WEEK 9: DNA SEQUENCING

MOLECULAR BIOLOGY AND DIAGNOSTICS | LECTURE

Prof. Justin Kim Vergara, RMT, MPH | College of Medical Laboratory Science | A.Y. 2022 - 2023

• DNA sequencing is a common molecular technique

CHEMICAL (MAXAM-GILBERT) SEQUENCING
that is used to determine the exact sequence of
bases in a DNA molecule.
• e.g. You wanted to know the exact nucleic acid • A direct manual sequencing method.
• One of the earliest sequencing methods that was
sequence of a particular gene. It carries the
developed.
information which is needed in the assembly of
• Uses different types of chemicals to know and to
protein or RNA molecule.. treat DNA.
• It is also used in investigation of functions of gene or • Developed in the early 1970s.
in gene expression. We should know what the exact • This method is already obsolete.
sequence of a particular DNA is. • Disadvantage: does not produce high throughput for
long fragments of DNA.
• Developed by Allan M. Maxam and Walter Gilbert
TYPES OF SEQUENCING • Efficient way to determine short runs of sequence
data.
• These are used to identify not only the exact • Maxam–Gilbert sequencing required a double- or
sequence of DNA but also in detection of mutations, single- stranded version of the DNA region to be
in typing organism, and identification of sequenced, with one end radioactively labeled.
polymorphism in the genes. o It requires a template.
1. Direct Sequencing
a) Manual Sequencing
b) Automated Sequencing
2. Pyrosequencing
3. Bisulfite DNA Sequencing
4. RNA Sequencing
5. Next-Generation Sequencing

DIRECT SEQUENCING

• Most definitive molecular method to identify genetic

lesions.
• Problem: you can only use this for short or medium
sized DNA. We cannot use it for Genomic DNA,
megabase DNA, or very long DNA.
• There are two approaches in direct sequencing,
namely:
1. Manual Sequencing
o Direct determination of the order, or
sequence, of nucleotides in a DNA polymer.
o We have to visually inspect the band in gel
electrophoresis.
o It involves gel electrophoresis.
o A very good method if you wanted to know
or identify mutation or polymorphisms
especially when looking for changes
affecting only one to two nucleotides.
o Usually used for single base changes. • Prepare four different tubes. For each tube, there is
2. Automated Fluorescent Sequencing a specific chemical used and each chemical has
o Fluorescent dyes used for sequencing have different ways on how to modify the DNA.
distinct “colors,” or peak wavelengths of • During the sequencing, we have the template
fluorescence emission, that can be (sample) which will be allocated into four different
distinguished by automated sequence. tubes.
o Uses advanced computer and software to • Add sample, plus:
yield a result, at the same time, for the o Dimethylsulphate
interpretation of the result. o Formic acid
o Uses a special type of dye which is o Hydrazine
fluorescent dye to check the exact o Hydrazine + Salt
sequence of DNA. • For each base modifier, there is a specific target to
o The dyes are used to identify the react:
nucleotides in the DNA. o Dimethylsulphate is used to methylate
Guanine.
▪ all of the sequences will be
restricted or cut on their Guanine,

