List of Figures and Information Required for Lab Report 1

RESULTS:

1. MOAP-1 sequence and underlined primer sequences

2. Forward and Reverse primer sequences and Tm
3. Agarose gel electrophoresis of PCR product (labelled gel picture)
4. Real Time PCR:
a. Labelled Picture of Real Time PCR amplification (RFU/Cycle)
b. Labelled Picture of Melt Curve
c. Labelled Picture of Melt Peak
d. Labelled table of Data (Excel), complete with DNA copy number, and log copy number
(refer to Tutorial 2) of standards and Unknown (Male and Female samples)
e. One sample calculation for getting DNA copy number (one of the standards, 50 ng)
f. One sample calculation for getting the log copy number (one of the standards, 50 ng)
g. Picture of Standard Curve (Cq versus Log copy number)
h. One sample calculation for getting the DNA copy number and quantity of the male and
female samples from the standard curve (refer to Tutorial 2)
5. Labelled pictures of dot-blot (antibody detection of EV71)
6. Table to summarize the results from Lab 1, Lab 2, and Lab 3

Lab 1 Lab 2 Lab 3 Conclusion:

Indicate Indicate Indicate +/- or Inconclusive
+/- or others +/- or others +/- or others
Male No. Female Male No. Female No. Male No. Female Male: ?
No. No. Female: ?
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DNA sequencing methods

and Next Generation DNA sequencing
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Molecular Diagnostics through DNA sequencing

•DNA sequences serves as molecular signatures of all living organisms

•It enables identification of pathogens, including bacteria and viruses
•It enables identification of gene mutations, deletion, insertion, and etc in human genome
•DNA sequencing technologies allow identification of DNA polymorphisms, including SNPs
•DNA sequences can be used to determine primary protein structure
•DNA sequencing can be used to identify DNA methylation
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Overview of DNA sequencing technologies

• Chemical sequencing : Maxam-Gilbert method

• Sequencing DNA using Di-deoxy chain termination method:
a) Sequencing single stranded DNA obtained through M13 bacteria-phage
b) Sequencing double stranded DNA through alkaline method
c) Sequencing double stranded DNA through PCR
• Pyro-sequencing
• Bisulfite DNA sequencing: Detection of DNA methylation
• RNA sequencing through cDNA and PCR amplification
• Next Generation DNA sequencing
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Molecular Diagnostics: Bio 2064

Maxam-Gilbert Chemical Sequencing Method

•DNA sequences are labeled at either 5’ or 3’ end with radioactive ATP labeled with gamma-P 32 using
Polynucleotide Kinase (5’ end labeling) or Terminal Transferase (3’ end labeling)
•Using chemicals to selectively modify or react with specific bases, resulting in DNA strand breaks
•Sequences are determined from 4 chemical reactions that lead to DNA strand breaks
•Disadvantages:
a) Relatively inefficient method for DNA sequencing, applicable to short DNA sequencing (<100 base pair)
b) Use of chemicals that are hazardous
c) Requires the use of DNA gel

References :https://www.nationaldiagnostics.com/electrophoresis/article/maxam-
gilbert-sequencing
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Maxam-Gilbert Chemical Sequencing Method

http://classroom.sdmesa.edu/eschmid/Lab17-Biol210.htm
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Maxam-Gilbert Chemical Sequencing Methods

http://classroom.sdmesa.edu/eschmid/Lab17-Biol210.htm
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Dideoxy DNA sequencing method: Sanger Sequencing method

• Using dideoxy nucleotide to terminate DNA polymerase chain reaction

• dNTPs are mixed with specific dideoxy nucleotides in the presence of
radioactive nucleotide (P32) to generate 4 chain termination reactions
• Terminated reactions generate DNA fragments of various length
which can be separated on DNA sequencing gel
• DNA sequence is read from bottom of gel
• Disadvantages:
1) Sequence relatively short DNA (<500 bp)
2) Requires the use of DNA gel
3) Band compression due to specific DNA sequences (GC rich)
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Generation of single stranded DNA for sequencing using M13 bactriophage

• Clone gene of interest in plasmid DNA

• Transform E.Coli with the plasmid DNA
• Infect E.Coli with M13 bacteriophage (helper phage)
• Isolation of packaged single stranded DNA from M13 bacteriophage
• Sequence single stranded plasmid DNA

