Chapter 5

5. Primer Designing &

Restriction Analysis
Goals:

 PCR primer construction & analysis

 Restriction digestion & mapping

Tools:
 Primer Analysis & Design
 NetPrimer
 Primer3Plus
 Primer-BLAST
 Restriction Mapping
 NEBcutter
 Webcutter
Primer Analysis & Design

A little something to get you in the mood…

Polymerase Chain Reaction (PCR)
1983-Kary Mullis
 very simple
 exponential amplification
 similar to natural DNA replication

The primary reagents, used in PCR are:

 Template DNA–DNA sequence to amplify
 DNA nucleotides–building blocks for new DNA
 Taq polymerase–heat stable enzyme catalyzes new DNA
 Primers–single-stranded DNA, ~20-50 nucleotides, complimentary to a short
region on either side of template DNA
Polymerase Chain Reaction (PCR)
1. Raise temperature (94-98),
denature DNA strands

2. Lower temp (50-65), anneal

primers

3. Increase temp (72-80), allow

time for extensions

4. Repeat process 25-40X

Things to consider for primer design…
 Primer-Dimer formation

 Secondary Structures in Primers

 Illegitimate Priming in Template DNA due to repeated sequences

 Incompatibility with PCR conditions

SOURCE: NCBI
Primer-Dimer formation

 Homology within a primer (self dimer) or between the sense and

anti-sense primer (cross dimer)
 Bonding of the two primers, increasing primer-dimer artifact and
reducing product yields
 Particularly problematic when the homology occurs at the 3' end of
either primer
SOURCE: NCBI
Self Dimer (example)
Internal
dimer

3’ end dimer

 The primer sequence is ATCAGCTGTAGAT

 It forms 2 dimers:
 Internal dimer where 3rd-8th bases of primer in 5‘3' (starting from 5') bond
with 6th-11th bases (starting from 3') when primer is placed in reverse direction
 3' end dimer where the last 3 bases (starting from 5') of primer placed in 5‘3'
direction bond with last three base (starting from 3') placed in reverse direction.

SOURCE: NCBI
Cross Dimer (example)

3’ cross dimer

 Sense primer sequence is ATCAGCTGTAGAT

 Anti-sense primer sequence is ATAGTGTAGAT
 Forms one cross dimer at the 3' end

SOURCE: NCBI
Secondary Structure in Primers
 Hairpin loop
 Formed when primer folds back upon itself

 Held in place by intramolecular bonding

 Can occur with as few as 3 consecutive homologous bases

 Stability measured by the free energy

The free energy of the loop is based upon the energy of the intramolecular bond and
the energy needed to twist the DNA to form the loop.
 If free energy >0, the loop is too unstable to interfere with the reaction

 If free energy <0, the loop could reduce the efficiency of amplification
Hairpin Loop (example)
3’ end hairpin

internal hairpin

 The primer sequence is ATCGATATTCGAAGAT

 It forms two hairpins:
 3' end hairpin where the primer folds back upon itself and first and last 3 bases bond together
 Internal hairpin where 2nd-5th and 9th-12th bases bond together

SOURCE: NCBI
Basic Primer Analysis & Design Software
 NetPrimer
 http://www.premierbiosoft.com/netprimer/

 Primer3Plus
 http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi

 Primer-BLAST
 http://www.ncbi.nlm.nih.gov/tools/primer-blast/
 NetPrimer
 http://www.premierbiosoft.com/netprimer/
 From PREMIER Biosoft
 Free
 Major features:
 Primer properties: Tm , molecular weight, GC%, optical activity (both in

nmol/A260 & µg/A260), DG, 3' end stability, DH, DS, and 5' end DG
 Secondary structures: Hairpins, dimers, cross dimers, palindromes, repeats and

runs
 Primer rating: Quantitative prediction of the efficiency of a primer

 Comprehensive report: Prints complete primer analysis for an individual

primer or primer pair

 Primer pairs: Analyze individual primers or primer pairs

 Comprehensive help: Details all the formulas and references used in primer

analysis algorithm
NetPrimer

Enter sequence here

NetPrimer—sense primer
NetPrimer—help
NetPrimer—theories & formulas
NetPrimer—antisense primer
NetPrimer—antisense hairpin

The most negative (i.e., most stable) DG

is used for calculating the rating.
NetPrimer—antisense dimer
NetPrimer—cross dimer
NetPrimer —3’ & 5’ stability
AN IDEAL PRIMER HAS A STABLE 5' END
AND AN UNSTABLE 3' END.

