Biotechnology Engineering

1. DNA Sequencing -I
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Lecture- 31

2. Introduction
DNA sequencing is the process of determining the sequence of
nucleotides in a DNA molecule.
First DNA Sequencing Techniques
1. Chain termination method or Sanger’s dideoxy method (1977)
2. Chemical degradation method or Maxam and Gilbert’s method
(1977)
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• Allan M. Maxam and Walter Gilbert, and Frederick Sanger and
coworkers in 1977 reported methods for DNA sequencing. Initially the
success of these methods was limited to the sequencing of some
prokaryotic and eukaryotic genes, and the complete sequencing of
some viral genomes.
• Further improvements in the sequencing methods and the combined
use of the sequencing methods and the recombinant DNA approaches
led to the taking up of the complete genome sequencing projects.
Human Genome Project, under the leadership of James D. Watson, was
launched in the year 1990 to sequence the entire human genome.
• Later, Celera Genomics, a private organization, also joined the race
of human genome sequencing, independently. Both the groups
published two draft sequences of human genome in 2001. Bacterial
artificial chromosome vectors played a very significant role in the
sequencing of the human genome.

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•Complete genome sequencing has been achieved for Haemophilus
influenzae (1995), Saccharomyces cerevisiae (1996), Methanococcus
jannaschii (1996), E. coli (1997), C. elegans (1998), Drosophila melanogaster
(2000), Arabidopsis thaliana (2000), rice (2005), poplar (2006) and many
other organisms.
•On 1st June, 2007 James D. Watson became the first human to receive his
personal haploid genome sequence and on 4th September in the same year
the complete diploid genome sequence of Craig Venter became available.
Presently the sequencing of thousands of human genomes is in progress in
different laboratories.
•The cost of the first human genome project was about $3 billion and the
project was completed in about 12 years. Presently a human genome can be
sequenced in 4-6 months with an estimated cost of $50, 000.
•Rapid advancements in sequencing techniques and approaches are giving
us a promise of significantly cutting down the time and cost of the human
genome sequencing. It is expected that in the next few years sequencing of a
complete human genome could be achieved in about two minutes at the cost
of $ 5, 000!

5. 1977
devised methods for DNA sequencing
Walter Gilbert
(Mar 21, 1932 - )
Frederick Sanger
(Aug 13, 1918 –Nov 19, 2013)
Nobel Prize in Chemistry 1980
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6. Sanger’s Dideoxy Method
Developed by Frederick Sanger and coworkers, it became the
most used method for sequencing DNA.
Frederick Sanger
– Was originally a protein chemist
– Made his first mark in sequencing proteins
– Made his second mark in sequencing DNA
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7. Principle of Sanger’s Method
The underlying principle of this method is that the incorporation of
a 2’,3’-ddNTP in place of its corresponding dNTP in the DNA
chain terminates DNA synthesis. This is because the ddNTPs
are nucleotide analogs that lack the 3’-OH group that is
necessary for phosphodiester bond formation and chain
elongation.
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8. NTP, dNTP and ddNTP
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9. Dideoxynucleotide
no hydroxyl group at 3’ end
prevents strand extension
CH2
O
OPPP
5’
3’
BASE
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10. Sanger’s Dideoxy Method
 DNA synthesis using deoxy- and dideoxynucleotides that
results in termination of synthesis at specific nucleotides
 Incorporation of di-deoxynucleotides into growing strand
terminates synthesis
 Requires a primer, DNA polymerase, a template, a mixture of
four dNTPs and a ddNTP, and detection system
 Synthesized strand sizes are determined using gel or capillary
electrophoresis
 Enzymatic method 10

11. Steps Involved in Sanger’s Method
• Four different reactions in separate tubes are carried out,
each having four dNTPs, small amount of single type of
ddNTP, primer, and DNA polymerase.
• ddNTPs are incorporated in place of dNTPs at different
positions during template-directed synthesis of a DNA strand
by a DNA polymerase and terminate DNA synthesis in a base
specific manner as these lack 3’-hydroxyl residues.
• After a suitable incubation period, DNA of each reaction
mixture is denatured, electrophoresed on a sequencing gel,
and subjected to autoradiography.
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12. Components of a Sequencing Reaction
1. Template DNA (single – stranded)
2. Primer (end-labeled primer)
3. Enzymes (Klenow fragment of Escherichia coli or
Sequenase)
4. Substrates (dNTPs and ddNTPs )
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13. Sequencing of DNA by the Sanger method
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15. Comparison with Maxam and Gilbert’s
Method
• Sanger’s Method
– Enzymatic
– Requires DNA synthesis
– Termination of chain
elongation
• Maxam & Gilbert’s Method
– Chemical
– Involves DNA degradation
– Breakage of DNA at different
nucleotides
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16. Applications of DNA Sequencing
• The DNA sequencing information is important for planning of
gene manipulation experiments.
• DNA sequencing is used for construction of restriction
endonuclease maps.
• It is used to find tandem repeats or inverted repeats for the
possibility of hairpin formation.
• DNA sequences can be used to study evolutionary
relationships of various organisms.
• Used to identify exons and introns
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