Reference:http://rzv054.rz.tu-bs.de/Biotech/SD/M13LiveCycle.jpg
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Dideoxy Sequencing (Sanger Sequencing Methods)

• In the presence of dNTPS, dideoxy nucleotide

(ddGTP, ddATP. ddCTP or ddTTP) and DNA polymerase,
chain termination will occur,
resulting in short fragments of DNA
• DNA fragments are separated in DNA sequencing gel
• The gel is subsequently transferred to Whatman paper and air dried
• Died gel is then exposed to X-ray film

Reference: http://hshgp.genome.washington.edu/teacher_resources/modules-view.htm
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Sequencing Gel System: Gel Tank and Comb

DNA sequencing gel

Shark Tooth Comb

http://www.geneq.com/images/products/jqt182.jpeg
References: http://www.cbsscientific.com/images/products/detail/sharktoothcombs.8.jpg
http://www.genetiks.org/genomics281.html
 Dideoxy Sequencing Using P32 (radioactive)

Reference: http://www.siumed.edu/~bbartholomew/course_material/methods_B.html:
Dideoxy Sequencing Using Fluorescence-labeled Dideoxy Nucleotide

http://compbio.pbworks.com/w/page/16252893/Genome%20Sequencing%20and%20Annotation
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Dideoxy Sequncing (Sanger Sequencing) using fluorescence dyes

Fluorescence Dideoxy
Nucleotide

Dideoxy Nucleotide

Capillary Electrophoresis

• Dideoxy nucleotide tagged with fluorescence

• Four fluorescence colors are used, one for each dideoxy-nucleotide
• Fluorescence labelled DNA fragments are separated on capillary tube
Dideoxy Sequencing Fluorescence DNA fragments

http://www.bio.davidson.edu/genomics/method/capillary.gif
http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-
References: technology/dna-sequencing-and-fragment-analysis/overview-of-dna-sequencing/faqs/data-
images.Par.64513.Image.310.171.1.gif.faqs_data_fig2_gif.gif

http://bio3400.nicerweb.com/Locked/media/ch19/19_28-dideoxynucleotides.jpg
 Capillary Gel Electrophoresis

Ref:https://www.mun.ca/biology/scarr/Capillary_electrophoresis.html

• Voltage is applied to load the sequencing reaction from micro well into capillary gel
• DNA fragments are separated based on size and short DNA fragment moves
faster than long DNA fragment inside the capillary gel
• Near the end of capillary gel, laser is used to excite the fluorophore, and the
emitted fluorescence is read by photometer
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Molecular Diagnostics: Bio 2064

Methylation Specific PCR (MCP) and Bisulfite Sequencing

• Bisulfite Reaction: When treated with Bisulfite (NaHSO3), Cytosine is converted to Uracil
• However, 5-Methycytosine is resistant to Bisulfite conversion
• Design specific primers to amplify the Bisulfite treated DNA (template), and clone PCR amplified DNA for sequencing

Reference: Alpha Bio-laboratory (http://alphabiolab.com/page2/page2.html)

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Molecular Diagnostics: Bio 2064

• Design primers
DNA Sequencing Approach • Primer walking to cover the entire length of DNA

Reference: Lilian Et Al. Quarterly Reviews of Biophysics 35, 2 (2002), pp. 169–200
Next Generation Sequencing
 FireFly Luciferase: Reporter Gene Assay
and Biosensor of ATP
• Firefly Luciferase is responsible for generation of light in Firefly
• Firefly Luciferase is a 62 Kda protein
• Firefly Luciferase gene has been used in gene reporter assays
• Firefly Luciferase is used to detect microbial contamination, which is a rich source of ATP
Cheese Sample: Microbial Contamination

http://insecta.blogfa.com/
https://www.mirusbio.com/products/accessories/firefly-luciferase-assay-kit
https://www.hindawi.com/journals/js/2016/1492467/fig3/
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Pyro-sequencing
adenosine 5´ phosphosulfate
(APS)

