gene sequencing by sanger method and pcr

1. Sanger method of sequencing
Gene sequencing

2. Procedure
• Denature sample in four different test tubes
• Annealing; the mechanism of attachment of
primer to the dna single stand
• Extension; by attachment of polymerase
attachment formation of new stand occurs
causing extension of existing stand
• Termination; adding 4 types one in each test
tube dd A, G, C ,T,ntp extension stops and results
no of short tandem repeats called STR’s

3. Steps continue….
• Gel electrophoreses
• Reading by autoradiography machine and use
of computer with software

4. Pcr steps
• Make Pcr mixture in test tube; include buffer
to control PH, Mgcl2 as polymerase cofactor
,labeeled dntp’s for extension dna sample dna
polymerase enzyme and primer
• Start pcr cycles
 denature at 92-94c for 30 seconds
 annealing at50-60c depend on melting
temperature of duplex for 30 seconds
Extension; at 72c for 45s

5. Con…..
• These cycle repeat many times to produce no
of dna copies
• Gel electrophoreses
• autoradiography

6. Applications
• Molecular identification
• Sequencing
• Genetic enginearing