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DNA SequencingDNA 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 methodB. 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, 5e, p. 378

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DNA Sequencing - Sanger Method

Campbell, 5e, p. 378

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DNA Sequencing - Sanger Method

Campbell, 5e, p. 371

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DNA Sequencing

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DNA Sequencing

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DNA Sequencing

Campbell, 5e, CD

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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 sequenceis read up from the bottom of the gel by identifying the lane givingthe 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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• structure of a dNTP

• structure of a ddNTP

O

BASE (A, T, G, C)

HO

OPOPOP C

O O O

O- O- O-

O-

OH

O O O

O

BASE (A, T, G, C)

OPOPOP C

O- O- O-

O-

OH

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Labeling methods

1. Labeling the nucleotidesS35dNTP or P32 dNTP Or Fluorescent

labeled dNTP2. Labeling the primersS35dNTP or P32 dNTP Or Fluorescent

labeled dNTP

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• DNA to be sequenced is cloned into the EcoRI siteimmediately adjacent to the primer binding site

EcoRI

Sal I

gene forampicillin

resistance

gene fortetracycline

resistance

Pst I

ori

primer binding site

pBR322

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5’3’ ||||||||||||||| 3’

• For sequencing• the DNA is denatured into single strands• the primer is hybridized to the template strand• DNA is synthesized using DNA polymerase

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5’3’ |||||||||||||||

ddATPDNAdNTPs

ddTTPDNAdNTPs

ddGTPDNAdNTPs

ddCTPDNAdNTPs

A T C A T G T C A T C A A G T C T A G C A C

T AT A G T AT A G T A C AT A G T A C A G T AT A G T A C A G T A G T T C AT A G T A C A G T A G T T C A G A

• All possible products of the reaction

containing ddATP

• Each fragment isterminated by a ddA

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5’3’ ||||||||||||||| A T C A T G T C A T C A A G T C T A G C A C

A T G C

TAG

TAC

AG

TAG

TTC

AG

ATC

GTG

Longer fragments

Shorter fragments

Sequenceof thestrand

that wassynthesized

3’

5’

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Manual methodAutomated method

Sanger dideoxy chain termination technique

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Automated DNA sequencing result

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3’

5’

Manual DNA sequencing result

20http://www.rvc.ac.uk/Extranet/DNA_1/6_Sequencing.htm

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• 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

600-800basesDesign primer at 3’ end

5’ 3’

DNA Template to be sequenced in a plasmid vector/PCR product (2kb)

Primer- 1

Primer- 2

600-800bases5’ 3’

Design primer at 3’ end

Primer- 2

600-800bases5’ 3’

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5’ 3’

5’ 3’

5’ 3’

Contig assembly

5’ 3’

Primer walking cont..

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

Sequence the insert

Contig assembly

Gene fragments in the plasmid vector.

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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 be removed (usually done using a molecular biology software eg. DNASIS, DNASTAR