1. DNA sequencing

2. How DNA synthesis takes place inside the living cell?

3. Can you think of a way to determine the DNA seq. in vitro?

4. The idea of DNA sequencing The use of ddNTPs + the normal dNTPs

5. There are 2 methods for DNA sequencing 1- Classic sequencing Using radioactivity 2- Modern sequencing Using fluorescence

6. The classic sequencing Sanger Chain termination method

7. Modern sequencing Chain termination method

8. Steps for DNA sequencing 1- DNA isolation and purification 2- PCR 3- Amplified DNA purification 4- Termination 5- Sequencing

9. 1- DNA isolation and purification Obtain high quality DNA to be sequenced 1- Conc. by spec * Calculation ……. 2- Integration

10. 2- Is PCR for sequencing different from the general PCR

11. 3- Amplified DNA purification

12. General PCR components

13. General PCR strategy

14. PCR Program

15. PCR objective

16. PCR for sequencing Components of PCR Template + terminator ready reaction mix.

17. The PCR program Hot start 2 min Den. 10 sec @ 94 o C Annealing 5 sec @ 50 o C Extension 4 min. @ 60 o C 25 cycles

18. Seq. PCR end product What are the characteristics of the resulted DNA?

19. How to handle the DNA after this point Purification Termination Sequencing

20. What happens inside the sequencer

21. Review over the sequencing process