INTRODUCTION HISTORY WHY GENOME SEQUENCE IS IMPORTANT METHODS FOR SEQUENCING MAXAM & GILBERT’S METHOD SANGER’S METHOD AUTOMATIC SEQUENCING SHOTGUN SEQUENCING APPLICATION CONCLUSION REFERENCE

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

2. SYNOPSIS
INTRODUCTION
HISTORY
WHY GENOME SEQUENCE IS IMPORTANT
METHODS FOR SEQUENCING
MAXAM & GILBERT’S METHOD
SANGER’S METHOD
AUTOMATIC SEQUENCING
SHOTGUN SEQUENCING
APPLICATION
CONCLUSION
REFERENCE

3. INTRODUCTION
DNA sequencing includes several methods and technologies that are
used for determining the order of the nucleotide bases—adenine,
guanine, cytosine, and thymine—in a molecule of DNA.
The sequence of DNA constitutes the heritable genetic information in
nuclei, plasmid, mitochondria, and chloroplast that forms the basis for
the developmental programs of all living organism.

4. Determination of DNA sequences has become useful for basic
biological research, other research branches utilizing DNA sequencing,
and in numerous applied fields such as diagnostic, biotechnology,
forensic biology.
The advent of DNA sequencing has significantly accelerated
biological research and discovery. The rapid speed of sequencing
attained with modern DNA sequencing technology has been
instrumental in the sequencing of the human genome, in the Human
Genome Project.

5. HISTORY
RNA sequencing was one of the earliest forms of nucleotide
sequencing. The major landmark of RNA sequencing is the MS2,
identified and published by Walter Fiers and his coworkers at the
University of Ghent (Ghent, Belgium), between 1972 and 1976.
Prior to the development of rapid DNA sequencing a number of
experiment was done by Frederick Sanger and Walter Gilbert and
Allan Maxam .

6. For instance, in 1973, Gilbert and Maxam reported
the sequence of 24 base pairs using a method known
as wandering-spot analysis sequence of the first
complete gene and the complete genome of
Bacteriophage
The chain-termination method developed by Sanger
and coworkers in 1977 soon became the method of
choice, owing to its relative ease and reliability

7. WHY GENOME SEQUENCE IS SO IMPORTANT
Study of DNA will answer:
Why some cells are prokaryotic and some eukaryotic?
why some yeasts divide by budding and other by binary fission?
How so many life forms?
What is the basis of evolutionary process at DNA level?
How the cell differentiation occurs at different age of life etc.?

8. Sequencing DNA will give answer to most of the
fundamental questions. Knowledge of every gene, its
transcript and the timing of transcript synthesis will be
useful.
Gene sequencing will be also useful to have control on more
complex, useful or quantitative traits, or to separate the
genetic and environmental components of particular
diseases

9. METHODS FOR SEQUENCING
Maxam and Gilberts methods
Sangers dideoxy nucleotide method
Shotgun sequencing method
Automatic sequencing method

10. MAXAM & GILBERT’S METHOD
In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA
sequencing method based on chemical modification of DNA and
subsequent cleavage at specific bases.
This was the readily available sequence method. In this method, end
labelled fragments are specifically cleaved by various chemical
treatment. The cleavage is allowed to occur at G, G + A, C + T and T
bases.

11. These four reaction are sufficient to deduce the sequence
of DNA fragment .
The method requires radioactive labeling at one 5' end of
the DNA (typically by a kinase reaction using gamma-32P
ATP) and purification of the DNA fragment to be
sequenced.
Chemical treatment generates breaks at a small proportion
of one or two of the four nucleotide bases in each of four
reactions (G, A+G, C, C+T).

12. Cleavage at the 3'-side of guanosines with a) dimethylsulfate b) , c) piperidine.

18. SANGER’S DIDEOXY NUCLEOTIDE METHOD
Dideoxynucleotide sequencing represents one method of sequencing DNA. It is
commonly called Sanger sequencing since Sanger devised the method.
This technique utilizes 2',3'-dideoxynucleotide triphosphat (ddNTP), molecules that
differ from deoxynucleotides by the having a hydrogen atom attached to the 3'
carbon rather than an OH group. These molecules terminate DNA chain elongation
because they cannot form a phosphodiester bond with the next deoxynucleotide
.
In order to perform the sequencing, one must first convert double stranded DNA
into single stranded DNA. This can be done by denaturing the double stranded DNA
with NaOH.

21. This figure is a representation of an acrylamide sequencing gel. Notice that the sequence
of the strand of DNA complementary to the sequenced strand is 5' to 3'
ACGCCCGAGTAGCCCAGATT while the sequence of the sequenced strand, 5' to 3',
is AATCTGGGCTACTCGGGCGT.

22. AUTOMATIC SEQUENCING
Automatic sequencers are developed on a method which is variant of dideoxy
nucleotide method.
Here a different fluorescent dye is tagged to oligonucleotide primer in each of the
four reaction tubes.
Four reaction mixtures are then pooled and electrophorosed together in single
polyacrylamide gel.
A high sensitivity fluorescence detector is placed near bottom of the tube and is
measure the amount of each flurophore as a function of time.
The sequence is determined from the order of peaks of four different dyes.

23. PROCEDURE

24. SHOTGUN SEQUENCING
The method developed and preferred by Celera is simply
called shotgun sequencing. This approach was developed
and perfected on prokaryotic genomes which are smaller in
size and contain less repetitive DNA
.
Shotgun sequencing randomly shears genomic DNA into
small pieces which are cloned into plasmids and sequenced
on both strands, thus eliminating the BAC step from the
HGP's approach.
Once the sequences are obtained, they are aligned and
assembled into finished sequence.

26. APPLICATION
• Humans/Animals
As more traditional pharmaceutical firms move from chemical
compounds to ones made from biological material, the importance of
genomic sequencing rises. Genome sequencing can potentially create
a new class of therapeutics in the form of personalized medicine and
eliminate certain side effects of current therapeutics.
• Cancer Research
Cancer research and diagnostics is perhaps the fastest-growing
application for genomic sequencing targets. Through initiatives like
the Cancer Genome Project, scientists hope to classify every cancer
according to their molecular markers and make this information
available to therapeutic and diagnostic researchers.

27. Microbial
Environmental samples can provide a rich survey of genomic diversity. Shotgun
Sanger sequencing and pyrosequencing are cutting-edge techniques designed to
procure unbiased samples of all genes from the members of the sequenced
communities.
Plant
Plant materials are used in food, clothing, building materials, energy production and
pharmaceuticals. The 454 can be used to discover biomarkers of plant species under
critical conditions such as temperature, disease and drought.
Biomass / Bio-fuel
• Biomass, including biofuels, biomaterials, and transgenic proteins, involves taking
DNA from organic life forms such as grasses, weeds, and trees and using them as the
building block for growth.
• The market for biomasses is predicted to significantly grow in coming years as
sustainability assumes a greater role for businesses and policy makers. There are
genes and their enzymes which are key in the production of biofuels.
•

29. REFERENCE
• MOLECULAR BIOLOGY OF THE GENE
BY WATSON
• CELL AND MOLECULAR BIOLOGY BY P.
K. GUPTA
• GENE BIOTECHNOLOGY BY S. N.
JOGDAND