DNA SEQUENCING USE OF DIDEOXYNUCLEOTIDES

Sanger sequencing technique • Dideoxynucleotides are modified

• Frederick Sanger - was an English Biochemist deoxynucleotides
born in Gloucestershire, United Kingdom (1918) • They are called as such because there is no
hydroxyl (OH) group on the third prime position
• Elucidated the complete structure of Insulin of the sugar. But instead there is a third prime
(1955) hydrogen bond.
• Because of the hydrogen bond present in the
• Received a Nobel Price for Chemistry for his dDNTPs, these are termed chain terminating
work in insulli(1958) nucleotides.
SANGER SEQUENCING
• Began working with DNA materials
➢ Uses 4 ddNTPs
• Led to the breakthrough of the di deoxy method • ddATP
workings. • dd TP
• ddGTP
• Received his second Nobel Price for this work in • ddTTP
DNA (1980) Shared his prize with Paul Berg and ➢ Remember, are devoid of 3' Hydroxyl Groups.
Walter Wilbert ➢ The Hydrogen bond on the 3' terminates the
formation of new nucleotide bonds.
• Used this method to complete the entire
genome of eX174 (Bacteriophage).

• Sanger died on November 19, 2013 in

Cambridge, United Kingdom

PROCESS OF WHOLE GENOME SEQUENCING (SANGER)

• Involves In Vitro DNA Synthesis.

• Use of DNA template strand
• Use of Primers
• Use of DNA Polymerase
• Use of Deoxynucleotides (dATP, dCTP, dGTP.
dTTP)
• Use of Dideoxynucleotides (ddNTPs)
Polyacrylamide Gel Electrophoresis
• Forms a gel network from acrylamide and bis-
acrylamide
• Separates the DNA fragments according to
molecular sizes
• Provides higher resolution in separating short
DNA fragment