1 | Molecular Biology and Diagnostics

Transcriber: admn
 o Formic acid is used to Protonate purine. • Dye first the primer. Usually primer put labels on 5’
▪ All of the DNA will be cut on their end. One of the labels is the fluorescent tag or
purine (adenine and guanine). fluorescent label.
o Hydrazine is used to split pyrimidine rings. • The 5’ end of primer should be labeled either
▪ All of the DNA will be cut on their radioactive label or fluorescent label.
pyrimidine (cytosine and thymine). • Modification of the DNA replication process
o Hydrazine + Salt is used to split only C • A short, synthetic, single-stranded DNA fragment
rings. (primer) complementary to sequences just 5ʹ to the
▪ Specifically cut on Cytosine rings. region of DNA to be sequenced.
• After treating with chemicals, add 10% Piperidine so • For detection of the products of the sequencing
that all single stranded DNA would break at specific reaction:
nucleotide.
• Base modifier + 10% piperidine= it would break at a 32P-labeled nucleotide or a fluorescent dye-
specific nucleotide. labeled nucleotide → 5’ end.
• After treatment, resolve the sample on gel
electrophoresis.
DIDEOXYNUCLEOTIDE (ddNTP)
MAXAM-GILBERT SEQUENCING RESULT
• Separated by PAGE (Polyacrylamide Gel • Difference of dNTP and ddNTP: Instead of hydroxyl
Electrophoresis) group, there is only hydrogen on the 3rd carbon of the
• Advantage of using Polyacrylamide instead of ddNTP.
agarose gel: It could separate single base changes, • Importance of the hydroxyl group on 3rd carbon
even single base changes could be detected using during DNA synthesis: binding of phosphate group
polyacrylamide gel. There is base separation or on the next nucleotide base.
differences in the bands even if there’s only one • But once there is loss of oxygenation on the 3rd
nucleotide base that is different. carbon, the phosphate group of the next nucleotide
• The sequence was inferred from the bands on the base will not bind. It will not form phosphodiester
film. bond.
• The lane in which that band appeared identified the • Lacks the hydroxyl group found on the 3ʹ ribose
nucleotide. carbon of the deoxynucleotides.
• Load the reaction mixture on each sample well. Load • Chain Termination
the sample on wells containing DMS, formic acid, o DNA synthesis will stop upon incorporation
hydrazine, hydrazine + salt. Then run. of a ddNTP into the growing DNA chain.
• After running in gel electrophoresis, it will produce
bands.
• Based on the bands formed, we could deduce what
is the exact sequence of the DNA.
• Cutting of DNA: 5’ to 3’.
• The shorter the DNA cut using chemicals, the farther
the migration will be.
• The first band that will migrate will be the 5’ end of
DNA.
• e.g. the band is placed in the 2nd lane: guanine and
adenine (purine). We couldn’t know if it was guanine
or adenine. To confirm, since the first well specifies
that the DNA has been broken at the Guanine, if the
band on the 2nd lane is Guanine, there should be a
band seen on both the 1st and 2nd well.
• But in the example, there is only band seen on 2nd
well but not on the 1st well, thus it is Adenine. The
first nucleotide on the 5’ end is Adenine.
• You have to deduce the sequence based on the
bands. Starting from the bottom going to the top.
From 5’ end to 3’ end.

DIDEOXY CHAIN TERMINATION (SANGER)
SEQUENCING
• Disadvantage of Maxam-Gilbert: exposure to
chemicals such as hydrazine and piperidine. Toxicity
of chemicals used is the reason for the development
of new method of sequencing.
• In Sanger sequencing, it uses the same components
of PCR. It has template, primer, enzyme that will
catalyze the addition of dNTPs.
• But instead of dNTPs, ddNTP will be added.
(dideoxynucleotide triphosphate).
• If you have the hydroxyl group on the 3rd carbon,
there will be binding between the hydroxyl group and
the phosphate group of the next nucleotide.
• If the 3rd carbon is deoxygenated, no nucleotide will
be added to the growing chain of the DNA.
DIDEOXY CHAIN TERMINATION (SANGER) PROCEDURE
1. 1:1 mixture of template and radioactively labeled
primer → four separate reaction tubes.
o aside from radioactive label, fluorescent
label can be use.
o make a mixture of the template, primer,
dNTPs, ddNTPs and enzyme.

