DNA Sequencing

By-
Dr. Dinesh C. Sharma
Head, Zoology
K.M. Govt. Girls P. G. College
Badalpur, G.B. Nagar
dr_dineshsharma@hotmail.com
“the process of determining the sequence of nucleotides (A, T, G, and C) in a piece of DNA”

DNA sequencing is the process of determining
the sequence of nucleotides (A, T, G, and C) in
the DNA. It includes method or technology that
is used to determine the order of the four bases:
adenine, thymine, guanine and cytosine.
Sequencing an entire genome of an organism
remains a complex task. It requires breaking
the DNA of the genome into many smaller
pieces, sequencing the pieces, and assembling
the sequences into a single long "consensus."
But, new methods developed over the past two
decades make it easier, genome sequencing is
now much faster and less expensive than it was
during the Human Genome Project.

Knowledge of DNA sequences has become
indispensable for basic biological research (Medical
diagnosis, biotechnology, forensic biology, virology,
biological systematics etc.).
Comparing healthy and mutated DNA sequences can
diagnose different diseases such as single gene
disorders, various cancers, characterize antibody
repertoire and can be used to guide patient treatment.
The first DNA sequences were obtained in the early
1970s by academic researchers using laborious methods
based on two-dimensional chromatography. Following
the development of fluorescence-based sequencing
methods with a DNA sequencer, DNA sequencing has
become easier and orders of magnitude faster

Two-dimensional chromatography- is a type of
chromatographic technique in which the
injected sample is separated by passing
through two different separation stages. Two
different chromatographic columns are
connected in sequence, and the effluent from
the first system is transferred onto the second
column. Typically the second column has a
different separation mechanism, so that bands
that are poorly resolved from the first column
may be completely separated in the second column. (For instance, a
C18 reversed-phase chromatography column may be followed by a
phenyl column.) Alternately, the two columns might run at different
temperatures. During the second stage of separation the rate at which
the separation occurs must be faster than the first stage, since there is
still only a single detector. The plane surface is amenable to sequential
development in two directions using two different solvents.

DNA sequencing methods
• The first method for determining DNA sequences involved a
location-specific primer extension strategy established by Ray
Wu at Cornell University in 1970. Between 1970 and 1973, Wu, R
Padmanabhan and colleagues demonstrated that this method can be
employed to determine any DNA sequence using synthetic
location-specific primers.
• Frederick Sanger then adopted this primer-extension strategy
to develop more rapid DNA sequencing methods at the MRC
Centre, Cambridge, UK and published a method for "DNA
sequencing with chain-terminating inhibitors" in 1977.
• Walter Gilbert and Allan Maxam at Harvard also
developed sequencing methods, including one for "DNA
sequencing by chemical degradation". In 1973, Gilbert and Maxam
reported the sequence of 24 basepairs using a method known as
wandering-spot analysis.

Sequencing of full genomes
• The first full DNA genome to be sequenced was that of
bacteriophage φX174 (5386bp) in 1977.
• Medical Research Council scientists deciphered the
complete DNA sequence of the Epstein-Barr virus in
1984, finding it contained 172,282 nucleotides.
• Leroy E. Hood's laboratory at the California Institute of Technology
announced the first semi-automated DNA sequencing machine in 1986.
• Applied Biosystems marketing of the first fully automated sequencing
machine, the ABI 370, in 1987 and by Dupont's Genesis 2000 which used a
novel fluorescent labeling technique enabling all four dideoxynucleotides to
be identified in a single lane.
• In 1995, Venter, Hamilton Smith, and colleagues at The Institute for
Genomic Research (TIGR) published the first complete genome of a free-living
organism, the bacterium Haemophilus influenza (contains 1,830,137 bases) and
its publication in the journal Science marked the first published use of whole-
genome shotgun sequencing, eliminating the need for initial mapping efforts.
• By 2001, shotgun sequencing methods had been used to produce a draft
sequence of the human genome

