INTRODUCTION DEFINITION HISTORY METHODS OF DNA SEQUENCING MAXAM GILBERT METHOD SANGERS METHOD AUTOMATED DNA SEQUENCER PYROSEQUENCING SHOTGUN SEQUENCING DNA MICROARRAY APPLICATION CONCLUSION REFRENCES

1. By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. S
Y
N
O
P
S
I
S
 INTRODUCTION
 DEFINITION
 HISTORY
 METHODS OF DNA SEQUENCING
MAXAM GILBERT METHOD
SANGERS METHOD
AUTOMATED DNA SEQUENCER
PYROSEQUENCING
SHOTGUN SEQUENCING
DNA MICROARRAY
 APPLICATION
 CONCLUSION
 REFRENCES

3. INTRODUCTION
• DNA sequencing is the determination of the
precise sequence of nucleotides in a sample of
DNA .
• DNA sequencing is important to understand the
function of genes, and basis of inherited disorders,
DNA cloning and gene manipulation .

4. DEFINITION
“DNA Sequencing means finding the
order of nucleotides (adenine, guanine,
cytosine, and thymine) on a piece of
DNA.”

5.  James Watson and Francis Crick published the first
description double-helix DNA structure in 1953.
 In 1977, Maxam and Gilbert was first developed DNA
sequencing technique by using chemical reagents.
 In 1980, Fredrick Sanger developed most widely
used method of DNA sequencing i.e., Chain
termination method.
 Leroy E. Hoods laboratory at the California Institute
of Technology and Smith announced the first semi-
automated DNA sequencing machine in 1986 .
HISTORY

6.  Pyrosequencing : developed by Mostafa Ronaghi
and Pål Nyrén at the Royal Institute of
Technology in Stockholm in 1996.
 DNA Microarray discovered by Shina et al 1995.

7. MAXAM GILBERT METHOD
It is based on chemical modification of
DNA and subsequent cleavage at specific
bases with toxic chemicals :-
Guanine (G) --> Dimethylsulfate
Guanine & Adenine (G+A) --> Formic Acid
Cytosine & Thymine (C+T) --> Hydrazine
Cytosine --> Hydrazine + 5M NaCl

9. SANGERS (CHAIN TERMINATION)
METHOD

12. • Chain termination (Sanger sequencing)
• 400 to 900 bp
• 99.9%
• 20 minutes to 3 hours
• $ 2400
• Long individual reads. Useful for many
applications.
• More expensive and impractical for larger
sequencing projects
1) Method
2) Read length
3) Accuracy
4) Time per run
5) Cost per 1 million
bases (in US $)
6) Advantages
7) Disadvantages

13. AUTOMATED DNA SEQUENCER

14. Advantage
 Rapid and Accurate
 Sequence up to 10,000 nucleotides per day.
 Used in human genome project.

15. PYROSEQUENCING
 Pyrosequencing is a method of DNA
sequencing based on the "sequencing by
synthesis" principle .
 It relies on the detection of pyrophosphate
release on nucleotide incorporation, rather than
chain termination with dideoxynucleotides .

17. 1) Method
2) Read length
3) Accuracy
4) Reads per run
5) Time per run
6) Cost per 1 million bases
(in US$)
7) Advantages
8) Disadvantages
• Pyrosequencing (454)
• 700 bp
• 99.9%
• 1 million
• 24 hours
• $10
• Long read size. Fast.
• Runs are expensive.
• Homopolymer errors.

18. Shotgun sequencing
 Shotgun sequencing is a sequencing method designed
for analysis of DNA sequences longer than 1000 base
pairs, up to and including entire chromosomes .
 In shotgun sequencing, DNA is broken up randomly into
numerous small segments, which are sequenced using the
chain termination method to obtain reads.

21. DNA MICROARRAY
 A DNA microarray (or biochip) is a collection of
microscopic DNA spots attached to a solid
surface .

23. MASS SPECTROMETRY
 MALDI-TOF MS has specifically been
investigated as an alternative method to gel
electrophoresis for visualizing DNA
fragments.
 The mass of each nucleotide is different
from the others and this difference is
detectable by mass spectrometry.

24. APPLICATIONS
1) Molecular Biotechnology
2) Human Genome Project
3) Clinical Application
4) Agriculture
5) Forensic Science
6) Genetic Engineering
7) Molecular Medicine

25. INSTITUTES FOR DNA
SEQUENCING
A) In India
1. Institute of Genomics and Integrative Biology (CSIR-IGIB),Delhi
2. National Botanical Research Institute (CSIR-NBRI), Lucknow
3. Central Drug Research Institute (CSIR-CDRI), Lucknow
4. Centre for Cellular & Molecular Biology (CSIR-CCMB), Hyderabad
5. National Aids Research Institute (NARI), Pune
B) In Abroad
1. The Whitehead Institute/MIT Center for Genome Research, Cambridge.
2. Genome Sequencing Center , Washington ,University School of Medicine, St.
Louis.
3. The Wellcome Trust Sanger Institute, Cambridge, England.
4. National Centre for Genome Sequence (Mexico).

26. REFRENCES
Books
• Biochemistry by Nelson and Cox, fifth edition .
• Gene Cloning and DNA Analysis by T.A. Brown,
sixth edition, A John Wiley and
Sons,Ltd,publication,2010.
• U Satyanarayan: Biotechnology
Internet
• Wikipedia.org/wiki/Nucelicacid