window shows the principle list of primer pairs. Open the advice curtain on the right side of the window by dragging (in the upper right corner) towards the left. Note that the top pair is annotated as the “Best choice. the lower pane currently shows a list of alternatives for the pair currently selected in the top pane.” 146 ● PrimerSelect tutorials Getting Started with Lasergene . • PrimerSelect ranked this primer pair based on criteria established in the Initial Conditions.2. as shown below. • Because (the Alternate Pairs palette tool) is active by default. PrimerSelect’s advice about each of the primer pairs now appears in the right column of the window. Primer Characteristics and Primer Locations dialogs located under the CONDITIONS menu.

4. Getting Started with Lasergene PrimerSelect • 147 . and possible secondary products. Note that some of the primer pairs have false primer sites. 5. Double-click the primer pair at the top of the window to make it active. Click each successive primer pair in the top pane and observe how the product bar in the lower pane becomes thinner. Viewing composition & hairpin reports Objective: To view reports showing the composition of the primers and information about possible hairpins. indicated by pink or light green arrows. denoting a decrease in the amount of product expected. (the Show Alternate Products palette tool) to change the lower pane into a display Click showing intended and false primer sites and potential products for the highlighted primer pair. denoted by dotted lines: Proceed to the next tutorial.3.

3. Select REPORT > Composition Summary to view the following table describing the composition of the primer pairs.1. Select REPORT > Primer Hairpins to view predicted primer hairpin structures in a window like the one below. • Other commands in the REPORT menu let you view self-dimers and pair dimers. 148 ● PrimerSelect tutorials Getting Started with Lasergene . Double-click the primer pair at the top of the window. 2.

Changing primer length Objective: To extend the length of a primer using the Upper Primer Workbench.4. Getting Started with Lasergene PrimerSelect • 149 . Proceed to the next tutorial. Leave the Located Primer Pairs and main PrimerSelect window (“Untitled”) open. When you are done viewing these windows. 1. With the top primer pair active. close them before continuing with the next tutorial. select EDIT > Work on Upper Primer to open the Upper Primer Workbench shown below.

Check the length in the header.3ºC. 3. The triangular “handles” at either end allow you to shorten or lengthen the sequence. Click the black triangle on the right side of the primer sequence and drag it one base to the left so that the length is shortened to 23 nucleotides. note the new. the primer sequence is shown in black. Click on the primer sequence in black. 2. (Show self-dimers). energetically favorable dimers that PrimerSelect has labeled with the warning “BAD!”. click the black triangle on the left side of the primer sequence and drag it to the left until the length has been extended to 31 nucleotides. Such primers may not amplify the region you want efficiently. Looking at the lower part of the window.8ºC. • The header in the upper left corner of the window now shows that the primer is 24 nucleotides long and has a melting temperature of 66. The header also shows that the melting temperature for the primer has increased to 77. • Below the ruler are messages showing that there are no dimers larger than 2 bp. and that the primer is able to form only one. About a third of the way down the Workbench. while a ruler near the bottom of the window shows the single possible annealing site as a short green bar. energetically unfavorable 2 bp hairpin.The palette tools hairpins) and (Show reading frames). (Show primer (Show false priming sites) are active by default. Then. adjusting as necessary until your header matches the one in the figure below. 150 ● PrimerSelect tutorials Getting Started with Lasergene .

3. but the duplicate sequence that appears below it in black now shows Ser where the Gly used to be. 6.3ºC). Ser appears in red to show that it is a mutation from the original primer sequence.Proceed to the next tutorial. In the Name field at the bottom of the workbench. Click S . A menu appears. which is very close to the melting temperature of the original 24 bp primer (66. you can modify a primer simply by clicking on any base and typing. note that the two “BAD!” dimers have been replaced by one unfavorable hairpin. but now the residues differ by only a single nucleotide. Getting Started with Lasergene PrimerSelect • 151 . • The Gly code in the original line of green text does not change. Proceed to the next tutorial. Objective: To improve the undesirable extended primer while keeping its sequence relatively unchanged. showing all four possible Glycine codons and the word Other. Introducing a primer mutation If desired. The original Ser residue had two substitutions compared to the original extended primer (TCT vs. presents a more “structured” method for modifying a primer. 4. The melting temperature has now been reduced to 63. • Looking at the bottom of the screen. Click the Ser residue and select AGT from the menu that appears. Click OK to save the mutant primer. This will be accomplished by changing a Glycine residue to Serine 1. GGT). 2. Click the Glycine (Gly) residue in frame 2. The check mark shows that GGT is the triplet encoding this particular Glycine residue. This tutorial. This makes a silent mutation to the Ser residue by changing the triplet that codes for Serine from TCT to AGT. however.3ºC. Select Other to open a menu with a list of amino acids.Serine. enter “Mutant Upper Primer”. written in green just below the primer. 5.