2 | Molecular Biology and Diagnostics

Transcriber: admn
2. Mixtures of all four dNTPs and one of the four
ddNTPs are then added to each tube, with a different
ddNTP in each of the four tubes.
o Increase ddNTPs: polymerization will
terminate too frequently early along the
template.
▪ The growing chain of DNA will
stop.
o Decrease ddNTPs: infrequent or no
termination.
o Ensure that the amount of ddNTP and
amount of dNTP that will be added for each
tube should be correct.\
o The ratio between the dNTP and ddNTP is
critical to generate a readable sequence.
o For the manual sanger sequencing, you
have to manually stop the reaction.
o After mixing the template, primer, dNTP,
ddNTP, that will react for 20 minutes. Then
manually, add the stop buffer.
3. Reaction begins after addition of DNA polymerase.
4. After 20 minutes, the reactions are terminated by
addition of a stop buffer.
o 20 mM EDTA- to chelate the calcium and
magnesium.
o Formamide- used to maintain denatured
products in order for the synthesis reaction to
occur.
o Gel loading dye/ Tracking Dye- it has two types:
o bromophenol blue (faster moving dye)
o xylene cyanol (slower moving dye)
• Prepare 4 reaction tubes. For each reaction tube, it
contains all of the four dNTP (dATP, dGTP, dCTP,
dTTP) + 1 ddNTP.
• For 1st tube, it specifies that it is only used for
adenine residues. To have a termination, use
ddATP.
• For 2nd tube, it has ddCTP so that the termination will
stop on Cytosine. Then we have ddGTP, and ddTTP.
• All of these reactions are carried out for equal time.
All have the same volume to provide consistent band
to know if you have more DNA or lesser DNA in the
sample.
• e.g. 5’--3’ (has the enzyme)
3’-A T C G-5’
(dTTP) (dATP) (dGTP) (ddCTP)
In ddCTP, the formation of DNA will stop. Once
ddCTP is added, the growing chain of DNA will stop.
• e.g. there is a reaction, all of the tube that contains
ddATP have fragments that ends with ddATP.
• In here, you will produce different sizes of fragments
of DNA. The fastest migrating amplicon will be the
smallest fragment. The longest fragment will be the
slowest or the migration will be near the sample well.
• To resolve these, we use polyacrylamide gel
electrophoresis.
POLYACRYLAMIDE GEL ELECTROPHORESIS
• We could be able to identify the bases present on
DNA by visually inspecting.
• The products of each of the four sequencing
reactions are loaded into adjacent lanes → Labeled
A, C, G or T.
• The fragment patterns are visualized by the signal on
the 32P-labeled primer (or incorporated
deoxynucleotide).
• All fragments from a given tube will end in the same
ddNTP.
o e.g. all the fragments synthesized in the
ddCTP tube end in C.
SEQUENCING LADDER
• We can use smaller polyacrylamide or larger
polyacrylamide. If there is a lot of fragments that will
be formed once you tested the sample, you have to
use longer gels. But if you have very short DNA to
sequence, use a smaller gel.
• Load the sample on the well, apply electricity so that
the substances will migrate towards the anode. The
fastest migrating band will be the first nucleotide
present in DNA. From 5’ end going to 3’ end.
• The four-lane gel electrophoresis pattern of the
products of the four sequencing reactions.
• The sequence is read from the bottom (smallest, 5ʹ-
most) to the top (largest, 3ʹ-most) fragments across
or within lanes.
CYCLE SEQUENCING
• Used for automated sanger sequencing.
• Uses PCR or thermal cycler to create sequences and
to know the exact sequences of DNA.
• Sample is the template itself.
• The sequencing reaction took place in a thermal
cycler.
• Timed manual starting and stopping of the
sequencing reactions were not necessary.
• Using heat-stable enzymes
o With in vitro removal of the exonuclease
activity.
o Using double stranded template.
3 | Molecular Biology and Diagnostics
Transcriber: admn
 o Used for catalyzation or to lengthen DNA.
AUTOMATED FLUORESCENT SEQUENCING
• It uses a double stranded template.
• Relies on stain used. Fluorescent dyes are used
instead of radioactive for safety.
• Universal systems combined automation of DNA
isolation of the template and setup of the sequencing
reactions.
• Electrophoresis and reading of the sequencing
ladder were also automated.
• Two detection system: by gel electrophoresis or
capillary electrophoresis
• A requirement for automated reading of the DNA
sequence ladder is the use of fluorescent dye to label
the primers or sequencing fragments.
o Fluorescein
o Rhodamine
o Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-
indacene)
o These three are dye derivatives that are
recognized by commercial detection
systems. Not all stains could be identified
by the automated fluorescent sequencing.
There are certain brands of dye that cannot