High-throughput sequencing (HTS) or "next-
generation" or "second-generation" sequencing
(NGS) methods
• On 26 October 1990, Roger Tsien, Pepi Ross, Margaret
Fahnestock and Allan J Johnston filed a patent describing
stepwise ("base-by-base") sequencing with removable 3'
blockers on DNA arrays (blots and single DNA molecules).
• In 1996, Pål Nyrén and his student Mostafa Ronaghi at the
Royal Institute of Technology in Stockholm published their
method of pyrosequencing (Pyrosequencing is a method of
DNA sequencing based on the "sequencing by synthesis"
principle, in which the sequencing is performed by detecting
the nucleotide incorporated by a DNA polymerase.
Pyrosequencing relies on light detection based on a chain
reaction when pyrophosphate is released. Hence, the name
pyrosequencing).

• On 1 April 1997, Pascal Mayer and Laurent Farinelli
submitted patents to the World Intellectual Property Organization
describing DNA colony sequencing. The DNA sample preparation
and random surface-polymerase chain reaction (PCR) arraying
methods described in this patent, coupled to Roger Tsien et al.'s
"base-by-base" sequencing method, is now implemented in
Illumina's Hi-Seq genome sequencers.
• In 1998, Phil Green and Brent Ewing of the University of
Washington described their phred quality score for sequencer
data analysis, which is still the most common metric for assessing
the accuracy of a sequencing platform.
• In 2000 Lynx Therapeutics published and marketed
massively parallel signature sequencing (MPSS). This method
incorporated a parallelized, adapter/ligation-mediated, bead-
based sequencing technology and served as the first
commercially available "next-generation"
sequencing method.

Maxam-Gilbert sequencing published a DNA sequencing
method in 1977 based on chemical modification of DNA and
subsequent cleavage at specific bases. Also known as chemical
sequencing, this method allowed purified samples of double-
stranded DNA to be used without further cloning.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end
of the DNA and purification of the DNA fragment to be sequenced.
Chemical treatment then generates breaks at a small proportion of one
or two of the four nucleotide bases in each of four reactions (G, A+G,
C, C+T). The concentration of the modifying chemicals is controlled
to introduce on average one modification per DNA molecule. Thus a
series of labeled fragments is generated, from the radiolabeled end to
the first "cut" site in each molecule. The fragments in the four reactions
are electrophoresed side by side in denaturing acrylamide gels for size
separation. To visualize the fragments, the gel is exposed to X-ray film
for autoradiography, yielding a series of dark bands each corresponding
to a radiolabeled DNA fragment, from which the sequence may be
inferred.

Maxam–Gilbert sequencing requires radioactive labeling at
one 5′ end of the DNA fragment to be sequenced (typically by
a kinase reaction using gamma-32P ATP) and purification of
the DNA.
Chemical treatment generates breaks at a small proportion of
one or two of the four nucleotide bases in each of four
reactions (G, A+G, C, C+T). For example, the purines (A+G)
are depurinated using formic acid, the guanines (and to some
extent the adenines) are methylated by dimethyl sulfate, and
the pyrimidines (C+T) are hydrolysed using hydrazine. The
addition of salt (sodium chloride) to the hydrazine reaction
inhibits the reaction of thymine for the C-only reaction. The
modified DNAs may then be cleaved by hot piperidine;
(CH2)5NH at the position of the modified base.
The concentration of the modifying chemicals is controlled to introduce on average
one modification per DNA molecule. Thus a series of labeled fragments is
generated, from the radiolabeled end to the first "cut" site in each molecule.
The fragments in the four reactions are electrophoresed side by side in denaturing
acrylamide gels for size separation. To visualize the fragments, the gel is exposed to
X-ray film for autoradiography, yielding a series of dark bands each showing the
location of identical radiolabeled DNA molecules. From presence and absence of
certain fragments the sequence may be inferred

https://www.youtube.com/watch?v=_B5Dj8PL4E0

5”- C T C G A G T G T A T C G A C -3”
3”- T C T C T C A C A T A G C T G -5”
| | | | | | | | | | | | | | |
1-Denturation @ 95O C
3”- T C T C T C A C A T A G C T G -5”
Four samples (A+G), G, (T+C) and C
A+G G T+C C