a useful way to evaluate whether primers you already have may be useful in a particular experiment. Double-click in the Name field to activate it. Double-click in the Sequence field to make it active. Besides adding primers to the catalog as above. Simply use any text-editing or spreadsheet program capable of saving files in text (. The new primer is now added to the catalog. Highlight the sequence. 1. 3. 6. Press the Tab key to enter a comment into the Note field. The new Sequence field will automatically be active. Macintosh) showing that it has passed initial secondary structure tests. and then type in a name. Windows) or bullet (•. and a chevron (>>. Select EDIT > Copy.Creating a new primer Objective: To create a new primer using the “mutated primer” sequence as a foundation within the Primer Catalog. 2. 9. Select EDIT > Paste to paste in the sequence you just copied.txt) format and use the Tab key to separate each field. 152 ● PrimerSelect tutorials Getting Started with Lasergene . 4. 8. Place the cursor at the end of the sequence and type ACGT. Select LOG > Primer Catalog to open the primer catalog for the current project. you may create catalogs manually outside PrimerSelect. • Note that the mutated upper primer created in the last tutorial is already present in the catalog. Press Return/Enter to create a line for entering a new primer. 5. To the left of the primer is a checkmark identifying it as active. 7. You can limit future primer searches to only those primers in the catalog. Close the Primer Catalog window to finish.

whose amplified products are displayed beneath the template sequence in the Block View. printed. simply click on it. select it and press Backspace (Win) or Delete (Mac). To add a primer pair to the list. Note: Close any open PrimerSelect projects and begin afresh before using this command. PrimerSelect considers all of the desired primer characteristics before sorting primers in the Located Primer Pairs window from best to worst. Matches from either search type may be opened in any Lasergene application. Getting Started with Lasergene PrimerSelect • 153 . Selecting a primer pair To retain information about multiple primer pairs found along the template sequence. The current pair. Creating an oligo request form To create an oligo request form with the active primers entered automatically.Further exploration Sorting primers by characteristic When you select LOCATE > Primers & Probes. select LOCATE > Sort Primers. the results will be combined with the results of any previous Primers & Probes search. select it from the Located Primer Pairs window and select LOCATE > Choose This Pair. To remove the check mark without discarding a pair from the list. Otherwise. Click elsewhere to make a new pair active. Additional commands in the NET SEARCH menu allow you to perform a text search of databases such as NCBI’s Entrez database. such as melting temperature. select LOG > Selected Primer Pairs. NET SEARCH menu commands may be used to search for similar sequences in BLAST databases. you may place your favorite primers in a special list. viewed with the browser. or saved. select LOG > Oligo Request Form > Generic or LOG > Oligo Request Form > IDT. the Located Primers & Probes window sorts the list of primers according to the specified criteria. To open the list. select LOCATE > Only Cataloged Primers. After using this command. Using only cataloged primers To perform a Primers & Probes search using only primers in the Primer Catalog. To re-sort primers according to a particular characteristic. is indicated by a check mark. BLAST and Entrez Searching In PrimerSelect. To discard a pair.

you may also open it directly using FILE > Enter Entrez Sequence. 154 ● Further exploration Getting Started with Lasergene .If you already know the locus name or accession number of a sequence in NCBI’s Entrez database.

Format the document as desired by using the EDIT menu. associated comments.EditSeq Overview EditSeq is a sequence editor and import/export tool. reverse complement. 10 Quick-Start Steps for using EditSeq Here are ten basic steps for using EditSeq: 1. 2. open an existing sequence from a variety of file formats. Use the SEARCH menu to locate ORFs or find sequence strings. Enter or edit data into the Sequence Pane and Comments Pane by typing. or open a sequence from a BLAST or text database. or move the cursor to a numerical position or line. and features in separate adjustable and editable panes. or by using the EDIT menu. You can trim a long sequence by specifying a portion of the sequence by coordinates. 3. A sequence saved using EditSeq in Lasergene DNA or protein sequence format may be opened by any other Lasergene module. Create a new EditSeq document or open/import an existing sequence file. or view statistics about any sequence or portion of a sequence. EditSeq shows the DNA or protein sequence. EditSeq’s search functions let you find positions and open reading frames instantly. Getting Started with Lasergene EditSeq • 155 . You can begin an EditSeq project with an empty document. You can complement. You may also translate a DNA sequence or back-translate a protein sequence using customized. expression-specific genetic codes. 4.