be used.
• Fluorescent dyes used for sequencing have distinct
“colors,” or peak wavelengths of fluorescence
emission.
• Fluorescent dye color rather than lane placement will
assign the fragments as ending in A, T, G, or C in the
sequencing ladder.
• It is mentioned that we could deduce the sequence
by just knowing what the length is and what are the
nucleotides included. The fastest migrating
nucleotide is the first nucleotide present in the DNA,
usually on the 5’ end.
• We can use gel electrophoresis to separate each of
the nucleotides or use capillary electrophoresis.
• In capillary electrophoresis, the separation is based
on the charge and size. The one that is first detected
by the computer will serve as the first nucleotide on
the 5’ end of DNA.
APPROACHES TO AUTOMATED SANGER SEQUENCING
• There are two approaches to automated fluorescent
sequencing:
a) DYE PRIMER- the primer itself is labeled
(in the 5’ end).
b) DYE TERMINATOR- the ddNTP is labeled.
• The goal of both approaches is to label the fragments
synthesized during the sequencing reaction
according to their terminal ddNTP.
• The type of fluorescent to be used should be specific
for the nucleotide that has been added.
o ddATP → A → Green dye
o ddCTP → C → Blue dye
o ddGTP → G → Black or Yellow dye
o ddTTP → T → Red dye
• Once it pass in the capillary tube, it has laser, and
detector.
• e.g. the 1st molecule that pass through the laser is
DNA that contains a label which transmit yellow.
o The molecule will pass through laser, it will
be activated or excited and will transmit
fluorescent light which is color yellow. Thus,
the fastest fragment has guanine. The first
nucleotide base present on the sequence is
Guanine which is near the 5’ end of primer.
▪ 5’-G
o e.g. The 2nd molecule fluoresces green
color, thus, the second fragment has
Adenine.
▪ 5’-GA
o e.g. Next the 3rd molecule, it fluoresce red.
thus it has Thymine.
▪ 5’-GAT
o The more that we add, the longer the
fragment gets.
A. DYE PRIMER SEQUENCING
• Uses four separate tubes. For each tube we have
primers which has different colors.
o e.g. 1st tube: blue= it has all fragments
that end with nucleotide Cytosine.
2nd tube: green= it has all fragments that
end with nucleotide Adenine.
3rd tube: yellow= it has all fragments that
end with nucleotide Guanine.
4th tube: red= it has all fragments that end
with nucleotide Thymine.
• There is a specific color that has been added to the
primer.
• PCR will be used in this approach (automated). No
need to use stop buffer.
• The four different fluorescent dyes are attached to
four separate aliquots of the primer.
• The dye molecules are attached covalently to the 5ʹ
end of the primer.
• The primer labeled with each “color” is added to four
separate reaction tubes, one each with ddATP,
ddCTP, ddGTP, or ddTTP.
Sequencing reaction components (addition of
nucleotides) + Heat stable polymerase (will catalyze
the addition of either ddNTP or dNTP) → Cycle
sequencing → 5’ labeling.
• The products of the sequencing reaction are then
labeled at the 5ʹ end, using the dye color associated
with the ddNTP at the end of the fragment.
B. DYE TERMINATOR SEQUENCING
• Primer is unlabeled.
• The ddNTP is labeled.
o ddTTP: red
o ddGTP: yellow
o ddATP: green
o ddCTP: blue
• Once ddNTP is added, there will be a termination on
the growing chain. We can identify what is the exact
nucleotide based on the fragments formed.
o e.g. -----green
-------green
----------yellow
------------red
---------------blue
The sequence formed is:
5’-AAGTC-3’
4 | Molecular Biology and Diagnostics
Transcriber: admn
 o Dye Primer- 5’ (the one labeled is primer).
o Dye Terminator- 3’ (the one labeled is
ddNTP).
• One of the four fluorescent dyes covalently attached
to each of the ddNTPs.
• All four sequencing reactions are performed in the
same tube.
Sequencing reaction components + Heat stable
polymerase → Cycle sequencing → 3’ labeling.
• The color of the dye corresponds to the ddNTP that
terminated the strand.
THE SEQUENCING LADDER
• Before loading in a gel or capillary instrument,
sequence ladders are cleaned.
o Clean the excess ddNTPs. There should
only be fragments that are labeled.
• Excess dye terminators are removed with:
o Column or Beads (solid phase isolation
technique)
o Ethanol precipitation
• Denaturing conditions (50°C to 60°C, formamide,
urea denaturing gel) are maintained so that the
fragments are resolved strictly according to size.
o used in order to maintain the size of
fragments.
o to prevent intra strand homology,
hybridization, etc.
• The ladders are heated to 95°C to 98°C for 2 to 5
minutes and placed on ice just before loading.
o If there is delay on loading in gel
electrophoresis, place on ice to maintain it