1
A+G
2
G
3
T+C
4
C
A-Add radioactive phosphate (p32) in all four
2-Chemical Treatment
p32

1
A+G
2
G
3
T+C
4
C
B-Add Formic Acid in 1: Formic acid breaks link
between a purine (A+G) and the deoxyribose to which it attached
5”- C T C
5”- C T C G A G T
5”- C T C G A
5”- C T C G
5”- C T C G A G T G T
5”- C T C G A G T G T A T C
5”- C T C G A G T G T A T C G
7
Fragment

1
A+G
2
G
3
T+C
4
C
C-Dimethyal Sulfate in 2: Methylation of
Guanine by DMS
5”- C T C
5”- C T C G A G T
5”- C T C G A
4
Fragment

1
A+G
2
G
3
T+C
4
C
D-Add Hydrazine in 3: The pyrimidines are
hydrolyzed by using hydrazine
5”- C T C G A G T G T A T C G A C
5”- C T C G A G T G T A T C G A
5”- C T C G A G T G T A T
5”- C T C G A G T G T A
5”- C T C G A G T G
5”- C T C G A G
5”- C T
5”- C
8
Fragment

1
A+G
2
G
3
T+C
4
C
E-Add Hydrazine and NaCl in 4: The addition of
NaCl to hydrazine reaction inhibits the reaction of thymine for –C
only reaction
5”- C
5”- C T
4
Fragment

1
A+G
2
G
3
T+C
4
C
F-Add Piperidine in all 4: The modified DNAs
cleaved by hot piperidine at the position of the modified base

3-Acrylamide Gel Electrophoresis
1
A+G
2
G
3
T+C
4
C
Anode
Cathode

• The negative charge of phosphate
backbone move the DNA
fragments towards the positively
charged anode
• Smaller DNA fragments migrate
more rapidly than larger DNA
fragments
Small
Large

5”- C T C
5”- C T C G A G T
5”- C T C G A
5”- C T C G
7

5”- C T C
5”- C T C G A G T
5”- C T C G A
4

5”- C T C G A G
5”- C T
5”- C
8

5”- C
5”- C T
4

5”- C T C
5”- C T C G A G T
5”- C T C G A
5”- C T C G
5”- C T C
5”- C T C G A G T
5”- C T C G A
5”- C T C G A G
5”- C T
5”- C 5”- C
5”- C T
C
C
C
C
T
T
T
T
G
G
G
G
A
A
A
C
A
G
C
T
A
T
G
T
G
A
G
C
T
C

Sanger sequencing
The
chain termination
method

The chain-termination method developed by Frederick Sanger and
coworkers in 1977. This method used fewer toxic chemicals and
lower amounts of radioactivity than the Maxam and Gilbert
method. Because of its comparative ease, the Sanger method was
soon automated and was the method used in the first generation
of DNA sequencers.
The Sanger method, in mass production form, is the technology
which produced the first human genome in 2001. In the Human
Genome Project, Sanger sequencing was used to determine the
sequences of many relatively small fragments of human DNA.
(These fragments weren't necessarily 900 bp or less, but
researchers were able to "walk" along each fragment using
multiple rounds of Sanger sequencing.) The fragments were
aligned based on overlapping portions to assemble the sequences
of larger regions of DNA and, eventually, entire chromosomes.
Although genomes are now typically sequenced using other
methods that are faster and less expensive, Sanger sequencing is
still in wide use for the sequencing of individual pieces of DNA,
such as fragments used in DNA cloning or generated
through polymerase chain reaction (PCR).
It was first commercialized by Applied Biosystems in 1986

