molecular biolpgy techniques

7/30/2019 molecular biolpgy techniques

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DNA Sequencing

Presented By:

Prince Chaubey

M.Phil. Botany


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DNA Sequencing

DNA Sequencing is a technique which isused to determine the nucleotidesequence of unknown DNA strand.

Techniques mainly used for DNAsequencing is:

a. Chain Termination Method

b. Chemical Degradation Method

c. Automated DNA Sequencing

d. Pyrosequencing


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CHEMICAL DEGRADATION METHOD:

Allan Maxam & Walter Gilbert (1976-1977)

developed chemical degradation technique

for DNA sequencing.

This method based on the chemical

modification of DNA & subsequent cleavageat specific bases.


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METHODS:The DNA strand to be sequenced must be radioactively labelled at

one end with 32P-phosphate group.

Chemical reagents are used to alter one or two bases in the DNA

strand.

An altered base can then be removed from the sugar phosphatebackbone.

The strand is then cleaved with piperdine at the sugar residuelacking the base.

Cleaved products are separated using high-resolution

polyacrylamide gel electrophoresis. Urea & high temperature of700C is maintained to prevent formation of secondary structure.

The sequence is read from the bottom to top of the gel.


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When basesare modified & destroyed by treatment,piperdine at 900 C is used to cleave phosphodiester

bond at the site.

Base modified Specific Modification

G Methylation of base by Dimethyl Sulphate at pH

8.0.Susceptible base cleave by alkali.A+G Treatment with piperidine formate at pH 2.0. Result

in removal of purine bases.

C+T Treatment with Hydrazine ,opens pyrimidine rings &

causes their removal from DNA.

C Treatment with Hydrazine + NaCl (1.5 M NaCl),only

cytosine react with Hydrazine.

A>C Treatment with 1.2 M NaCl at high temperature (900

c) gives strong cleavage at A, less at C


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CHAIN TERMINATIONMETHOD:

Fred Sanger & his colleagues devised chaintermination method.

This method based on the principle that singlestranded DNA molecules that differ in length by

just a single nucleotide can be separated by oneanother by PAGE.

They used 2,3-dideoxynucleotides.

This method can lead to sequencing of 300

bases per reaction.


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Dideoxy Nucleotides

Lack an -OH group at

the 3-carbon position.

Incorporation of a

dideoxynucleotide to

growing DNA strand

terminates its further

extension.

They are added in

small proportion. Prevent further DNA

synthesis .
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Chain-Termination Method:

In the Sanger method, the ss-DNA is used as a template.

ss-DNA is split into four aliquots, each containing DNA polymerase , Primers,certain percentage of dideoxynucleoside triphosphate (ddNTP) analogs to one ofthe four nucleotides (ATP, CTP, GTP or TTP) & each of the four dNTPs (dATP,dTTP, dGTP, dCTP).

The mixture are loaded into separated lanes of a gel & electrophoresis is used toseparate the DNA fragments.

Each reaction will end up containing a mixture of different lengths of DNA

strands, all ending with the nucleotide that was dideoxy labeled for that reaction.

This method lead to sequencing of 300 bases per reaction.


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Newly synthesized DNA is

Radiolabeled by using

Radiolabeled

Primers, or

RadiolabeledNucleotides, or

Radiolabeleddideoxnucleotides


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Automated DNA sequencing:

It is a modified form of chain termination method.

In this method, four dideoxynucleotides (ddNTPs) are

labelled with four different types of fluorescent dyes.

All four individual sequencing reactions (eachcontaining a different ddNTPs) combine into a single

reaction that could be analyzed on single lane of gel.

As many as 650 bases can be read automatically from asingle reaction.


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PYROSEQUENCING:

Pyrosequencing method determine which of thefour bases is incorporated at each step in the

copying of a DNA template.

If one of the four bases is incorporated thenpyrophosphate is released & this is detected in

an enzyme cascade that emits light.

Pyrosequencing is of two types:

a. Solid phase

b. Liquid phase

PYROSEQUENCING I lid


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PYROSEQUENCING: In solid

phase


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Pyrosequencing: In liquid phase


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REFERENCES:

Gene Cloning and DNA analysis; T.ABrown (Fifth edition)

Principles of gene manipulation andgenomics; S.B. Primrose and R.M Twyman(

Seventh edition)

Whole Genome Sequencing; Sachin Kumar

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molecular biolpgy techniques