DNA Sequencing

Presented By:
Prince Chaubey M.Phil. Botany

DNA Sequencing
DNA Sequencing is a technique which is used to determine the nucleotide sequence of unknown DNA strand. Techniques mainly used for DNA sequencing is: a. Chain Termination Method b. Chemical Degradation Method c. Automated DNA Sequencing d. Pyrosequencing

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 cleavage at specific bases.

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 phosphate backbone.

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

Cleaved products are separated using high-resolution polyacrylamide gel electrophoresis. Urea & high temperature of 700C is maintained to prevent formation of secondary structure.

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

Base modified
G
A+G C+T C A>C

Specific Modification
Methylation of base by Dimethyl Sulphate at pH 8.0.Susceptible base cleave by alkali.
Treatment with piperidine formate at pH 2.0. Result in removal of purine bases. Treatment with Hydrazine ,opens pyrimidine rings & causes their removal from DNA. Treatment with Hydrazine + NaCl (1.5 M NaCl),only cytosine react with Hydrazine. Treatment with 1.2 M NaCl at high temperature (900 c) gives strong cleavage at A, less at C

When bases are modified & destroyed by treatment, piperdine at 900 C is used to cleave phosphodiester bond at the site.

CHAIN TERMINATION METHOD:

Fred Sanger & his colleagues devised chain termination method. This method based on the principle that single stranded DNA molecules that differ in length by just a single nucleotide can be separated by one another by PAGE.

They used 2,3-dideoxynucleotides.

This method can lead to sequencing of 300 bases per reaction.

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 .

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 of the 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 to separate 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.

Newly synthesized DNA is Radiolabeled by using

Radiolabeled Primers, or Radiolabeled Nucleotides, or Radiolabeled dideoxnucleotides

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 (each containing 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 a single reaction.

PYROSEQUENCING:
Pyrosequencing method determine which of the four bases is incorporated at each step in the copying of a DNA template. If one of the four bases is incorporated then pyrophosphate is released & this is detected in an enzyme cascade that emits light.

Pyrosequencing is of two types: a. Solid phase b. Liquid phase

PYROSEQUENCING: In solid phase

Pyrosequencing: In liquid phase

REFERENCES:
Gene Cloning and DNA analysis; T.A Brown (Fifth edition) Principles of gene manipulation and genomics; S.B. Primrose and R.M Twyman( Seventh edition) Whole Genome Sequencing; Sachin Kumar