Requirement for Sanger sequencing
Sanger sequencing make many copies of a target DNA
region. Its raw material are similar to the requirement of
DNA replication in an organism, or for polymerase chain
reaction (PCR), which copies DNA in vitro.
They include:
• A DNA polymerase enzyme
• A primer, acts as a "starter" for the DNA polymerase
• The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
• The template DNA to be sequenced
Sanger sequencing reaction also contains a unique
ingredient:
• Dideoxy nucleotide (dd), or chain-terminating,
versions of all four nucleotides (ddATP, ddTTP, ddCTP,
ddGTP), each labeled with a different color of
dye

Dideoxy nucleotides lack a hydroxyl
group on the 3’ carbon of the sugar
ring. In a regular nucleotide, the 3’
hydroxyl group acts as a “hook,"
allowing a new nucleotide to be added
to an existing chain.
Once a dideoxy nucleotide has been
added to the chain, there is no
hydroxyl available and no further
nucleotides can be added.
The chain ends with the dideoxy
nucleotide, which is marked with a
particular color of dye depending on
the base (A, T, C or G) that it carries.
normal
deoxynucleotidetriphosphates
(dNTPs)
modified
di-deoxynucleotidetriphosphates
(ddNTPs),

Fluorescent modified di-deoxynucleotidetriphosphates (ddNTPs), molecules

1-Amplification of desired DNA sequence
2-Denturation of DNA by heating @ 95O C

3” 5”
5” 3”
| | | | | | | | | |
| | | | | | | | | |
| | | | | | | | | |
| | | | | | | | | |
| | | | | | | | | |
| | | | | | | | | |
1-Amplification of desired DNA sequence

3” 5”
5” 3”
| | | | | | | | | |
2-Denturation of DNA by heating @ 95O C
3” 5”
5” 3”
Heat Heat
To produce a complimentary strand and the template strand for DNA sequencing

3” T A C G C A T A 5”
T A T 3”
| | |
4-The Primed DNA is then dispersed equally among for vessels
<------- Template strand
<------- Primer
3” T A C G C A T A 5”
T A T 3”
| | |
3-A Primer is then annealed to the 5” end of DNA
<------- Primer

5-DNA polymerase is added to all 4 reaction vessels
DNA P DNA P DNA P DNA P
6-Add all four (dATP, dTTP, dGTP, dCTP) to each vessels
A,G,C,T A,G,C,T A,G,C,T A,G,C,T

7-Modifeid ddNTP are added to reaction vessels
ddATP ddTTP ddGTP ddTTP
AT
G C
3” T A T G C A T A 5”
T A T 3”
| | |
<------- Primer

3” T A T G C A T A 5”
T A T 3”
| | |
<------- Primer
A
T
C
G
DNA
Polymerase
ddATP
8- The DNA polymerase attaches the dNTP to the template
strand at the primer normally until ddNTP base is pared. As
ddNTP attached the chain termination occur.

3” T A T G C A T A 5”
T A T 3”
| | |
<------- Primer
A T 3”
9-Once the ddNTP is based paired , the sequence is
terminated because ddNTP lacks the –OH group at 3’ carbon
ddATP ddTTP ddGTP ddCTP
10-As a result of chain termination, DNA fragments of
different length are formed in all four vessels

A T G C
Polyacrylamide Gel
Electrophoresis is
used to sequence
DNA

A CGT
• DNA migrates form
the –ve pole towards
the +ve pole, due to
–ve charge impaired
by phosphate back
bone
• Smaller (lighter)
DNA fragments
migrate more
rapidly than larger
DNA fragments
• As a result of this
different bands are
observed on plate Small & lighter
Large & Heavy

A CGT
The sequence is read form
the bottom of the plate
T
C
A
T
G
G
T
A
T
T
C
A
C
G
G
A
T
A
G
T
C
G
A