• dNTP nucleotides are added in certain order until one of the dNTP is incorporated by DNA polymerase
• Incorporated nucleotide frees up two inorganic phosphate (PPi) which reacts with Adenosine 5’ phosphosulfate (APS) by Sulfurylase
to generate ATP
• Free ATP and Luciferin are used by Luciferase enzyme to generate OxyLuciferin and light
• The light is recorded by CCD camera as “signal’ for incorporation of the nucleotide into DNA, and the intensity and identity of the
nucleotide are recorded
• Apyrase is used to degrade unincorporated nucleotides, and ATP so that the next sequencing cycle can continue
Ref: http://www.clinchem.org/content/55/5/856/F1.expansion
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Roche /454 FLX Pyro-sequencer

• Generate genomic DNA fragments

•“Flush ends” of DNA fragments,
and ligate it to linker/adapter
• Oligonucleotide complementary to the
linker/adapter-DNA fragment are purified
and selected through binding to
oligonucleotide—conjugated beads
(‘Capture Beads)

.
Reference;:Mardis ER Annu. Rev. Genom. Human Genet. 2008.9:387-402
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• PCR reactions are carried to amplify DNA

fragments and captured the DNA fragments
on Capture beads
• Eventually, the beads would capture
multiple copies of DNA strands
• Each bead is then subjected to pyro-
sequencing

Reference: Mardis ER Annu. Rev. Genom. Human Genet. 2008.9:387-402.

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Molecular Diagnostics: Bio 2064

• Up to 400,000 beads obtained in parallel

• Each bead/well contain multiple copies of single stranded DNA from a single cloned ssDNA
• Nucleotide solution (T/C/G/A) is loaded into each well containing bead
• Light emitted is recorder on CCD camera
• Will have problem reading homo-polymers (>6 of the nucleotide)
• Susceptible to base insertion or deletion error during reading
• Average read length is about 250 nucleotides
• During 8 hour run, could produce up to 100MB quality data
Reference: Mardis ER Annu. Rev. Genom. Human Genet. 2008.9:387-402.
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Molecular DIAgnostics: Bio 2064

Illumina Genome Analyzer

• Fragmented genomic DNA fragments are ligated to adapter linker
• Single stranded DNAs are attached to a surface inside a flow cell
• In the flow cells, there is dense lawn of primer that binds to the single stranded DNA
• Single stranded DNAs are amplified through bridge amplification
• DNA polymerase incorporates one base only after all four fluorescence-tag
bases (TACG) with blocked 3’-OH are added.
• The fluorescence from the incorporated base is captured by CCD camera
• The sequence is extended after the blocked 3’OH is removed to allow
incorporation of next nucleotide .The approach used is Sequencing-By-Synthesis
with allows discrete read length of 25-25 base, depending on number of user defined cycles
• Millions of DNA fragments can be sequenced in parallel using the system
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Molecular Diagnostics: Bio 2064

Illumina Genome Analyzer

Reference: Mardis ER Annu. Rev. Genom. Human Genet. 2008.9:387-402

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Molecular Diagnostics: Bio 2064

Illumina Genome Analyzer

https://www.youtube.com/watch?v=9YxExTSwgPM

Reference: Mardis ER Annu. Rev. Genom. Human Genet. 2008.9:387-402

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Applied Biosystem:SOLid Sequencer

• Ligate Dibase probe (Fluorescence labeled) to the primer to extended the sequence
• Once Dibase probe binds, to the template, excite the dye, and fluorescence image is taken,
and the fluorescence probe is cleaved off to allow next ligation cycle to occur for sequencing
• Dibase probes are ligated to primer with different length (n-1, n-2, n-3, and n-4) which bind to
cover the entire DNA template
• Read length is about 25-35 base pairs, and each sequencing produce 2-4 Gb of DNA sequencing Data
Reading Sequencing information

http://www.scielo.org.za/scielo.php?script=sci_arttext&pid=S0038-23532012000600016
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Applied Biosystem: SOLid Sequencer

Reference: Mardis ER Annu. Rev. Genom. Human Genet. 2008.9:387-402

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Summary
• DNA sequencing technologies have evolved over the years from
chemical, dideoxy sequencing to next generation sequencing
• Chemical and dideoxy sequencing yield short DNA sequence data
(100-500 bp) due to limitation of gel resolution
• Next generation sequencing using technologies without having the need
to use gel for separation of DNA fragments
• Next generation sequencing using Pyro-sequencing that is
based on light or fluorescence technologies
• Amplification of DNA fragments (million of copies) allow sensitive and high through-put
sequencing (Mb-Gb)