1. DNA SEQUENCING
By
Mallappa. Shalavadi,
Department of Pharmacology,
HSK College of Pharmacy,
Bagalkot.

2. Contents:
1. Introduction.
2. Methods of sequencing.
 Conventional DNA sequencing method.
 Cycle sequencing.
 Automated DNA sequencing.
 Pyrosequencing.

3. INTRODUCTION:
The information content of DNA is encoded in
the form of four bases (A,G,C and T) and the
process of determining sequence of these
bases in a given DNA molecule is referred to as
DNA sequencing.
DNA fragments can be analyzed to determine
the nucleotide sequence of DNA and to
determine the distribution and location of
restriction sites.

4. Fundamental reasons for knowing the sequence
of DNA molecule:
 To charecterise the newly cloned DNA.
 For predictions about its fuctions.
 To facilitate manipulation of the molecule.
 To confirm the identity of a clone or a
mutation.
 To check the fidility of newly created
mutation and ligation junction.
 Screening tool to identify polymorphisms and
mutation in genes of particular interest.

5.  To confirm the product of a PCR.
METHODS OF DNA SEQUENCING:
(A)Conventional DNA sequencing methods
1. Chemical degradation method.
2. Chain termination method.
(B) Cycle sequencing.
(C) Automated DNA sequencing.
(D) Pyrosequencing.

6. (A)Conventional DNA sequencing methods:
1. Chemical degradation method
[ Maxam and Gilbert’s method]
• This method involves the base specific
chemical cleavage of an end labeled DNA
segment to generates a set of labeled
molecules.
PRINCIPLE:
• The partially cleaved DNA fragment is
subjected to five separate chemical reactions.
• Each of which is specific for a particular base.

7. • The resulting fragment terminate at that
specific base followed by high resolution gel
electrophoresis and detection of the labeled
fragments by autoradiography
METHOD
• Labeled DNA at one end with 32P.
• DNA copies are divided in to 4 samples.
• Each samples treated with a chemical that
specifically destroys one or two of the 4 base
in DNA.

8. Different types of base specific reactions used in
the Chemical degradation method.
Reagents Base Specific modification
Dimethyl Sulphate G Methylation of N7 renders the C8-C9
(pH) bond susceptible to cleavage.
Piperidine formate A+G Weakens the glycosidic bond of
adenine and guanine residues by
protonaing nitrogen atoms in the purin
rings resulting in depurination.
Hydrazine C+T Opens pyrimidine rings, which recyclize
in a five membered form which is
valnerable.
Hydrazine + 1.5 M Nacl C Only cytocin reacts with Hydrazine.

9. • Results in series of
labeled fragments.
• Length is depends on
the distance of
distroyed base from the
labeled end of molecule.
• If G is 3,6 and 9 base
away from the labeled
end then treatment of
DNA strand with
chemical that cleave at
G will generates labeled
fragments 2, 5 and 8
base length.
• Acrylamide gel
electrophoresis.
• Gel is autoradiographed.
• Read out sequence.

10. • T
• G
• A
• C
• G
• C
• T
• G
• A
• T

11. 2. Chain Termination Method
[Sanger’s Dideoxy method]
• Developed by Frederic’s Sanger.
• Common method.
• Involves controlled synthesis of DNA to
generate fragments terminating at specific
point.
PRINCIPLE
• Replacement of dNTPs with 2’, 3’ dideoxy
NTPs in the DNA chain terminates DNA
synthesis.

12. • This is because
these ddNTPs
are nucleotide
analogues that
lacks the 3’ OH
group that is
necessary for
phosphodiester
bond formation
and chain
elongation.

13. METHOD
• Primer is labeled so that newely
synthesised DNA can be
detected.
• When the primer extended DNA
polymerase occasionally inserts a
ddNTPs instead of dNTPs.
• No further elongation.
• In ddA reaction all cains ends
with ddA.
• DNA chains in in each reaction
are seperated by polyacrylamide
gel electrophoresis.
• Sequence are determined by
autoradiogram by reading
sequencing ladder from bottom
to top to give the sequence in 5’-
3’ orientation.

