dna sequencing is the one of the most important technique in today's biotech field and in this ppt I cover up the most imporatant techniques of DNA sequencing methods

1. GURU GHASIDASVISHWAVIDYALAYA
BILASPUR,CHHATTISGARH
Department of Biotechnology
Subject : Molecular Diagnostic
Topic : Nucleic Acid Sequencing
Guided by
Dr. Dhananjay Shukla
Assistant Professor Submitted by
Parth Sharma
MSc II semester
Roll no. 22043125

2. Introduction
• Nucleic acids are genetic material of all living organisms. They are polymers of
nucleotides which is composed of pentose sugar (5-carbon sugar), phosphate
group, and nitrogenous base.
• Nucleotides are ATP, GTP, CTP,TTP and UTP.
• Nucleic acid sequencing is the method to analysing nucleic acids DNA, RNA which
quickly determine the sequence of bases in nucleic acid. It allow the genetic
information to be read and providing information of gene structure.
• To study of evolutionary analysis between species or populations.
• DNA sequencing can reveal changes in a gene that may cause a disease.
• Used in medicine including diagnosis and treatment of diseases and epidemiology
studies.
• It can be helpful to improve food quality and sustainable agriculture.

3. Nucleic acid sequencing can be divided in
to three group :
First generation sequencing
o like Maxam & Gilbert’s sequencing and Sanger sequencing
Second generation / next generation sequencing
o Pyrosequencing, Reversible chain terminator sequencing and sequencing by
ligation
 Third generation sequencing
o Single molecule real time sequencing

4. Maxam & Gilbert’s chemical degradation
method :
• This method was developed by Allan Maxam andWalter Gilbert in 1977.
• It is based on nucleobase specific partial base chemical modification of DNA.
o Dimethyl sulfate – adds methyl group in guanine
o Formic acid – breaks glycosidic bond between adenine and guanine.
o Hydrazine – acts on thymine and cytosine and split there pyrimidine ring.
o Hydrazine + NaCl – modify cytosine.
o Pyperdine used to break sugar phosphate bond at modified nucleotide.

5. Procedure
PCR amplification
Denaturation
End labeling by radioactive phosphate at 5’ end
Chemical degradation
Gel electrophoresis
Autoradiography
Detection
Dimethly sulfate formic acid hydrazine hydrazine + NaCl
Figure 1: Diagram of Maxam-Gilbert’s chemical
degradation method.

6. Sanger sequencing
• This method was discovered by Fredrick
Sanger & colleagues in 1977.
• This method is based on the ability of a
DNA polymerase to extend primers, after
hybridized to the template that is to be
extended until a chain termination
nucleotides are incorporated.
• This method uses dideoxy nucleotides to
terminate the DNA replication.
• Dideoxy nucleotides lack hydroxyl group
at 3’ carbon so phosphodiester bond will
not form and synthesis of new strand will
be terminated.
Figure 2: (A) a dideoxy nucleotide lacking hydroxyl
group at 3’ carbon. (B) a normal deoxy nucleotide
containing hydroxyl group at 3’ carbon.

7. Procedure :
Denaturation
Primer annealing
Chain termination reaction
Gel electrophoresis
Autoradiography
Determination Figure 3 : Process of Sanger sequencing and a autoradiograph
showing different bands of DNA and representing the sequences
from bottom to top.

8. Automated sanger sequencing
ddNTPs labelling with fluorescent dye
Mixing of DNA fragments and ddNTPs in a single
tube
Primer annealing
Chain termination reaction
Gel electrophoresis
Detection by ion laser beam and photomultiplier
Figure 4 : Process of automated Sanger sequencing.

9. Second generation / Next generation sequencing
• NGS technologies permit the millions of sequencing reactions in parallel on the same
solid surface which may be beads or glass slide.
• NGS begins with the preparation of a library of DNA fragments that have been
immobilized on a solid support, in such a way that the individual sequencing reactions
can be carried out side-by-side in an array format.
• There are three steps to the preparation of this library:
1 Breakage of the starting DNA into fragments of sizes that are suitable for the
sequencing method being used.
2 Immobilization of the fragments on to the solid support.
3 Amplification of the immobilized fragments

10. Pyrosequencing
• Pyrosequencing is a type of
sequencing of synthesis technique in
which nucleotides are sequenced
during the addition.
• In this method nucleotides are not
labelled, instead the sequences are
read by detecting flashes of light, due
to release of pyrophosphate during
chain elongation.
• The pyrophosphate generated by the
incorporation of a nucleotide is
combined with adenosine-5′-
phosphosulfate by the enzyme ATP
sulfurylase to form ATP.
• ATP drives the conversion of luciferin
to oxyluciferin by the enzyme
luciferase, which generates light that
is recorded by a photon detector.
Figure 5 : Reaction showing release of pyrophosphate.

11. Figure 6: Reaction showing the emission of light

12. Procedure :
Adaptor ligation
Addition of dNTPs
Light emission
Removal of remaining dNTPs
Detection of nucleotide, observation by
pyrogram
Addition of next dNTP Figure : 7 Process showing pyrosequencing.

13. Figure 8 : A Pyrogram

14. Reversible termination sequencing
• A modified nucleotide is used to
terminate the strand synthesis, this
nucleotide is fluorescently labelled,
but this modified nucleotide can be
removed. So that it is known as
reversible termination reaction.
• The capping of the 3′ carbon of the
deoxyribose sugar with a chemical
group that blocks subsequent addition
of nucleotides.
• The fluorescently labelled nucleotide
join the complementary strand it
shows fluorophore which is detected
by the detector present on flow cell.
• Removal of this blocked chemical
group converting the position to a 3′ -
hydroxyl group, which allows further
extension to occur.
Figure 9 : A blocked fluorescently labelled nucleotide

15. Procedure :
Denaturation
Oligonucleotide attachment
Attachment to flow cell
Primer annealing
Addition of nucleotide
Removal of blocked chemical group and
fluorescent dye
Addition of next nucleotide
Figure 10: Process of reversible terminator
sequencing.