being single stranded and to maintain its
structure.
o If there’s a secondary structure, it will affect
the migration speed and it will lower the
quality of sequence.
1. Remove excess ddNTPs.
2. Denature
3. Heat
ELECTROPHORESIS
• Types: gel or capillary electrophoresis.
• Gel electrophoresis: read based on lane assignment.
• Capillary electrophoresis: read based on fluorescent
dye.
• The four sets of sequencing products in each
reaction are loaded onto a single gel lane or capillary.
• The fluorescent dye colors → distinguish which
nucleotide is at the end of each fragment.
• The migrating fragments pass a laser beam and a
detector in the automated sequencer.
Laser → Excitation of Dye → Fluorescence →
Detector
• The detector converts the fluorescence to an
electrical signal.
• The electrical signal will be converted into
electropherogram.
ELECTROPHEROGRAM
• We check for the fluorescent peaks formed.
• Each electrical signal signifies a specific nucleotide
depending on what color has fluoresced or excited.
• The sequencing software reads, or “calls,” the bases
from the smallest (fastest-migrating) fragments that
first pass the detector to the largest based on the dye
emission wavelength. (Base calling)
• The electropherogram is a series of peaks of the four
fluorescent dyes as the bands of the sequencing
ladder migrate by the detector.
• The first to migrate is the shortest fragment. It is the
nearest at the 5’ end.
• e.g. (based on the image)
5’-AGGCCACCCTGAGGTGCTGGGCCCTG-3’
SEQUENCE INTERPRETATION
• Base calling is the process of identification of bases
in a sequence by sequencing software.
• Interpretation of sequencing data from a dye
terminator reaction depends on the quality of the
electropherogram.
o An electropherogram is dependent on the
quality of template.
• Failure to clean the sequencing ladder properly:
→ Dye Blobs- bright flashes of
fluorescence. (Due to excess ddNTPs)
• Poor starting material
→ Poor-quality sequence that cannot be
read accurately.
• **When the base call is not clear, the letter “N" will
replace A, C, T, or G
• There should be a single peak at a single location.
good sequence quality bad sequence quality
o S= G and C (both has 3 hydrogen bonds).
o Y= A, C, T (represents pyrimidine)
5 | Molecular Biology and Diagnostics
Transcriber: admn
 o These sequences can be used to identify
mutation of polymorphism.
reference sequence test sequence complementary
• If you wanted to know if there is mutation (or gene
lesion) or polymorphism.
o e.g. in test sequence, instead of Guanine,
there is Thymine. (Should be black but
there’s red, this is a mutation).
• To check for mutation, since DNA is double
stranded, confirm if both of the strands are mutated.
• Check for its complementary.
o e.g. In complementary, instead of Cytosine,
there is Adenine. Thus, both of the strands
have been mutated.
• Software programs can compare two sequences or
test sequences with reference sequences to identify
mutations or polymorphisms.
reference sequence
test sequence
• If there is deletion or insertion, it could be checked
through the electropherogram.
• Heterozygous deletions or insertions affect all
positions of the sequence downstream of the
mutation.
• Chain termination using Sanger sequencing still
became the widely used method to determine DNA
sequence.
• e.g. in COVID-19, they compare its sequence to the
sequence of SARS-CoV-1. They also tried to isolate
corona virus from bats. There is more than 90%
same sequence of corona virus from bats in SARS-
CoV-2. The sequence of SARS-CoV-2 from bat
corona virus is highly similar compared to SARS-
CoV-1. Thus, there is a high possibility that SARS-
CoV-2 came from bats.
PYROSEQUENCING
• Yields the same information as chain termination but
when it comes to throughput capacity or its ability to
identify the sequence itself, it is lesser compared to
chain termination.
• Designed to determine a DNA sequence without
having to make a sequencing ladder.
• Relies on the generation of light (luminescence)
when nucleotides are added to a growing strand of
DNA.
• Addition of nucleotides on the growing strand of DNA
and relies on the formation of phosphodiester bond
between dNTP and the previous sequence where it
is added.
• Do not need to use any of the ddNTP.
• Uses enzymes. (Enzyme-driven method)
• The pyrosequencing reaction mix consists of:
o Single-stranded DNA template
o Sequencing primer
o Sulfurylase
o Luciferase
o Two substrates adenosine 5
phosphosulfate (APS)
o Luciferin
STEPS IN PYROSEQUENCING
1. One of the four dNTPs → Reaction mix.
2. If complementary to the template, DNA polymerase
extends the primer.
3. Pyrophosphate (PPi) is released.
4. The PPi is converted to ATP by sulfurylase.
