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2. 2 VARIANTS OF PCR APPLICATIONS OF PCR MECHANICS OF PCR WHAT IS PCR? PRIMER DESIGN

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

4. 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. 5 SELECTIVE DNA ISOLATION High amounts of pure DNA is supplied, enabling DNA analysis from small starting samples DNA sequencing to determine unknown PCR- amplified sequences AMPLIFICATION OF DNA Used to analyze extremely small samples QUANTIFICATIO N Estimation of the amount of a given sequence present in a sample Real time PCR – measures amplification after each cycle

6. 6 FORENSIC S Using DNA sequencing, possible to determine if two skin samples at different crime scenes belong to the same person Paternity tests ANCIENT DNA DNA can be extracted and analyzed from ancient remains. Challenges – amplified DNA is authentic DNA Risk of contamination because of high sensitivity. DISEASE DIAGNOSI S Early diagnosis of malignant diseases Applications in microbiology Viral DNA

7. 7 DENATURATIONANNEALINGEXTENSIONREPEATFINAL EXTENSIONHOLD

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

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 10

12. MULTIPLEX - PCR REVERSE TRANSCRIPTASE PCR QUANTITATIVE PCR Multiple primer sets Several amplified sequences From RNA to cDNA From cDNA to amplified DNA Precise sample quantification Using fluorescence 12

13. Sample QPCR Readout 13

14. 14 LIGASE CHAIN REACTION BACTERIAL CLONING

15. 15 LACKS SELF - CORRECTION HIGH SENSITIVITY SHORT DNA SEQUENCES

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