Professional Documents
Culture Documents
1
DNA Sequencing
Campbell, 3
5e, p. 378
DNA Sequencing - Sanger Method
Campbell, 4
5e, p. 378
DNA Sequencing - Sanger Method
5
Campbell, 5e, p. 371
DNA Sequencing
6
DNA Sequencing
7
DNA Sequencing
8
Campbell, 5e, CD
Procedure for the Sanger dideoxy chain termination technique
9
Composition of a DNA sequencing reaction
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
11
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
12
primer binding site
gene for
gene for tetracycline
ampicillin resistance
resistance EcoRI
Pst I
Sal I
pBR322
ori
5’ 3’
|||||||||||||||
3’
14
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
15
5’
|||||||||||||||
3’ ATCATGTCATCAAGTCTAGCAC
A T G C
3’
TAGTACAGTAGTTCAGATCGTG
Longer fragments
Sequence
of the
strand
that was
synthesized
Shorter fragments
16
5’
Sanger dideoxy chain termination technique
17
Automated DNA sequencing result
18
Manual DNA sequencing result
3’
5’ 19
20
http://www.rvc.ac.uk/Extranet/DNA_1/6_Sequencing.htm
• DNA Sequencing by the Enzymatic
Method
21
• 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.
22
• 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.
23
Cycle sequencing
Cycle sequencing
24
Strategy for DNA sequencing
25
Primer walking
Primer- 1
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
26
Primer walking cont..
Contig assembly
5’ 3’
5’ 3’
5’ 3’
5’ 3’
DNA sequence of 2kb DNA
27
Shotgun sequencing
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