DNA Sequencing
Dr. M. Ravichandran/ Dr P. Lalitha
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DNA Sequencing
Two main methods
1. Sanger dideoxynucleotide chain termination method
(Commonly used method)
A. Manual method
B. Automated method
2. Chemical cleavage method (Maxam and Gilbert method)
Not used nowadays
Use of the technique:
Provides the order of the nucleotides in a given DNA
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DNA Sequencing - Sanger Method
Campbell, 3
5e, p. 378
DNA Sequencing - Sanger Method
Campbell, 4
5e, p. 378
DNA Sequencing - Sanger Method
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Campbell, 5e, p. 371
DNA Sequencing
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DNA Sequencing
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DNA Sequencing
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Campbell, 5e, CD
Procedure for the Sanger dideoxy chain termination technique
• DNA to be sequenced is cloned into a vector, adjacent to a primer site
• Four reactions are set up -- each containing one of four ddNTPs
• DNA is synthesized in the presence of the ddNTPs, giving rise to sets
of DNA products representing all of the possible size fragments
for the unknown sequence
• The fragments are resolved by gel electrophoresis and the sequence
is read up from the bottom of the gel by identifying the lane giving
the next larger size fragment
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Composition of a DNA sequencing reaction
• dNTP – dATP, dCTP, dGTP, dTTP
• One type of ddNTP in each type of reaction
• DNA polymerase (Taq polymerase can also
be used- cycle sequencing)
• One primer
• Template DNA
• Labeling- Radioactive and non radioactive
labeling
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O O O
BASE (A, T, G, C)
P O P O P O C
O- O
O- O- O-
OH
• structure of a dNTP
HO
O O O
BASE (A, T, G, C)
P O P O P O C
O- O
O- O- O-
• structure of a ddNTP OH
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Labeling methods
1. Labeling the nucleotides
S35dNTP or P32 dNTP Or Fluorescent
labeled dNTP
2. Labeling the primers
S35dNTP or P32 dNTP Or Fluorescent
labeled dNTP
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primer binding site
gene for
gene for tetracycline
ampicillin resistance
resistance EcoRI
Pst I
Sal I
pBR322
ori
• DNA to be sequenced is cloned into the EcoRI site
immediately adjacent to the primer binding site
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• For sequencing
• the DNA is denatured into single strands
• the primer is hybridized to the template strand
• DNA is synthesized using DNA polymerase
5’ 3’
|||||||||||||||
3’
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ddATP ddTTP ddGTP ddCTP
DNA DNA DNA DNA
dNTPs dNTPs dNTPs dNTPs
5’
|||||||||||||||
3’ ATCATGTCATCAAGTCTAGCAC
TA
• Each fragment is
TAGTA terminated by a ddA
• All possible products TAGTACA
of the reaction
containing ddATP TAGTACAGTA
TAGTACAGTAGTTCA
TAGTACAGTAGTTCAGA
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5’
|||||||||||||||
3’ ATCATGTCATCAAGTCTAGCAC
A T G C
3’
TAGTACAGTAGTTCAGATCGTG
Longer fragments
Sequence
of the
strand
that was
synthesized
Shorter fragments
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5’
Sanger dideoxy chain termination technique
Automated method Manual method
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Automated DNA sequencing result
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Manual DNA sequencing result
3’
5’ 19
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[Link]
• DNA Sequencing by the Enzymatic
Method
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• The predominate method used nowadays for DNA sequencing is an
enzymatic technique, known as dideoxy sequencing or the Sanger
method (to distinguish it from the chemical Maxam/Gilbert sequencing
method). The DNA to be sequenced is used as a template for in vitro
synthesis by DNA polymerase. Not only are all four normal
deoxynucleotide triphosphates present within the reaction mixture
(dATP, dTTP, dGTP, dCTP), but also present are dideoxynucleotides,
one of each type (eg. ddATP) per sequencing reaction.
Dideoxynucleotides are deoxynucleotides which lack a hydroxyl
groups at both the 2' and 3' positions, therefore cannot extend the chain
by linking to another nucleotide. They therefore act as chain
terminators.
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• Typically, in manual sequencing, four reactions are set up, one for each
of the four dideoxy chain terminators to be used. In addition, either the
primer, used to start the reaction, or one of the normal
deoxynucleotides, is labelled; this could be through a radioactive atom
or through a fluorescent tag. The dideoxynucleotide is present at a
concentration about 200-fold less than its competing nucleotide. There
is therefore a competition between deoxynucleotides and
dideoxynucleotides (eg. here for dA and ddA) for incorporation into
the growing chain leading to a statistical representation of lengths of
DNA which correspond to the first 200-500 residues complementary to
the template. Four separate reactions are run and these are loaded and
their components separated within four separate lanes of a denaturing
gel by electrophoresis. Labelled bands will appear at each location
where the dideoxynucleotide brought that particular elongation
reactions to a halt. Thus, one can read the sequence directly eg. from
the autoradiograph, from the bottom to the top.
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Cycle sequencing
Cycle sequencing
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Strategy for DNA sequencing
1. A "directed cloning" strategy, carefully preparing ordered overlapping
fragments.
2. A second strategy known as "primer walking" requires very fast and
accurate analysis of sequence reads since each sequencing reaction uses
information from the previous read.
3. A third strategy, know as "shotgun sequencing" takes maximum
advantage of the speed and low cost of automated sequencing, but relies
totally on software to assembly a jumble of sequence reads into a coherent
and accurate contig.
The Institute for Genomic Research (TIGR) has demonstrated the power
and utility of the shotgun approach by determining the complete genomic
sequences of Haemophilus influenzae , Methanococcus jannaschii , and
Mycoplasma genitalium.
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Primer walking
Primer- 1
DNA Template to be sequenced in a plasmid vector/PCR product (2kb)
5’ 3’
600-800bases
Design primer at 3’ end
Primer- 2
5’ 3’
600-800bases
Design primer at 3’ end
Primer- 2
5’ 3’
600-800bases
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Primer walking cont..
Contig assembly
5’ 3’
5’ 3’
5’ 3’
5’ 3’
DNA sequence of 2kb DNA
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Shotgun sequencing
DNA Template to be sequenced in a plasmid vector, YAC or BAC ( 20 kb)
Insert cut in to small fragments by restriction enzymes
Clone in a plasmid vector
Gene fragments in the plasmid
vector.
Sequence the insert
Contig assembly 28
Contig assembly
• By a DNA sequencing reaction we can get upto 600-800 bases of DNA sequence
• Both the forward and reverse strand can be sequenced separately using specific
primers
• In the case of 3kb DNA, DNA has to be sequenced as a stretch of 600-800 bases in
five sequencing reactions. The resulting sequences has to be contig-assembled as
shown in the picture above using software. eg. BIOEDIT, DNASIS, DNASTAR
• Before contig-assembly, vector sequence present in the sequence should be29
removed (usually done using a molecular biology software eg. DNASIS, DNASTAR