Create or edit feature annotations using the FEATURES menu. EditSeq tutorials The tutorials in this chapter should be followed in order. Print your results if desired.com). You may also translate a DNA sequence or reverse translate a protein sequence. Opening a subset of a sequence All Lasergene applications allow you to utilize a subset of a sequence if so desired. 6. The Further Exploration section at the end of the chapter highlights optional features you may wish to use as you gain experience with the software. 9. Save or export the sequence document. and exit from EditSeq.5. as they are designed to walk you through an example EditSeq project from start to finish. Search online for additional sequences using the NET SEARCH menu. 156 ● EditSeq tutorials Getting Started with Lasergene . Objective: To open a portion of an existing sequence file. 7. please contact a DNASTAR representative via telephone (608) 258-7420 or e-mail (support@dnastar. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign\Histone Sequences Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign\Histone Sequences Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo MegAlign:Histone Sequences 1. 8. 10. Launch EditSeq. Use the GOODIES menu to view DNA statistics or protein statistics for any highlighted portion of a sequence. Use the GOODIES menu to open a new sequence window containing the complement or inverse of a highlighted portion of sequence. If you have any difficulties or questions.

Locate Tethis21. Getting Started with Lasergene EditSeq • 157 . Click OK to return to the Open dialog. Select FILE > Open. From the Open dialog. Type 50 and 850 into the 5’ and 3’ end boxes. 5. • Note that the sequence length is 906 bp.2. Click Open to open the sequence in EditSeq. 3.seq from the Histone Sequences folder. Click on the sequence to highlight it. 7. 6. • 8. • Single-clicking a sequence lets you use the Set Ends feature and also causes sequence information to be displayed in the lower half of the dialog. Note that the sequence range is now 801 bp. 4. as shown below. click Set Ends.

• The upper right corner of the header shows that 801 bp of the original sequence has been opened in the upper pane of the window. The Search DNA dialog appears. Locating Open Reading Frames Objectives: To locate potential ORFs. • The top pane contains the sequence. Proceed to the next tutorial. and the bottom pane lists the features (annotations) included in the 50-850 bp range of the original sequence. the middle pane contains the sequence comments. 158 ● EditSeq tutorials Getting Started with Lasergene . 1. Select SEARCH > Find ORF.

which can be viewed in the Features Pane at the bottom of the EditSeq window. 4. we’ve typed “histone binding region”). Adding a feature with a translation Features are annotations that document points of interest in the sequence. then update an obsolete translation. Click on the box marked CDS at the beginning of the feature description to view the feature name options 3. EditSeq locates and highlights the first ORF in the Sequence Pane. Type a note about the feature if you wish. With bases 345-455 still highlighted. Proceed to the next tutorial. Assume that this portion of the sequence is an exon of interest. 2.2. the bottom pane in the EditSeq window. select FEATURES > New Feature with Translation. Insert the cursor between the quotation marks following the “/function” field. The current sequence already contains features. Getting Started with Lasergene EditSeq • 159 . (In the example below. Select GENE > Exon. The coordinates for the highlighted ORF appear in the EditSeq header. Click Find Next to close the dialog and locate the first ORF. • 3. The new feature and translation appear at the bottom of the Features Pane. EditSeq also lets you add your own features to a sequence. 1. Objectives: Create a new feature and an associated translation for a segment of the sequence. Click (Find Next) at the bottom left of the EditSeq window until you have located and highlighted the ORF at position 345-455. 5. Click directly on the word “/note” to display a menu of alternative qualifiers and choose “/function” from the list. Click anywhere outside the note area to complete the procedure.

Select FEATURES > Update Translation or FEATURES > Update All Translations to update the feature translation. Proceed to the next tutorial. Copying and translating sequences Objective: To copy the ORF and its associated feature into a new EditSeq document. Copy the ORF using EDIT > Copy. then to translate the ORF. The translation in the new feature turns red to notify you that changes have been made to the sequence. the translation will again appear black. The translation is now obsolete.6. 1. which now runs from 345-458. • 7. 160 ● EditSeq tutorials Getting Started with Lasergene . Highlight the modified ORF. After updating. by double-clicking anywhere on that feature in the Feature Pane (bottom). 2. Click anywhere in the Sequence Pane between bases 345-455 and then type three random bases.

Getting Started with Lasergene EditSeq • 161 . The lower pane of the translation window displays statistics about the translated sequence. viewed using a browser. matches may be opened in any Lasergene application. click No (Win) or Don’t Save (Mac). 4. • Note that the feature you created earlier has also been pasted into the document. • 7. Select the ORF using EDIT > Select All. Create a blank EditSeq document using FILE > New > New DNA. You may also use this menu to perform a text search of the databases. Searching a BLAST database In all Lasergene modules. features wholly included in the copied portion are also included. you can use the NET SEARCH menu to search for similar sequences in BLAST databases like that of NCBI. When you copy and paste sequence. In each application. 6. Features only partially included in the copied sequence are excluded. 5. or downloaded. Select GOODIES > Translate DNA to translate the sequence in a new protein sequence window. including the NCBI Entrez database. Paste the ORF into the blank document using EDIT > Paste. When prompted to save the document.3. Close all open EditSeq windows by selecting FILE > Close.

