DNA sequencing

Vaishnavi Rao
 Sequence Masters
• 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
 The 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
 The Maxam-
Gilbert Method
 Sequence Masters
• Fred Sanger, 1958
– Was originally a protein
chemist
– Made his first mark in
sequencing proteins
– Made his second mark in
sequencing RNA
• 1980 dideoxy
sequencing
 The Sanger Method
• Random incorporation of a dideoxynucleoside
triphosphate into a growing strand of DNA
• Requires DNA polymerase I.. Why?
• Requires a cloning vector with initial primer
(M13, high yield bacteriophage, modified by
adding: beta-galactosidase screening,
polylinker)
• Uses 32P-deoxynucleoside triphosphates
 Sanger
Method
Sequencing
Gel
 Comparison
• Sanger Method • Maxam Gilbert Method
– Enzymatic – Chemical
– Requires DNA – Requires DNA
synthesis – Requires long stretches
– Termination of chain of DNA
elongation – Breaks DNA at different
nucleotides