DNA sequencing methods have evolved significantly over time. Early methods like the Maxam-Gilbert chemical method and Sanger chain termination method involved laborious gel electrophoresis. Later developments led to automated Sanger sequencing using fluorescence detection. Next-generation sequencing methods like Illumina sequencing by synthesis and 454 pyrosequencing enabled massively parallel sequencing of many DNA fragments simultaneously. These new methods produce vast amounts of sequence data at lower cost and are used widely in research and applications such as agriculture, medicine, forensics, and more.

1. MUTHUKUMAR M
2017601705
DEPARTMENT OF CROP PHYSIOLOGY
TNAU, COIMBATORE
DNA SEQUENCING METHODS AND ITS
APPLICATIONS

2. OVER ALL VIEW
PRINCIPLE OF DNA SEQUENCING
HISTORY OF SEQUENCING
DNA SEQUENCING METHODS
1) Maxam/Gilbert chemical sequencing
2) Sanger chain termination sequencing
3) AUTOMATED SEQUENCING
4) PYRROSEQUENCING
APPLICATIONS

3. • Determination of the precise order of nucleotides within a DNA
molecule is popularly known as DNA sequencing.
• There are three main requirements for this to be achieved:
I. DNA fragments need to be prepared in a form suitable for
sequencing.
II. The technique used must achieve the aim of presenting each
base in turn in a form suitable for identification.
III. The detection method must permit rapid and accurate
identification of the bases.
PRINCIPLE OF DNA SEQUENCING

4. 1. 1975 The first complete DNA genome to be sequenced is that of
bacteriophage X174
2. 1977 - Allan Maxam and Walter Gilbert publish "DNA
sequencing by chemical degradation".
3. 1977 - Fred Sanger, independently, publishes "DNA sequencing
by enzymatic synthesis".
4. 1980 Fred Sanger and Wally Gilbert receive the Nobel Prize in
Chemistry
HISTORY OF SEQUENCING

5. 1. Maxam – Gilbert sequencing
2. Chain-termination methods
3. Dye-terminator sequencing
4. Automation and sample preparation
5. Large scale sequencing strategies
6. New sequencing methods
a. Lynx therapeutics' massively parallel signature
sequencing (MPSS)
b. Polony sequencing
c. Pyrosequencing
d. Illumina (Solexa) sequencing
e. SOLiD sequencing
f. DNA nanoball sequencing
DNA SEQUENCING METHODS

6. 1. Maxam – Gilbert sequencing/Chemical Cleavage Method
1. This method uses double-stranded DNA samples.
2. Involves modification of the bases in DNA followed by chemical base-specific cleavage.
3. Sequence DNA fragments containing upto ~500 nucleotides in length.
Stages
1. The double-stranded fragment to be sequenced is isolated and
radioactively labeled at the 5’-ends
with 32P.
2. The fragment is then cut with restriction enzyme and thus the label is
removed from one end.
3. The fragment of DNA with one end labeled is denatured.
4. Four identical samples of these end-labeled DNA restriction fragments are
subjected to chemical cleavage at different chemical nucleotides.
5.There are four specific sets of chemical reactions that selectively cut the
DNA backbone at G, A+G, C+T, or C residues.
G only: Dimethyl sulphate(DMS and piperidine
A+G : DMS, piperidine
C+T : Hydrazine, piperidine
C only : Hydrazine, alkali, piperidine

7. Contd.,, 6. For each labeled chain to be broken only
once, the reactions are controlled.
7. The labeled subfragments created by the
four reactions have the 32P label at one end
and the chemical cleavage point at the
other end.
8. The reaction products are separated by
polyacrylamide gel electrophoresis which is
based on size. Smallest fragment goes
fastest.

8. 9. The labeled fragments in the gel are
visualized by autoradiography.
10. The sequence is read from bottom
to top of the gel.

9. Advantages Disadvantages
 No premature termination due to
DNA sequencing. So, no problem
with polymerase to synthesize
DNA.
 Stretches of DNA can be sequenced
which can not be done with
enzymatic method.
 Not widely used.
 Use of radioactivity and toxic
chemicals.