§ Unstable 3’ = limits bonding to false priming

sites.
 The lower this value, numerically, the more
liable the primer is to show secondary bands.

 less negative = less false priming.

§ Stable 5’ = called the GC Clamp, it increases
bonding to the target site.
 The lower this value, numerically, the more
efficient is the primer.
 more negative = better bonding.
NetPrimer—rating

The rating of a primer provides a quick way of

measuring the predicted efficiency of a primer as well
as choosing between closely matched primers.
 The higher the rating of a primer, the higher its
amplification efficiency.
 NetPrimer—DG
DG = DH – T * DS = free energy of the primer
DH = enthalpy (internal energy) of primer
T = temperature
DS = entropy (unavailable energy) of primer
Example: primer sequence = ATTCGCGGATTAGCCGAT
DG = -154500 cal/mol – (298.15 * -403 cal/°K/mol) = -34.35 kcal/mol

Rating = 100 + [(DG dimer * 1.8) + (DG hairpin * 1.4)]

Example: 100 + [(-10.36 kcal/mol * 1.8) + (-3.28 * 1.4)]

100 + [-18.648 + -4.592]
100 + -23.24
76.76
 The higher the rating, the better!
NetPrimer—practice primers
Rank these primers with attention to rating, 5’ end DG, and 3’
end stability

1. atgtgcgaggagaaagtgct
2. acaaaccctggacttgcatc
3. cgacttgtcccaggtgtttt
4. ctgaaaccattggcacacac
5. ggctgtgaacatggacattg
6. ggctgaagccaaagctacac
NetPrimer

 Ideal for checking primers

 To create primers, try Primer3Plus

Primer3Plus
 http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi

 Select primer pairs to detect a given template sequence

 Targets and included/excluded regions can be specified

 Steve Rozen and Helen J. Skaletsky (2000)

Primer3 on the WWW for general users and for biologist programmers. In:
Krawetz S, Misener S (eds)
 Bioinformatics Methods and Protocols: Methods in Molecular Biology. Human
Press, Totowa, NJ, pp 365-386
Primer3Plus
Primer3Plus

Design PCR primers to amplify sub region of the sequence

(600bp-2600bp) with product size 1800bp-2000bp.
Primer3Plus—getting started

click here to retrieve sample

sequence, then copy/paste into box
Primer3Plus Design PCR primers to amplify sub region of the sequence (600bp-2600bp)
with product size 1800bp-2000bp.
Primer3Plus Design PCR primers to amplify sub region of the sequence
(600bp-2600bp) with product size 1800bp-2000bp.
Primer3Plus—results
Primer3Plus—results
Primer3Plus—results
Primer3Plus—Primer3Manager
Primer3Plus—check primers
Primer3Plus—check primers
Primer3Plus—primer info
Primer3Plus—BLAST primers
Primer3Plus—BLAST primers
Primer3Plus—check w/NetPrimer

How good are these primers? Analyze

with NetPrimer!
Primer3Plus—NetPrimer sense

Left (F) primer

Primer3Plus—NetPrimer sense
Primer3Plus—NetPrimer antisense

Right (R) primer

Primer3Plus—NetPrimer antisense
Primer-BLAST
 http://www.ncbi.nlm.nih.gov/tools/primer-blast/

 Combines primer design (Primer3) and a specificity check (BLAST)

 Can also be used w/pre-designed primers

 ref: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412702/
Primer Design Tips
 RT-PCR (to avoid unwanted amplification of genomic DNA)
 Primer pair should span an intron

