Explanation about sequencing of DNA by different techniques.

2. What is the.........
....In DNA
No. Of base pairs ?
Sequence ?
Length ?

3. WHAT IS DNA SEQUENCING.....
 DNA sequencing refers to sequencing methods for determining the order of the
nucleotide bases - adenine, guanine, cytosine, and thymine - in a molecule of DNA.
 Founder of DNA sequencing was SANGER and GILBERT
 Knowledge of DNA sequences has become indispensable for basic biological
research, other research branches utilizing DNA sequencing, and in numerous
applied fields such as:
• diagnostic,
• biotechnology,
• forensic biology and
• biological systematics
 Fifteen years elapsed between the discovery of the DNA double helix in 1953 and
the first experimental determination of a DNA sequence. This delay was caused by
several factors that made the problem intimidating.

4. HISTORY
1953
Discovery of
DNA double
helix by James
Watson and
Francis Crick.
E.Coli alanine
tRNA was
the first
nucleic acid
molecule to
be
sequenced.
1965
1970
Discovery
of type II
restriction
enzyme
Maxum and
gilbert
experiment
1970
1981
Sanger
experiment
Polymerase
chain
reaction
1983

5.  Gene isolation
 Sequence characterization
 Forensics
 Gene gene interaction
 Gene protein interaction
 Cloning
NEED OF DNA SEQUENCING

6. METHODS
Basic Sequencing methods
 Chemical cleavage method (Maxam and Gilbert,
1977)
 Enzymatic method (Sanger, 1981)
Generation Sequencing methods
 Pyrosequencing
 Next generation

7. CHEMICAL CLEAVAGE METHOD
 Allan Maxam and Walter Gilbert developed a method for
sequencing single-stranded DNA.
 This method is based on chemical modification of DNA
and subsequent cleavage at specific nitrogenous bases.
 That are the two-step catalytic process involving
piperidine and two chemicals that selectively attack
purines and pyrimidines

8. PRINCIPLE
 The two-step catalytic process involving Piperidine and two
chemicals that selectively attack purines and pyrimidines.
 Purines will react with dimethyl sulphate and pyrimidines will react
with hydrazine and remove the bases from DNA. Piperidine is also
used to create a cut on glycoside bond and form the fragments of
the DNA.
 It is followed by the purification of DNA fragments that to be
sequenced and labelled with radioactive material.
 The fragments in four reaction are arranged side by side in gel
electrophoresis 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 radiolabelled DNA fragment from which the sequence may be
inferred.

9. STEPS INVOLVED
 End labelling
 Restriction enzyme digestion
 Denaturation
 Chemical degradation
 Gel electrophoresis
 Autoradiography
 Sequence determination

10. WORKING

13. LIMITATION AND APPLICATION
 Limitation
 Chemical can be toxic which can lead to mutation.
 Advantage
 Forensic lab
 Agricultural

14.  Developed by Fredrick sanger in 1977. And got novel prize in 1980.
 Method to find the DNA sequence of unknown DNA strand.
 Also called chain termination method.
 Also called Dideoxy sequencing.
 Both linear DNA and circular DNA can be sequenced using the dideoxy DNA
sequencing method.
 Linear DNA fragments can be generated, for example, by cutting plasmid DNA
with a restriction enzyme or enzymes, or by using the polymerase chain
reaction.
Sanger method

15. PRINCIPLE
 Chain termination DNA sequencing is based on the principle that single
stranded DNA molecules that differ in lenght by just a single nucleotide.
 The chain-termination method uses special, modified substrates called
2’,3’-dideoxynucleotides (ddNTPs), which lack the 3’-hydroxyl group on
their sugar moiety as well as the 2’-hydroxyl.
 DNA polymerase will incorporate a 2’,3’-dideoxynucleotide at the 3’ end of
a growing polynucleotide chain.
 But once incorporated, the lack of a 3’-hydroxyl group prevents the
addition of further nucleotides, causing elongation to terminate.
 the chain-terminating nucleotides will generate a nested set of
polynucleotide fragments, all sharing the same 5’ end but differing in their
lengths and hence their 3’ ends.
 The length of the fragments therefore specifies the position of Cs in the
template strand

16. REQUIREMENTS
 Single stranded DNA
 Primer
 DNA polymerase
 Di-Deoxynucleotide
 Denaturing of template DNA- Heat is used to denature and
separate DNA.
 Addition of a prmer. Primer is about 15-20 nucleotide was
added to the denatured DNA.
 Addition of DNA polymerase. This enzyme bind to a primer to
synthesis new strand of DNA by base pairing complamentary
nucleotides to the complementary DNA.
 Elongation of strand. By adding dNTPs and ddNTPs

17. DI-DEOXY NUCLEOTIDE

19. ddATP + ddA
four dNTPs dAdGdCdTdGdCdCdCd
ddCTP + dAdGddC
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP + dAddG
four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCd
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCd
A
C
G
T
In a sequencing reaction, a large number of template/primer pairs are present, which leads to the synthesis of
DNA fragments stopped at all possible positions along the DNA template strand by the incorporation of a
dideoxynucleotide.