10. Chain Termination method
 single-stranded DNA.
 Also known as dideoxy sequencing method because it involves the
use of analogue of normal nucleotide 2’,3’-dideoxynucleoside
triphosphates (ddNTPs). These are chain terminating nucleotides
lacking 3’-OH ends.
 This method is based upon the incorporation of ddNTPs into a
growing DNA strand to stop chain elongation.
Figure: Structure of NTP,
dNTP, and ddNTP

11. Stages
1.The DNA to be sequenced is called the template DNA. It is prepared as a single-
stranded DNA after being spliced into M13 vector DNA. Infected E. coli host cells
release phage particles which contains single-stranded recombinant DNA that
includes the sample DNA. This DNA sample is then extracted from phage for
sequencing purpose.
2. A synthetic 5’-end-labeled oligodeoxynucleotide is used as the primer.
3. The template DNA is hybridized to the primer.
4. The primer elongation is performed in four separate polymerization reaction
mixtures. Each mixture contains
- 4 normal deoxynucleotides (dNTPs) in higher concentration
- a low concentration of the each of the 4 ddNTPs.

12. 5. There is initiation of DNA synthesis by adding enzyme DNA polymerase
since the enzyme cannot distinguish between the normal nucleotides and
their analogues.
6. The strand synthesis continues until a ddNTP is added. The chain
elongation ceases on the incorporation of a ddNTP because it lacks a 3’-
OH group which prevents addition of the next nucleotide.
7. There is a result of mixture of terminated fragments, all of different
lengths.
8. Denature DNA fragments.
9. Each of the four mixtures are run together on a polyacrylamide gel for
electrphoresis.
10. The separated fragments are then visualized by autography.
11. From the position of the bands of the resulting autoradiogram, the
sequence of the original DNA template strand can be read directly.

13. 1. A labeled primer is used to initiate DNA
synthesis.
2. The addition of four different dideoxy
nucleotides randomly arrests synthesis

14. Advantages Disadvantages
 Most popular method.
 Simpler and quicker allowing
large output. Within an hour
the primer-annealing and
sequencing reactions can be
completed.
 Yielding of poor results owing
to secondary structure in the
DNA as sometimes DNA
polymerases terminate chain
elongation prematurely.
 The sequence is obtained not
from the original DNA molecule
but from an enzymatic copy. So,
there is a chance of
incorporation of wrong bases.
 The dideoxy method is good
only for 500-750bp reactions
 Expensive

15. Automated DNA sequencing
1. Principle of automated DNA sequencing is same as Sanger’s method but the
detection is different.
2. ddNTPs are labeled by incorporation of a fluorescent dye
3. 384 DNA samples in a single batch (run) in up to 24 runs a day.
4. capillary electrophoresis for size separation, detection and recording of dye
fluorescence, and data output as fluorescent peak trace chromatograms.
Electropherogram with peaks representing the bands on the sequencing gel

16. Clone-by-Clone
Ordered Approach

17. • Fred Sanger in 1982
• DNA is broken into fragments followed by sequencing at random and
assembling together the overlaps
Whole Genome Shotgun Sequencing

18. •High throughput sequencing technique that can
collect a large amount of data at a fast rate.
•Works by partially digesting a genome or big
strand of DNA into small overlapping fragments
•These small fragments are sequenced and
fragments that overlap are matched together.
Shotgun or 454 sequencing
a. The genome is fragmented and the fragments are
denatured.
b. Fragments are amplified and assigned to beads.
One fragment per one microbead.
c. Each bead is placed in the wells of a fiber optic
slide.
d. Packing beads placed in all the wells.
Steps Behind 454 sequencing

19. • Solution of one nucleoside is
flooded onto tray.
• If base added is next in the
sequence, it will be added to the
single stranded DNA on the
bead.
• When a nucleoside is added to
DNA, 2 phosphates are given
out
• Enzymes in packing beads
convert phosphate groups to
ATP and then the ATP to light
energy.
• Computer and camera detect
light in a certain well as a
certain base is added to the tray.
• Base is washed off and process is
repeated with another base.
• End product is large amount of
fragments sequenced.