5”A G C T G AT A G G C A C T T AT G GT A CT 3”
T
C
A
T
G
G
T
A
T
T
C
A
C
G
G
A
T
A
G
T
C
G
A
3”T C G A C T AT C C GT G A AT A C C AT G A 5”

Method of Sanger sequencing
• The DNA sample is divided into four separate
sequencing reactions, containing all four of the
standard deoxynucleotides (dNTP, A,C,G,T) and
the DNA polymerase.
• To each reaction is added only one of the four
dideoxynucleotides (ddNTP ddATP, ddTTP,
ddGTP, ddCTP), while the other added
nucleotides are ordinary ones (dN).
• The ddNTP concentration should be
approximately 100-fold higher than that of the
corresponding dNTP (e.g. 0.5mM ddTTP :
0.005mM dTTP) to allow enough fragments to
be produced while still transcribing the
complete sequence (but the concentration of
ddNTP also depends on the desired length of
sequence)

• Four separate reactions are needed in this process
to test all four ddNTPs. Following rounds of
template DNA extension from the bound primer,
the resulting DNA fragments are heat denatured
and separated by size using gel electrophoresis.
• In the original publication of 1977, the formation of
base-paired loops of ssDNA was a cause of serious
difficulty in resolving bands at some locations. This
is frequently performed using a denaturing
polyacrylamide-urea gel with each of the four
reactions run in one of four individual lanes (lanes
A, T, G, C). The DNA bands may then be visualized
by autoradiography or UV light and the DNA
sequence can be directly read off the X-ray film or
gel image.

• DNA fragments are labelled
with a radioactive or
fluorescent tag on the
primer , in the new DNA
strand with a labeled dNTP,
or with a labeled ddNTP.
• Chain-termination methods
have greatly simplified DNA
sequencing. For example,
chain-termination-based
kits are commercially
available that contain the
reagents needed for
sequencing, pre-aliquoted
and ready to use.

Dye-terminator sequencing
utilizes labelling of the chain terminator ddNTPs,
which permits sequencing in a single
reaction, rather than four reactions
as in the labelled-primer method. In dye-
terminator sequencing, each of the four
dideoxynucleotide chain terminators is labelled
with fluorescent dyes, each of which emit light at
different wavelengths.
Owing to its greater expediency and speed, dye-
terminator sequencing is now the mainstay in
automated sequencing.
Its limitations include dye effects due to differences in the incorporation of the
dye-labelled chain terminators into the DNA fragment, resulting in unequal peak
heights and shapes in the electronic DNA sequence trace chromatogram after
capillary electrophoresis. This problem has been addressed with the use of
modified DNA polymerase enzyme systems and dyes that minimize incorporation
variability, as well as methods for eliminating "dye blobs". The dye-terminator
sequencing method, along with automated high-throughput DNA sequence
analyzers, was used for the vast majority of sequencing projects.

Automated DNA-sequencing instruments (DNA
sequencers) can sequence up to 384 DNA samples
in a single batch. Batch runs may occur up to 24
times a day. DNA sequencers separate strands by
size (or length) using capillary electrophoresis,
they detect and record dye fluorescence, and
output data as fluorescent peak trace
chromatograms. Sequencing reactions
(thermocycling and labelling), cleanup and re-
suspension of samples in a buffer solution are
performed separately, before loading samples onto
the sequencer. A number of commercial and non-
commercial software packages can trim low-
quality DNA traces automatically. These programs
score the quality of each peak and remove low-
quality base peaks (which are generally located at
the ends of the sequence). The accuracy of such
algorithms is inferior to visual examination by a
human operator, but is adequate for automated
processing of large sequence data sets.

