DNA sequencing2.pdf

DNA SEQUENCING

DNA sequencing

ÖDetermination of nucleotide sequence

the determination of the precise sequence of nucleotides in a

sample of DNA

Ö Two similar methods:

1. Maxam and Gilbert method

2. Sanger method

Ö They depend on the production of a mixture of oligonucleotides labeled

either radioactively or fluorescein, with one common end and differing in

length by a single nucleotide at the other end

Ö This mixture of oligonucleotides is separated by high resolution

electrophoresis on polyacrilamide gels and the position of the bands

determined

Maxam-Gilbert

• Walter Gilbert

– Harvard physicist

– Knew James Watson

– Became intrigued with

the biological side

– Became a biophysicist

• Allan Maxam

The Maxam-Gilbert

Technique

• Principle - Chemical Degradation

of Purines

– Purines (A, G) damaged by

dimethylsulfate

– Methylation of base

– Heat releases base

– Alkali cleaves G

– Dilute acid cleave A>G

Maxam-Gilbert

Technique

• Principle

Chemical Degradation

of Pyrimidines

– Pyrimidines (C, T) are

damaged by hydrazine

– Piperidine cleaves the

backbone

– 2 M NaCl inhibits the

reaction with T