Or
 One of the primers should be at exon-exon junction

 SNP issues
 May cause mismatch, so pick primers outside of this region

 qPCR
 Specificity of amplification (amount of PCR product = fluor intensity)
Primer-BLAST

click here to retrieve

sample sequence, then
copy/paste into box
Primer-BLAST results
HSLS MolBio Primer Design Tools
Finding Primer Resources…

search.HSLS.MolBio
More Primer Databases
Review Questions
1. What is a primer? Describe the importance of primers.
2. Discus the roles of universal and specific primers
3. How do you design primers ? What are the conditions to consider
for designing primers ?
4. Discuss Primer specificity and efficiency
Restriction Mapping

www.biologyreference.com
Restriction Mapping—for your sequence

 Determine the # of restriction sites

 Determine the nucleotide position of each cut
 List the enzymes that do not cut
 List the enzymes that cut only once
 Graphical representation of the restriction sites
 Textual representation of the restriction sites
Restriction Mapping Tools

 NEBcutter
 http://tools.neb.com/NEBcutter2/index.php

 Webcutter
 http://bio.biomedicine.gu.se/cutter2/
NEBcutter V2.0
 From New England BioLabs
 Free
 Major features:
 Takes a DNA sequence and finds the large, non-overlapping open reading frames

using the E. coli genetic code and the sites for all Type II and commercially
available Type III restriction enzymes that cut the sequence just once.
 By default, only enzymes from NEB are used, but other sets may be chosen.

 Further options appear in the output.

 Maximum size of input file = 1 MB; maximum sequence length = 300 KB.
NEBcutter
NEBcutter—program guide
NEBcutter
NEBcutter—help
NEBcutter—getting started

click here to retrieve sample sequence,

then copy/paste into box
NEBcutter—restriction map
NEBcutter—cutters
NEBcutter—zoom in
NEBcutter—zoom in more
NEBcutter—zoom in more
NEBcutter—custom digestion