Challenges
• Poor quality in the first 15-40 bases of the sequence due
to primer binding and deteriorating quality of sequencing
traces after 700-900 bases. Base calling software such as
Phred typically provides an estimate of quality to aid in
trimming of low-quality regions of sequences.
• In cases where DNA fragments are cloned before
sequencing, the resulting sequence may contain parts of
the cloning vector. In contrast, PCR-based cloning and
next-generation sequencing technologies based on
pyrosequencing often avoid using cloning vectors.
• One-step Sanger sequencing (combined amplification and
sequencing) methods such as Ampliseq and SeqSharp
have been developed that allow rapid sequencing of
target genes without cloning or prior amplification.
• Current methods can directly sequence only relatively
short (300-1000 nucleotides long) DNA fragments in a
single reaction.
• The main obstacle to sequencing DNA fragments above
this size limit is insufficient power of separation for
resolving large DNA fragments that differ in length by only
one nucleotide.

Microfluidic Sanger sequencing
Microfluidic Sanger sequencing is a lab-on-a-chip
application for DNA sequencing, in which the
Sanger sequencing steps (thermal cycling, sample
purification, and capillary electrophoresis) are
integrated on a wafer-scale chip using nanoliter-
scale sample volumes.
This technology generates long and accurate
sequence reads, while obviating many of the
significant shortcomings of the conventional
Sanger method (e.g. high consumption of
expensive reagents, reliance on expensive
equipment, personnel-intensive manipulations,
etc.) by integrating and automating the Sanger
sequencing steps.

In its modern inception, high-throughput genome
sequencing involves
• fragmenting the genome into small single-stranded
pieces,
• followed by amplification of the fragments by Polymerase
Chain Reaction (PCR).
Adopting the Sanger method, each DNA fragment is
irreversibly terminated with the incorporation of a
fluorescently labeled dideoxy chain-terminating nucleotide,
thereby producing a DNA “ladder” of fragments that each
differ in length by one base and bear a base-specific
fluorescent label at the terminal base.
Amplified base ladders are then separated by Capillary
Array Electrophoresis (CAE) with automated, in situ “finish-
line” detection of the fluorescently labeled ssDNA
fragments, which provides an ordered sequence of the
fragments. These sequence reads are then computer
assembled into overlapping or contiguous sequences
(termed "contigs") which resemble the full genomic
sequence once fully assembled.

Applications of microfluidic sequencing technologies
• Single nucleotide polymorphism (SNP) detection,
• Single-strand conformation polymorphism (SSCP)
• Heteroduplex analysis, and
• Short tandem repeat (STR) analysis.
Resolving DNA fragments according to differences in size
and/or conformation is the most critical step in studying
these features of the genome
A single-nucleotide polymorphism (SNP) is a substitution
of a single nucleotide that occurs at a specific position in
the genome, where each variation is present at a level of
more than 1% in the population.
Heteroduplex analysis (HDA) is a method in biochemistry
used to detect point mutations in DNA since 1992.
Heteroduplexes are dsDNA molecules that have one or
more mismatched pairs, on the other hand homoduplexes
are dsDNA which are perfectly paired

Next-generation sequencing
The most recent set of DNA sequencing technologies are
collectively referred to as next-generation sequencing.
There are a variety of next-generation sequencing techniques that
use different technologies. However, most share a common set of
features that distinguish them from Sanger sequencing:
• Highly parallel: many sequencing reactions take place at the
same time
• Micro scale: reactions are tiny and many can be done at once on
a chip
• Fast: because reactions are done in parallel, results are ready
much faster
• Low-cost: sequencing a genome is cheaper than with Sanger
sequencing
• Shorter length: reads typically range from 50-700 nucleotides in
length
Conceptually, next-generation sequencing is kind of like running a
very large number of tiny Sanger sequencing reactions in parallel.
This parallelization and small scale, large quantities of DNA can be
sequenced much more quickly and cheaply with next-generation
methods than with Sanger sequencing.
For example, in 2001, the cost of sequencing a human genome was
almost $100 million In 2015, it was just $1245.

https://www.khanacademy.org/science/high-school-biology/hs-molecular-
genetics/hs-biotechnology/a/dna-sequencing
https://www.youtube.com/watch?v=_B5Dj8PL4E0
https://en.wikipedia.org/wiki/DNA_sequencing

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