14. (B) Cycle sequencing
• Dideoxy mediated sequencing reactions using
PCR and end labeled primers.
• Also called thermal DNA sequencing or linear
amplification of DNA sequencing.
• Involves heating reaction mixture to 940 C to
denature the template.
• Cooling below the melting temperature of
primer to allow annealing and repeating the
sequencing reaction.
• This procedure can be repeated untill one of
reaction components is exhausted.

15. • 4 seperate
amlification
reactions are set
up .
• Each having the
same primer and
different ddNTP.
• 2 cycling
programs are
used in cycle
sequencing.

16. I- program
• Reaction mixtures are subjected to 15-40
cycles of conventional PCR cycling
- Determination of the ds DNA strand.
- Annealing of a labeled sequencing primer to
its target sequence.
- Extention of the anneal primer by a
thermostable DNA polymerase.
• Finally termination of extended strand is
done by the incorporation of ddNTP.
• Results in double stranded hybrid.

17. • This hybrid is denatured during the first step
of the next cycle there by liberating the
template strand for another round of priming
extension and termination.
• Therefore the radio labeled chain terminated
products accumulate in a linear fashion.
II- program
• Annealing step is eliminated so no further
extension of primer is possible.
• Radio labeled products are finally resolved on
a polyacrylamide gel and visualized by
autoradiography.

18. (C) Automated DNA sequencing
• Key advantage is automated data collection in
an easy way and in lesser time.
• Florescence technique is used for detection of
DNA bands.
Types of florescence labeling systems
1. FOUR REACTION/ ONE GE SYSTEM
• Fluorescent primer are used with non labeled
ddNTPs.
• Different fluorescence dye are used for 4
chain exyention reaction.

19. • Resulting DNA
strands are
seperated in 4
different lane in
electrophoresis.
• Detected by
fluorescence
detector.

20. 2. ONE REACTION/ ONE GEL SYSTEM
• 4 ddNTPs are labeled with different
fluorophor.
• Chain extention reaction is carried in a single
tube.
• Resulting fragment is subjected to gel
electrophoresis in a single lane.
• The tag is incorporated in DNA molecule.
• Leads to termination and attachment of
fluorophor at the end of DNA molecules.
• Gel is illuminated with argon beam snd
detected by photomultiplyer.

23. (D) Pyrosequencing
• More rapid minisequqncing method.
• Not require electrophoresis or any other
fragment seperation.
• Determine which of the 4 bases is incorporate
at each step in the copying of DNA templete.
• ddNTPs are not required.
• As the new strad is being made, the order in
which the dNTPs are incorporated is detected.
• So the sequence can read as the reaction
proceeds.

24. • In reaction all 4 dNTPs are not added at one
time.
• Each dNTP is added individually in a
sequential manner.
• If perticular dNTP are not incorporated then it
is rapidly degraded by nucleotidase or by
washed before addition of next dNTP.
• Incorporation leads release of pyrophosphate
which is detected in an enzyme cascade that
emits light.

26. TYPES OF PYROSEQUENCING
1. SOLID PHASE SEQUENCING
• Template and primer are immobilized on
solid support.
• All dNTPs are added stepwise and
incorporation of particular dNTP is detected
by addition of ATP sulfurylase and luciferase.
• A washing step is carried out after addition of
each dNTP for removal of the excess
sustrates.

28. REFERANCE
1. Genetic Engineering by Smitha Rasthogi and
N. Pathak.
2. I Genetics 2nd ed by Peter J. Russell

29. 2. LIQUID PHASE
PYROSEQUEN
CING
• All reagents
with DNA
template are
added in well
of a micro
titer plate.
• Further steps
similar to
solid phase
sequencing.

30. Referance
1. Genetics Engineering by S. Rasthogi and N.
Pathak.
2. I Genetics 2nd ed by Peter J. Russell.
3. www.google. Com