20. Sequencing gels are read from bottom to
top (5′ to 3′).
This is the result of polyacrylamide gel which separates the
molecules on the basis of length of DNA fragment

21. LIMITATIONS
 Not all DNA polymerases can be used for sequencing because some have
5′→3′ exonuclease activity and 3′→5′ exonuclease.
 Requires only single stranded DNA template.
After 4 cycles

22.  Pyrosequencing does not require electrophoresis or any other fragment
separation procedure and so is more rapid than chain termination sequencing.
 The advantage with pyrosequencing is that it can be automated in a massively
parallel manner that enables hundreds of thousands of sequences to be
obtained at once, perhaps as much as 1000 Mb in a single run.
 Sequence is therefore produced much more quickly than is possible by the
chain termination method.
 Pyrosequencing involves detection of pulses of chemiluminescence
Pyrosequencing

23. PRINCIPLE
 For the solution-based version of Pyrosequencing, the single-strand DNA
(ssDNA) template is hybridized to a sequencing primer and incubated with the
enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with
the substrates adenosine 5´ phosphosulfate (APS) and luciferin.

24. REQUIREMENTS
 ssDNA template
 DNA polymerase
 Sulfurylase enzyme
 Luciferase enzyme
 Primer
 Apyrase
 Adenosine 5` phosphosulfate (APS)
 Luciferine
 Make a solution which contains the single-strand DNA (ssDNA) template is
hybridized to a sequencing primer and incubated with the enzymes DNA
polymerase, ATP sulfurylase, luciferase and apyrase, and with the
substrates adenosine 5´ phosphosulfate (APS) and luciferin.
STEPS INVOLVED

25.  The addition of one of the four deoxynucleotide triphosphates (dNTPs)
initiates the second step. DNA polymerase incorporates the correct,
complementary dNTPs onto the template. This incorporation
releases pyrophosphate (PPi).
 ATP sulfurylase converts PPi to ATP in the presence of adenosine 5´
phosphosulfate. This ATP acts as a substrate for the luciferase-mediated
conversion of luciferin to oxyluciferin that generates visible light in
amounts that are proportional to the amount of ATP. The light produced
in the luciferase-catalyzed reaction is detected by a camera and
analyzed in a pyrogram.
 Unincorporated nucleotides and ATP are degraded by the apyrase, and
the reaction can restart with another nucleotide
MECHANISM

26.  In pyrosequencing nucleotide removal is performed in two different ways:
 (i) the solidphase pyrosequencing, which utilizes a three coupled enzymatic
procedure with washing steps
 (ii) the liquid-phase pyrosequencing technique, which employs a cascade of
four enzymes with no washing steps
 SOLID PHASE
The biotin-labeled DNA template with
annealed primer is immobilized to streptavidin
coated magnetic beads
The immobilized primed single stranded DNA
is incubated with three enzymes: DNA
polymerase, ATP sulfurylase and luciferase.
After each nucleotide addition to the reaction
mixture, the DNA template is immobilized by a
magnet system and the unincorporated
nucleotides are removed by a washing step.

28.  LIQUID PHASE
The apyrase shows high catalytic activity and
low amounts of this enzyme in the
Pyrosequencing reaction system efficiently
degrade the unincorporated nucleoside
triphosphates to nucleoside diphosphates and
subsequently to nucleoside monophosphate.
In the liquid-phase Pyrosequencing method,
the sequencing primer is hybridized to a
single-stranded DNA template and mixed with
the enzymes, DNA polymerase, ATP
sulfurylase, luciferase and apyrase, and the
substrates APS and luciferin. The four dNTPs
are added to the reaction mixture
sequentially.

29. USES AND APPLICATIONS
 Tackling Human Disease :- DNA Sequencing technology can help decode the
complexities of human disease. The sequencing technology to quickly identify
microbes and help stem outbreaks and epidemics. Cancer, which is a
multitude of different diseases with many characteristics, is the focus of
a recent effort from the National Cancer Institute (NCI).
 Uncovering the secrets of our past :-Next generation sequencing
allows what was previously technically impossible. DNA derived from
human specimens thousands of years old can now be sequenced to
unveil insights into our evolutionary past.
 Conserving our wildlife
 Parental testing
 Learning about the science behind ancient medicine