16. Sequencing by ligation :
• This procedure requires oligonucleotides that are usually 8 (octamers) or 9
(nonamers).
• They contain a known (fixed) nucleotide at a specific (query) position and any
nucleotide in the other positions. They are tagged at their 3′ ends with the same
fluorescent dye.
• The anchor primer binds to its complementary sequence in the adaptor at the 3′
end of the single-stranded template DNA.
• Nonamers with fixed nucleotides A, G, C, and T at the same query position, is
added and incubated for a brief period withT4 DNA ligase.
• If the nonamer sequence is exactly complementary to the template sequence,
then T4 DNA ligase will join the nonamer to the anchor primer. The identity of the
nucleotide in position 1 is determined by the fluorescence produced.
Figure 11 : Different nonamers with fixed nucleotides at different query position.

17. Procedure
Denaturation
Addition of anchor primer
Addition of nonamer and incubation
withT4 DNA ligase
Detection of fluorescent
Addition of next nonamer
Figure 12 : Representation of the first three cycles of
sequencing by ligation of a template sequence.

18. Ion semiconductor sequencing
• Ion semiconductor sequencing is based on the detection of hydrogen ions that are
released during the polymerization of DNA.
• Incorporation of a dNTP into a growing DNA strand involves the formation of
a covalent bond and the release of pyrophosphate and a positively charged
hydrogen ion.
• Semiconductor chip have microwells that contain many copies of one single-
stranded template DNA molecule to be sequenced and DNA polymerase are
sequentially flooded with unmodified dNTP.
• The hydrogen ion that is released in the reaction changes the pH of the solution,
which is detected by an ISFET (Ion Sensitive Field EffectorTransistor).

19. Procedure :
DNA fragmentation
Addition of oligonucleotides
Oligonucleotides are attached to
beads, present on microwells
Primer attachment
Detection of nucleotides Figure 12 (A) showing oligonucleotide attached with
bead and template DNA. (B): graph showing changes in
pH according to dNTPs.

20. Whole genome sequencing – Short gun
sequencing method :
• DNA is broken up randomly into numerous small segments, which are
sequenced using the chain termination method to obtain reads.
• Multiple overlapping reads for the target DNA are obtained by performing
several rounds of this fragmentation and sequencing.
• Computer programs then use the overlapping ends of different reads to
assemble them into a continuous sequence.
Figure 13 : different overlapping
fragments

21. Procedure :
DNA fragmentation
Gel electrophoresis for the separation
of small and large fragments
Cloning of the fragments by using
plasmid and BAC vector
Sequencing by computer programers
Figure 14 : process of whole genome short gun
sequencing

22. Third generation sequencing :
• Third generation sequencing techniques remove the biasness of second
generation techniques.
• Third -generation sequencing technologies do not require amplification step and
are capable of sequencing single DNA molecule in real time.
• Third gen sequencing techniques currently in the developmental stage several
companies trying to develop techniques for sequencing one is PacificBioscience.
• PacBio developed the sequencing platform of single molecule real time
sequencing (SMRT), based on the properties of zero-mode waveguides.

23. Single molecule real time sequencing (SMRT)
• This technology for sequencing is based upon two key inventions: phospholinked
nucleotides and zero-mode waveguides (ZMW).
• The key feature of ZMW is that it only permits light to illuminate the bottom of a well
where the immobilized template and DNA polymerase are present.
• A zero-mode waveguide is an optical waveguide that guides light energy into a volume
that is small in all dimensions compared to the wavelength of the light.
• A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single
molecule of DNA as a template.
• Each of the four DNA bases is attached to one of four different fluorescent dyes. When a
nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and
diffuses out of the observation area of the ZMW where its fluorescence is no longer
observable.
• A detector detects the fluorescent signal of the nucleotide incorporation, and the base
call is made according to the corresponding fluorescence of the dye.

24. Figure 16 : SMRT cell with zero mode waveguide

25. Procedure :
Attachment of DNA polymerase, single
strand DNA in ZMW
Attachment of four different
fluorescent dye to different dNTPs
Incorporation of nucleotide and
fluorescent tag is cleaved
Detection
figure :15 procedure of SMRT sequencing

26. Conclusion :
• Sequencing technology has revolutionized each and every field of medical science
and life sciences imparting numerous benefits in terms of massive parallel
sequencing.
• Earlier, high cost of sequencing was also the barrier and now the reduced cost by
several folds is enormously attracting the researchers and making it feasible to
plan their research based on sequencing.
• In coming years sequencing of individual genomes could be performed, which will
be helpful in different nutritional intake and better treatment and therapeutics for
each individual.

27. Figure 17 : A graph showing the history of sequencing technology

28. References :
1. The Applications of Gene Cloning and DNA Analysis in Research; T.A. BROWN,
Seventh Edition; Chapter 10, Sequencing Genes and Genomes, 175-200.
2. Molecular Biotechnology Principles and Applications of Recombinant DNA;
Bernard R. Glick and Jack J. Pasternak; 4th edition; Chapter 4 DNA sequencing
techniques, 117-132.
3. Microbial Technology for Welfare of Society; Pankaj Kumar Arora; Chapter 15,
Next-Generation Sequencing and Its Application: Empowering in Public Health
Beyond Reality, 313-341.