Get digestion map with SmlI and XbaI

NEBcutter—select enzymes
NEBcutter—custom digestion map

View gel
NEBcutter—agarose gel view
NEBcutter—ORF sequence

Find restriction enzymes that will

excise the selected portion of the
sequence.
NEBcutter—ORF sequence
NEBcutter—flanking sites
NEBcutter—ORF sequence
NEBcutter—silent mutagenesis
NEBcutter—excise a user-defined sequence
NEBcutter—excise a user-defined sequence
NEBcutter—enzyme information
NEBcutter—enzyme information
NEBcutter—REBASE enzyme page
REBASE—the restriction enzyme database
NEBcutter—enzyme information
NEBcutter—methylation sensitivity
NEBcutter—generate a vector map
NEBcutter—generate a vector map
NEBcutter—generate a vector map
 Sample DNA Sequence
You have cloned this mouse sequence:
Answer the questions on the
following page using NEBcutter.
TGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT
GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCA
AACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTG
CCAAATAAATCATCCTCATGTCC
ATTGGCGGATCCTGACTACACGCTGTCTTTCTGGCGGAATGGGAAAGTCCAGCACTGCCGCATCCACTCCCG
GCAGGATGCT
GGGACTCCTAAGTTCTTCTTGACAGATAACCTTGTCTTTGACTCTCTCTATGACCTCATCACACATTATC
AGCAAGTACCCCTGCGCTGCAATGAGTTTGAGATGCGCCTTTCAGAGCCTGTTCCACAGACGAATGCCCA
TGAGAGCAAAGAGTGGTACCACGCAAGCCTGACTAGAGCTCAGGCTGAACATATGCTGATGCGAGTGCCC
CGGGATGGGGCCTTCCTGGTGCGGAAACGCAATGAGCCTAACTCATATGCCATCTCTTTCCGGGCTGAGG
GAAAGATCAAGCACTGCCGAGTACAGCAGGAAGGCCAGACAGTGATGCTGGGGAACTCTGAGTTTGACAG
CCTGGTTGACCTCATCAGCTACTATGAGAAGCACCCCCTGTACCGCAAAATGAAGCTACGCTACCCCATC
AACGAGGAGGCACTGGAGAAGATCGGGACAGCTGAACCCGATTATGGGGCACTATACGAGGGCCGCAACC
CTGGTTTCTATGTGGAGGCAAACCCTATGCCAACTTTCAAGTGTGCAGTAAAAGCCCTCTTCGACTACAA
GGCCCAGAGAGAGGATGAGCTGACCTTCACCAAGAGTGCCATCATCCAGAATGTGGAAAAGCAAGATGGT
GGCTGGTGGCGAGGGGACTATGGTGGGAAGAAGCAGCTGTGGTTCCCCTCAAACTATGTGGAAGAGATGA
TCAATCCAGCAGTCCTAGAGCCTGAGAGGGAGCACCTGGATGAGAACAGCCCACTGGGGGACTTGCTGCG
AGGGGTCTTAGATGTGCCAGCTTGTCAGATCGCCATCCGTCCTGAGGGCAAAAACAACCGGCTCTTCGTC
TTCTCCATCAGCATGCCATCAGTGGCTCAGTGGTCCCTGGATGTTGCAGCTGACTCACAGGAGGAGTTAC
AGGACTGGGTGAAAAAGATCCGTGAAGTTGCCCAGACTGCAGATGCCAGGCTCACTGAGGGAAAGATGAT
GGAGAGGAGGAAGAAGATCGCCTTGGAGCTCTCCGAGCTTGTGGTCTACTGCCGGCCCGTTCCCTTTGAT
GAAGAGAAGATTGGCACAGAACGTGCTTGTTACCGGGACATGTCCTCCTTTCCGGAAACCAAGGCTGAGA
AGTATGTGAACAAGGCCAAAGGCAAGAAGTTCCTCCAGTACAACCGGCTGCAGCTCTCGCGCATCTACCC
TAAGGGCCAGAGGCTAGACTCCTCCAATTATGACCCTCTGCCCATGTGGATCTGCGGTAGCCAGCTTGTA
GCACTCAATTTCCAGACCCCAGACAAGCCTATGCAGATGAACCAGGCCCTCTTCATGGCTGGTGGGCATT
GTGGCTATGTGCTGCAGCCAAGCACCATGAGAGACGAAGCCTTTGACCCCTTTGATAAGAGCAGTCTCCG
AGGTCTGGAACCCTGTGTCATTTGCATTGAGGTGCTGGGGGCCAGGCATCTGCCGAAGAATGGCCGGGGT
ATTGTGTGTCCTTTTGTGGAGATTGAGGTGGCTGGGGCTGAGTACGACAGCACCAAGCAAAAGACGGAGT
TTGTAGTGGACAACGGACTGAACCCTGTGTGGCCTGCTAAGCCCTTCCACTTCCAGATCAGTAACCCAGA
GTTTGCCTTTCTGCGCTTTGTGGTGTATGAGGAAGACATGTTTAGTGACCAGAACTTCTTGGCTCAGGCT
ACTTTCCCAGTAAAAGGCCTGAAGACAGGATATAGAGCAGTGCCTTTGAAGAACAACTACAGTGAAGACC
TGGAGTTGGCCTCCCTGCTCATCAAGATTGACATTTTCCCTGCTAAGGAGAACGGTGACCTCAGTCCTTT
CAGTGGCATATCCCTAAGGGAACGGGCCTCAGATGCCTCCAGCCAGCTGTTCCATGTCCGGGCCCGGGAA
GGGTCCTTTGAAGCCAGATACCAGCAGCCATTTGAAGATTTCCGCATCTCGCAGGAGCATCTAGCAGACC
ATTTTGACAGTCGGGAACGAAGGGCCCCAAGAAGGACTCGGGTCAATGGAGACAACCGCCTCTAGTCAGA
CCCCACCTAGTTGGAGAGCAGCAGGTGCTGTCCACCTGTGGAATGCCATGAACTGGGTTCTCTGGGAGCT
GTCTACTGTAAAGCCTTCTTGGTCTCACAGCCTGGAGCCTGGATTCCAGCAGTGAAGGCTAGACAAAACC
AAGCCATTAATGATATGTATTGTTTTGGGCCTCCCTGCCCAGCTCTGGGTGAAGGCAAAAAACTGTACTG
TGTCTCGAATTAAGCACACACATCTGGCCCTGAATGTGGAGGTGGGTCCTTCCATCTTGGGCCAGGAGTA
GGGCTGAAGCCCCTTGGAAAGAGAAGTTGCCTCAGTTGGTGGCATAGGAGGTCTCAAGGAGCTGCTGACA
CATTCCTGAAAGAGGAGAAGGAGAAGGAGGAGGAGCCTTGGTGGGCCAGGGAAACAAAGTTTACATTGTC
CTGTAGCTTTAAAACCACAGGGTGAAAGAGTAAATGCCCTGCAGTTTGGCCCTGGAGCCAGGACAGAGGA
ATGCAGGGCCTATAATGAGAAGGCTCTGCTCTGCCCATGGAGGAAGACACAGCACAAGGGCACATTGCCC
ATGGCTGGGTACACTACCCAGCCTGAAAGATACAGGGGATCATGATAAAAATAGCAGTATTAATTTTTTT
TTCTTCTCAGTGGTATTGTAACTAAGTTATTCTGTCCTGCTCCTCACCTTGGAAGGGAAGACCCAGCACA
GAGCCTTTGGGAACAGCAGCTCTATGGGGTGTTGTACTGGGAGAGGGCACTGTCAAGAAGGGTGGAGGGG
CAGGAAGAGAGAAGAGCAATGTCTACCCTGGTGAGCTTTTTTGTTTTTATGACAAAGACGACTCGATATG
CTTCCCCTTAGGAATGGAGATATAGGTAAGTGGAGTCAGGCAGTAGGTACCAAATTAAGCTGCTGCTTGG
TGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT
GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCA
AACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTG
CCAAATAAATCATCCTCATGTCC
Sample Exercises
1. What is the %GC content of this Sequence?

