Dr Frederick Sanger
Sanger’s Method: Whenever ddNTP comes in the DNA synthesis, further synthesis of DNA stops due to non-formation of 3’-5’ phospodiester linkage as in ddNTP , there is 3’
H (Instead of 3’OH)
Structure of any ddNTP- (dideoxy ribose is a pentose sugar witoxygen atom removed from each 2’ and 3’ position.
It must include the following reagents:
- Single strand DNA which needs to be sequenced.
- A primer with a free 3’-OH.
- DNA polymerase
- dNTPs
- ddNTPs (1 % of total dNTPs)
Method:
- Primer extension in 4 different tubes each containing a specific ddNTP at low concentration.
- Termination at the point where ddNTP is incorporated
- Gel electrophoresis.
- Autoradiography-+reading of gel sequence.
