DNA

1. DNA SEQUENCING

2. What Is DNA Sequencing?
The term DNA Sequencing refers to method for
determining the order of the nucleotide bases
Adenine, Guanine, Cytosine and Thymine in a
molecule of DNA

3. T
o determine the order of the nucleotide bases adenine, guanine,
cytosine, and thymine in a molecule of DNA two methods
were used
1. Sanger; Chain Termination Sequencing method
2.Maxam and Gilbert; Chemical Sequencing method These
two methods are most popular conventional methods
Robotics and automated sequencing are based on these
methods

4. Disadvantages Of Maxam & Gilbert
Chemical Degradation Method
• Requires lots of purified DNA and many
intermediate purification step
• It requires extensive use of hazardous
chemicals
• It is difficult to scale up and cannot be used to
analyze more than 500bp

6. SANGERS METHOD OF DNA
SEQUENCING
• The chain terminator method is more efficient
and uses fewer toxic chemicals and lower
amount of radioactivity than the method of
Maxam and Gilbert.
• The key principle of the Sangers method was
use of dideoxynucleotides triphosphate
(ddNTPs) as DNA chain terminator

10. Chain termination method of DNA
sequencing
• It involves following components:
1. Primer
2.DNA template
3.DNA polymerase
4.dNTPs(A,T,G,C)
5. ddNTPs
• It involves following 4 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Poly acrylamide gel electrophoresis

11. • The Methods (Procedure ):-
1. Before the DNA can be sequenced, it has to be
denatured into single strands using heat.
2. Next a primer is annealed to one of the template
strands. This primer is specifically constructed so that
its 3' end is located next to the DNA sequence of
interest. Either this primer or one of the nucleotides
should be radioactively or fluorescently labeled so
that the final product can be detected on a gel. Once
the primer is attached to the DNA, the solution is
divided into four tubes labeled "G", "A", "T" and "C".

12. • ‘’G’’ tubes : all four dNTP’s, ddGTP and DNA
polymerase
• ‘’
A’’ tubes : all four dNTP’s, ddATP and DNA
polymerase
• ‘’T’’ tubes : all four dNTP’s, ddTTP and DNA
polymerase
• ‘’C’’ tubes : all four dNTP’s, ddCTP and DNA
polymerase

14. • Mixture first heated so that DNA strands separate
(96⁰C)
• Then temperature get lower so that short length DNA
sequence – a primer can bind to the template
DNA(50⁰C)
• Temperature raised to (60⁰C- 65⁰C)to enable the DNA
Polymerase enzyme to bind to the short section of
double stranded DNA.
• DNA polymerase to synthesizes new DNA. DNA
Polymerase will continue adding Nucleotides to chain
until it happens to add a dideoxy nucleotide instead of
normal one

17. Uses of Sangers sequence
• Region up to about 900 basepairs in length
sequenced using this method
• In Human Genome project sanger sequencing
was used to determine the sequence of
relatively small fragments of human DNA
• Sanger sequencing is still in wide use for the
sequencing of individual pieces of DNA, such
as fragments used in DNA cloning

18. • Advantage of Basic method
• 1:-Improvement diagnosis of disease.
• 2:- Identifying suspects .
• Disadvantage :
• 1:-Whole genome can not be sequenced at
once .
• 2:- Very slow and time consuming

20. • Applications of DNA
Sequencing
•Forensics: to help identify individuals because
each individual has a different genetic sequence
•Medicine: can be used to help detect the genes
which are linked to various genetic disorders such
as muscular dystrophy.
•Agriculture: The mapping and sequencing of a
genome of microorganisms has helped to make
them useful for crops and food plants.

21. Maxam–Gilbert sequencing is a method of DNA sequencing
developed by Allan Maxam and Walter Gilbert in 1976–1977.
This method is based on nucleobase-specific partial chemical
modification of DNA and subsequent cleavage of the DNA
backbone at sites adjacent to the modified nucleotides.

22. Maxam Gilbert Sequencing: Process Summarized
1. Label 5’- end of DNA
2. Aliquot DNA sample in 4 tubes
3. Perform base modification reaction
4. Perform Cleavage reaction
5. Perform Gel Electrophoresis
6. Perform Autoradiography
7. Interpret results

23. I. Chemical Modification of DNA; radioactive labeling at one 5'
end of the DNA (typically by a kinase reaction using
gamma-32P ATP)
II. Purification of the DNA fragment to be sequenced
III. Chemical treatment generates breaks in DNA
IV.Run on the gel

24. Chemical Modification and Cleavage
Poly nucleotide Kinase radioactive label at one
5' end of the DNA using gamma-32P
5′ G A C G T G C A A C G A A 3′
32P 5′ G A C G T G C A A C G A A 3′

25. Chemical Modification and Cleavage
Base Modification using Dimethyl sulphate
– Purine
• Adenine
• Guanine
– Only DMS------- G
– DMS+ Formic acid-------G+A
Cleavage of Sugar Phosphate backbone using
Piperidine

26. Base modification using Hydrazine
– Pyrimidine
• Cytosine
• Thymine
– Hydrazine----- C+T
– Hydrazine + NaCl--------C
Cleavage of Sugar Phosphate backbone using
Piperidine

29. An Example for
Maxam-Gilbert
Sequencing

30. 1. 454 Pyrosequencing
Pyrosequencing is based on the 'sequencing by synthesis'
principle, where a complementary strand is synthesized in the
presence of polymerase enzyme.

32. It initially uses the emulsion PCR technique to construct the
colonies required for sequencing and removes the
complementary strand.
Next, a ssDNA sequencing primer hybridizes to the end of
the strand (primer-binding region), then the four different
dNTPs are then sequentially made to flow in and out of the
wells over the colonies.
When the correct dNTP is enzymatically incorporated into
the strand, it causes release of pyrophosphate

33. In the presence of ATP sulfurylase and adenosine, the
pyrophosphate is converted into ATP
.
This ATP molecule is used for luciferase-catalysed conversion
of luciferin to oxyluciferin, which produces light that can be
detected with a camera.
The relative intensity of light is proportional to the amount of
base added (i.e. a peak of twice the intensity indicates two
identical bases have been added in succession).

34. Pyrosequencing, developed by 454 Life Sciences, was one of
the early successes of Next-generation sequencing; indeed,
454 Life Sciences produced the first commercially available
Next-generation sequencer.
However, the method was eclipsed by other technologies
and, in 2013, new owners Roche announced the closure of
454 Life Sciences and the discontinuation of the 454
pyrosequencing platform.