2. How many restriction enzymes cut this sequence only once?

3. If you cut the sequence with Kpn I and Hinc II, how many DNA fragments will
be generated?

4. How many open reading frames (ORF) are present?

5. Find the restriction enzymes with compatible ends that can be used to excise
the largest ORF.
Sample Exercises Hints (NEBcutter)
1. What is the %GC content of this Sequence?
 See top left of page (after entering sequence info)
2. How many restriction enzymes cut this sequence only once?
 Select for single cutters
3. If you cut the sequence with Kpn I and Hinc II, how many DNA fragments
will be generated?
 Select Custom digest, then View gel
4. How many open reading frames (ORF) are present?
 Select ORF summary
5. Find the restriction enzymes with compatible ends that can be used to
excise the largest ORF.
 Select the ORF, then locate multiple cutters, cut positions
Webcutter 2.0
 http://bio.biomedicine.gu.se/cutter2/
 Free
 Major features:
 Rainbow cutters Highlight your favorite enzymes in color or boldface for easy at-a-
glance identification
 Silent cutters Find sites which may be introduced by silent mutagenesis of your coding
sequence
 Sequence uploads Input sequences directly into Webcutter from a file on your hard
drive without needing to cut-and-paste
 Degenerate sequences Analyze restriction maps of sequences containing ambiguous
nucleotides like N, Y, and R.
 Circular sequences Choose whether to treat your sequence as linear or circular
 Enzyme info Click into the wealth of references and ordering information at New
England BioLabs' REBASE, directly from your restriction map results
Webcutter

find alternate versions of the DNA which will

translate into the same amino acid sequence,
but contains a new restriction site
Webcutter

Mutate CCGGGT to CCCGGG to introduce Sma I cutting site without

changing translation
Webcutter—silent mutagenesis

click here to retrieve sample

sequence, then copy/paste into box
Webcutter—results
Webcutter—specific restriction enzymes
Thank you!
Any questions?

Alemayehu Letebo
MSc in Biotechnology
Phone:+251964404740
Email: alemayehu.letebo@amu.edu.et
Sequence Manipulation

www.vam.ac.uk/images/image/44010-large.jpg
Sequence Manipulation Tools

 READSEQ
 http://www-bimas.cit.nih.gov/molbio/readseq/
 Sequence Manipulation Suite
 http://www.bioinformatics.org/sms2/
READSEQ

Format your sequence any way you want

READSEQ—change formats

click here to retrieve sample sequence,

then copy/paste into box
READSEQ—FASTAGenBank

FASTA GenBank
Sequence Manipulation Suite
SMS—filter DNA
removes non-DNA characters from text
SMS—reverse complement
converts DNA to its reverse and/or complement
counterpart
SMS—group DNA
adjusts the spacing of DNA sequences and adds
numbering
SMS—primer map creates a map of the annealing positions of PCR
primers
SMS—DNA pattern find
locates regions that match a sequence of interest
SMS—DNA stats
finds # of occurrences of each residue
SMS—translate
converts DNA sequence into protein