5. Luminescent signal by luciferase-catalyzed
conversion of luciferin to oxyluciferin.
6. The generation of a signal indicates which nucleotide
is the next correct base in the sequence.
• Relies on the formation of phosphodiester bond
between the growing chain of DNA and dNTP that
has been added.
• A dNTP will be added on the growing chain of DNA,
then check if the dNTP is complementary.
• If the dNTP is complementary or not. If the dNTP is
complementary (e.g. Guanine and the dNTP added
is Cytosine) it will proceed on pyrosequencing. But if
the dNTP is non complementary (e.g. Guanine and
dNTP is Thymine) the process of pyrosequencing
will not continue.
• If the nucleotide is complementary to the base in the
template strand, the DNA polymerase will extend the
primer. Polymerase enzyme will catalyze the
phosphodiester bond between the two to grow DNA.
• Once there is phosphodiester bond, it will release
pyrophosphate. The pyrophosphate will be
converted to ATP using Sulfurylase enzyme. The
ATP generated will be used for the production of
light.
• To produce luminescence signal, there is Luciferase
catalyzed conversion of Lucefirin to Oxyluciferin.
ATP is needed in order for Lucefirin to become
Oxyluciferin. Light is also produced.
• This process is being repeated as the dNTP has
been added to the growing chain of the DNA. If there
is a phosphodiester bond formed, pyrophosphate is
also produced.
• Pyrophosphate is produced if there is
complementary between the template and the dNTP
that has been added. if non complementary, no
pyrophosphate is produced, no light is produced.
• The light production will be converted into electrical
signal.
6 | Molecular Biology and Diagnostics
Transcriber: admn
PYROGRAM
• Produce peaks if light is generated.
• Consist of peaks of luminescence associated with
the addition of the complementary nucleotide.
• The nucleotide sequence is called based on the
order of nucleotide bases introduced to the
sequencing reaction and the peak heights.
• Useful in mutation detection, infectious disease
typing and DNA methylation analysis.
STEPS IN BISULFIDE SEQUENCING
• Nucleotide is added (predetermined). If there is
peak, it has that nucleotide base.
o e.g. (based on image) G is added, there is
peak, meaning it has G on its sequence. C
is added, there is peak, there is Cytosine. T
is added but there is no peak, meaning it
has no T in the sequence.
o if there is double peak or double height, the
sequence contain a repeated nucleotide
(e.g. GG)
o 5’-GCAGGCCT-3’
o Base calling is based on the nucleotide
bases introduced to the sequencing
reaction.
BISULFITE DNA SEQUENCING
• Also called as Methylation-Specific Sequencing
• Detecting if there is any methylated cytosine
residues.
• Cytosine can be methylated.
• Chain termination sequencing designed to detect
methylated cytosine nucleotides.
• Exposing of sequence using bisulfite.
• A methylated cytosine will resist changes. But if it is
unmethylated or it does not have methyl group on its
structure, Cytosine will be converted to Uracil once
bisulfite has been added.
• Purpose: to check whether cytosine is methylated or
not.
• Methylation of cytosine residues will become 5’
methyl cytosine and is very important to regulation of
gene expression.
• Methylation of cytosine controls the gene
expression.
• If there is methylation, it can change the activity of
the DNA segment without changing the sequence.
• In methylation, the cytosine is still cytosine but is
methylated, there is different activity of DNA and
resists any conversion.
• Once unmethylated cytosine is amplified, uracil will
be read as thymine.
• In base calling, if cytosine is methylated, the color of
the peak will remain as blue. If cytosine is
unmethylated, the peak will be color red.
1. 2 to 4 μg of genomic DNA is cut with restriction
enzymes to facilitate denaturation.
o Since genomic DNA is very large, we have
to turn it into smaller pieces.
2. Restriction digestion products are resolved on an
agarose gel.
o There is only a specific part of genomic
DNA that we want to study.
o e.g. we want only the 600bp, we cut the
genomic DNA to the part that has 600 bp
since that is the target or interest.
3. Fragments of the size of interest are purified from the
gel.
4. The DNA is denatured with heat.
o so that dsDNA will be ssDNA.
5. Exposure to bisulfite solution (sodium bisulfite,
NaOH, and hydroquinone) for 16 to 20 hours.
o Bisulfite converts non methylated cytosine
to uracil and methylated cytosine will
remain as cytosine.
o Never overexpose the sample in bisulfite
solution since it can result in strand
cleavage. We can lose the region we want
to identify.
o Strand cleavage- target size can be cut off.
(e.g. instead of 600 bp, only 300 bp was