Highlight the entire sequence using EDIT > Select All.Objective: To search for matches to the Tethis21 sequence on the BLAST server at the National Center for Biotechnology Information. The Blast Query dialog appears.seq from the Histone Sequences folder. 162 ● EditSeq tutorials Getting Started with Lasergene . 2. view. The data for this tutorial can be found in the following location: Windows Vista: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign\Histone Sequences Windows XP/2000: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data\Demo MegAlign\Histone Sequences Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene:Demo MegAlign:Histone Sequences Note: Make sure your computer has access to the Internet before beginning this tutorial. 4. save. Keep the default settings and click OK to begin the search. • The upper pane contains the names of possible matches in order of probability. open Tethis21. EditSeq displays search results in a BLAST search results window. • The lower pane contains the alignment of the query to the highlighted database entry. Select NET SEARCH > BLAST Selection. • The buttons at the top of the window are used to open. or print the sequence matches. Select FILE > Open. 1. 3.

The web browser displays a database entry like the one below. Getting Started with Lasergene EditSeq • 163 .Viewing sequences located by online searches You may view more information about a sequence such as its length before downloading the sequence and its related features and comments. Click Launch Browser. Objective: To view detailed information about a particular sequence by opening its Entrez entry. Highlight any sequence in the BLAST search results window. 1. 2.

3. 2. A Save dialog appears. After viewing the entry. Saving sequences located by online searches Objective: To save sequences 5-6 from the Searching a BLAST database tutorial as EditSeq documents. close it and continue with the next topic. Highlight the sequence fifth from the top in the BLAST search results window and click Batch Save. Proceed to the next tutorial. 1. Select Next from the Save list box and type “2” into the text box. 164 ● EditSeq tutorials Getting Started with Lasergene .

Further exploration Importing and exporting sequences • To open a sequence file created with an outside software program. select FILE > Export All As One.3. FastA file or DNA Multi-seq file format. Size. Click OK. use drag and drop or select FILE > Import. Getting Started with Lasergene EditSeq • 165 . FastA file or GCG file. • To make a sequence easier to read. • To save a sequence as a GenBank Flat File. group it into blocks using EDIT > Layout. use FILE > Export. Changing layout options • To optimize fonts. sizes and cases select EDIT > Font. select EDIT > Use 3 Letter Codes. To Uppercase. • Click Set Location to select a different location. • To save all open windows of a particular type (DNA or protein) into a single file having a GenBank Flat File. Sequences are saved under their locus names using the Lasergene DNA file extension. or Reverse Case. • The default location for downloading sequences is specified using EDIT/EDITSEQ > Preferences. To Lowercase. • The default folder appears under the Save menu. • To display amino acids using their three-letter code designations.

If you already know the locus name or accession number of a sequence in NCBI’s Entrez database. viewed with the browser. To hear a tone. printed. select GOODIES > Reverse Complement. EditSeq can search BLAST databases for sequences similar to your own.Proofreading a sequence • To listen to a sequence as you type. Matches from either search type may be opened in any Lasergene application. or Slower. • To translate a highlighted portion of DNA sequence or reverse translate a highlighted portion of protein sequence. select SPEECH > Speed. • To optimize playback speed. or saved. select GOODIES>Reverse Sequence. Complementing. instead. • To translate or reverse translate using a genetic code other than the Standard Genetic Code. select SPEECH > Tones. select SPEECH > Proofread Sequence. 166 ● Further exploration Getting Started with Lasergene . Faster. See Performing a text search for an example of a text search. reversing and translating sequence • To create a new window containing the complement of a highlighted portion of sequence. Additional commands in the NET SEARCH menu allow you to perform a text search of databases such as NCBI’s Entrez database. select GOODIES > Translate DNA or GOODIES > Reverse Translate Protein. you may also open it directly using FILE > Open Entrez Sequence. or to playback a sequence that has already been entered. Searching BLAST and text databases As discussed earlier in this chapter. • To hear the sequence read via a speech synthesizer. • To reverse the order of a selected sequence. The genetic code file can be edited using GOODIES > Edit Selected Code. select SPEECH > Windows Voice/Macintosh Voice. select GOODIES > Genetic Codes.

Getting Started with Lasergene • 167 .