20. Method
Single-molecule
real-time
sequencing
(Pacific Bio)
Ion
semiconductor
(Ion Torrent
sequencing)
Pyrosequencing
(454)
Sequencing by
synthesis
(Illumina)
Sequencing by
ligation (SOLiD
sequencing)
Chain
termination
(Sanger
sequencing)
Read length 30 bp up to 400 bp 700 bp 50 to 300 bp 50-150 bp 400 to 900 bp
Reads per run 50.000 up to 80 million 1 million up to 3 billion 1.2 to 1.4 billion N/A
Cost per 1
million bases (in
US$) $0.33-$1.00 $1 $10 $0.05 to $0.15 $0.13 $2400
NGS – comparison of basic parameters
Examples of recently developed or developing technologies:
454 sequencing – pyrosequencing (Roche)
- complementary strand synthesis
Illumina – sequencing by synthesis
- complementary strand synthesis
SOLiD - Sequencing by Oligonucleotide Ligation and Detection
- ligation of labelled oligonucleotides
Oxford nanopore technology
- exonuclease degradation, el. current changes detection

21. Next-Generation DNA Sequencing
• Illumina Genome Analyzer
The steps are as follows:
1. Prepare genomic DNA.
2. Attach DNA to surface
3. Bridge amplification.
4. Fragment becomes double stranded.
5. Denature the double stranded molecules.
6. Complete amplification.
7. Determine first base.
8. Image first base.
9. Determine second base.
10. Image second base.
11. Sequence reads over multiple cycles.
12. Align data.
Illumina Hi-seq produces approximately 1.6
billion short reads (18–150bp)

22. Illumina – sequencing by synthesis (Solexa)

23. Illumina – sequencing by synthesis (Solexa)

24. Illumina – sequencing by synthesis (Solexa)

25. Illumina – sequencing by synthesis (Solexa)

26. Pyrosequencing
Pyrosequencing is a real-time DNA-sequencing method based on the detection
of the Ppi released during the DNA polymerization reaction
1. dNTP is tried in the nucleic acid polymerization reaction. PPi is released when
one of the deoxynucleotides (dATP, dCTP, dGTP, or dTTP) is incorporated into
the chain extension by DNA polymerase.
2. This liberated PPi is then converted into ATP by ATP sulphurylase and light is
emitted via the firefly luciferase that catalyses luciferin into oxyluciferin.
3. The amount of light emitted can be measured by an avalanche photodiode,
photomultiplier tube, or with a charged-coupled device camera.

27. Advantages Disadvantages
(i) This sequencing technique dispenses
with the need for labelled primers,
labelled nucleotides, and gel
electrophoresis ;
(ii) detection is in real time with a cycle
time of approximately 2 min (solid-
phase)
(iii) the sequencing reactions occur at
room temperature and physiological pH
(iv) this method is cost-effective when
compared with the traditional methods
(v) the method is easily adapted for
multiplexed sample processing
(vi) short chains can be satisfactorily
sequenced
• (i) in the solidphase approach the
template must be washed
completely after each nucleotide
addition, resulting in a decreased
signal due to loss of templates
• (ii) in the liquidphase approach
the apyrase activity is decreased
in later cycles due to
accumulation of intermediate
products. Non-specific
interaction between apyrase and
DNA was observed; this results in
loss of nucleotide-degrading
activity

28. Applications of DNA Sequencing
1. DNA sequencing plays vital role in the field of agriculture. The mapping and
sequencing of the whole genome of microorganisms has allowed the agriculturists to
make them useful for the crops and food plants.
• specific genes of bacteria have been used in some food plants to increase their
resistance against insects and pests, as a result the productivity and nutritional value
of the plants may increase.
2. In medical research, DNA sequencing can be used to detect the genes which are
associated with some hereditary or acquired diseases.
3. In forensic science, DNA sequencing is used to identify the criminals by finding
some proof from the criminal scene in the form of hair, nail, skin, or blood
samples. DNA sequencing can also be used to determine the paternity of the child.
4. DNA sequencing information is important for planning the procedure and
method of gene manipulation.
5. It is used for construction of restriction endonuclease maps.
6. It is used to find tandem repeats or inverted repeat for the possibility of hairpin
formations.

29. 8. DNA sequences can be used to find a polypeptide sequence from the data bank or
to compare with DNA sequences from other organisms for phylogenetic analysis.
9. DNA sequencing is used to construct the molecular evolution map.
10. Last but not least, it is useful in identifying exons and introns.
11. DNA sequencing discovers intra and inter species variations.
12. It can characterize the transcriptome of a cell.
13. It can identify DNA bindinig sites for proteins.
14. Metagenomic applications: It uses whole sample DNA/RNA with wide range of
applications i.e phylogenetics/comparative/ functional analysis in addition to
environmental profiling.
Applications of DNA Sequencing

30. References
1. Indu Ravi, Mamta Baunthiyal, Jyoti Saxena (2014),Advances in biotechnology, springer
pub., pp: 11-25.
2. Anjana Munshi (2012), DNA Sequencing – Methods and Applications, intech open
science publication