cut).
6. During the incubation with bisulfite, the cytosines in
the reaction are deaminated, converting them to
uracils, whereas the 5-methylcytosines are
unchanged.
7. Purification of treated DNA.
8. Resuspended for use as a template for PCR.
• The PCR amplicons are then sequenced by:
1. Sanger sequencing
o Unmethylated cytosines will
appear as red (thymine) instead of
blue (cytosine) peaks on the
electropherogram.
2. Pyrosequencing
o The relative light intensity of
consecutive T and C additions to
the reaction mix provide a
quantitative degree of
methylation.
• Other methods:
7 | Molecular Biology and Diagnostics
Transcriber: admn
 o Methylation-sensitive restriction enzymes
o Primers → converted or nonconverted
sequences.
RNA SEQUENCING
• Purpose: to know the sequence of RNA transcript or
mRNA.
• Early methods to sequence RNA made use of
ribonucleases to cut end-labeled RNA at specific
nucleotides.
• Another approach was to infer mRNA sequence from
amino acid sequence.
• Disadvantage of using RNA transcript instead of
using RNA, there can be errors. mRNA is transcript
and converted to complementary DNA. During the
conversion, there can be errors.
• Direct Sequencing of RNA
o We use polydT oligomer.
o mRNA contains polyA tail which is used as
base.
o mRNA is captured by immobilized polydT
oligomers.
▪ it will capture polyA tail.
▪ If there is no polyA tail, treat the
RNA with polymerase enzyme to
generate polyA tail. Once there is
polyA tail, we can now sequence
the RNA.
o Four reversibly dye-labeled nucleotides are
then sequentially added.
▪ uses nucleotide to the growing
chain of RNA to find its
complementary, by that, we will
know the sequence of RNA.
o An image is taken, the extension inhibitors
are cleaved, and alternating C, T, A, or G
nucleotides are added, with imaging,
cleavage, and rinsing between each
nucleotide addition.
NEXT-GENERATION SEQUENCING
• Has been used widely for automated.
• Used for whole genome sequencing.
• Offers ultra-high throughput, fast detection speed.
• Also called as Massive Parallel Sequencing
• Designed to sequence large numbers of templates
carrying millions of bases simultaneously.
• NGS technologies include the following:
o Pyrosequencing
o Reversible dye terminator sequencing
o Ion-conductance sequencing
o Single-molecule sequencing
o Sequencing by ligation
• Powerful computer data assembly systems are
required to organize the massive amounts of
sequence information that are generated.
o fully automated, supercomputer is needed.
• NGS requires strong computer support as well as
terabytes of storage space to accommodate large
raw data sets.
• Three Basic steps:
1. Library preparation- whole genome plotter
should be identified first (location). Identify
location of important sequences on whole
genome. The color designates what
specific sequence it is.
2. Sequencing- sequencing is based on the
library.
3. Data analysis- using computer
• There are multiple spots and determine what specific
sequence is that. It will be combined, and the
computer will read what is the exact sequence of the
genome by scanning the spots.
GENOMIC ARRAY TECHNOLOGY
• Another way to sequence whole genome.
• It is a hybridization analysis.
• Simultaneous study of large numbers of targets (or
samples).
• Arrays are applied to gene (DNA) amplification or
deletion on comparative genome hybridization
arrays and to gene-expression (RNA or protein)
analysis on expression arrays.
• There are several approaches to array technology:
o Macroarrays
o Microarrays (most common)
▪ If you want to study the expression
of genes at once.
▪ It involves the placing of
thousands of gene sequences in
exact location or known location in
the glass slide.
▪ The glass slide used is called as
gene chip which contains
thousands of gene sequences.
8 | Molecular Biology and Diagnostics
Transcriber: admn
 ▪ A sample that contains DNA or
RNA is placed in contact with the
gene chip and read with computer.
o High-density oligonucleotide arrays
o Microelectronic arrays
• Next generation sequencing and Genomic array are
used by reference laboratories.

PHOTOLITHOGRAPHY

• A technique if you want to synthesize DNA

oligonucleotides in glass slide.
• First, activate the parts, there should be hydration.
There should be hydroxyl group by exposing it to UV
light at 365 nm.
• At 365 nm. the parts that we want to grow will be
exposed. The parts that we do not want to grow will
be masked.
• Once activated, the growing of nucleotides will start.
• It will grow depending on the sequences or template.
• This is repeated procedure until we generate the
desired sequences for each spot.
• Also used for whole genome.

9 | Molecular Biology and Diagnostics

Transcriber: admn