PCR AND PRIMER DESIGN

1. PCR AND PRIMER DESIGN BIOL1220 – SYNTHETIC BIOLOGY

2. PRIMER DESIGN

3. WHAT IS PCR? Repeated heated and cooling What is polymerase? What is chain reaction? Developed by Kary Mullis Few DNA strands to millions of copies

4. 'My God, you could use this to isolate a fragment of DNA from a complex piece of DNA, from its context. KARY MULLIS

5. USES OF PCR

6. PRACTICAL APPLICATIONS

7. MECHANICS OF PCR

9. Making a PCR reaction • Taq DNA polymerase      • dNTP mixture • PCR buffer (salts, loading dye, etc) • MgCl2 • ddH2O • DNA template • Primers http://upload.wikimedia.org/wikipedia/commons/8/81/PCR_tubes.png http://www-che.syr.edu/faculty/boddy_group/pages/thermocycler.jpg http://www1.qiagen.com/Products/Pcr/TaqSystem/TaqPcrCore.aspx

10. Primer Design • PRIMER LENGTH • Optimal length – 18 – 22 bp • MELTING TEMP • 52 – 58 C • GC content determines temp • NO SELF COMPLEMENTING • Formation of hairloop structures • If 3’ ends complement, form primer dimers • AVOID C OR G RUNS • Harder to separate

12. COMMON VARIATIONS OF PCR

13. Sample QPCR Readout

14. ALTERNATIVES TO PCR LIGASE CHAIN REACTION BACTERIAL CLONING

15. DISADVANTAGES OF PCR SHORT DNA SEQUENCES HIGH SENSITIVITY LACKS SELF - CORRECTION

16. THANK YOU