Polymerase Chain Reaction

1. Polymerase Chain Reaction

2. Problem Supposeyouhaveapatientwithan infectionoraheritable disease. Youwanttoknowwhichinfectionor diseaseitisand….. youwanttoknowitfastand..... fromaslittlematerialaspossible Isthatfeasable?

3. WhichDNAtechnologiesareavailable? • Southernblot • Insituhybridization • Sequencing • PCR

4. Whatisrelevantinmedicaldiagnostics? • SSS • Speed thefasterthe better • Sensitivity samplesize • Specificity reliabilityofdiagnosis

5. Which type of DNA can beusedforwhat?genes-repeatDNA:centromereDNAtelomereDNACArepeats-junkDNA

6. WhichtypeofDNAcanbeusedforwhat? • TypeofDNA: Junk ??? Repeat identification Gene diagnosisofdisease ‘foreign’DNA detectionofinfection CA-repeatsturnouttobeusefulforidentification ofindividuals.WhatisaCArepeat?Why? Mutationsingenescanleadtohereditarydiseases. PCR (incombinationwithsequencing)helpstodetect such mutations. PCRcanamplifynon-selfDNAassistindetecting infectionsofviruses,bacteriaorparasites.

7. WhatisaCA-repeats • CA-repeatsturnouttobeusefulforidentification ofindividuals. 5’ 3’ ---------- CACACACACACACA------------ Primer primer Fixed location in the genome but the length Differs among individuals

8. PrincipalofaPCRassayonCArepeats CArepeatsvary inlengthfrom 4to40bp and canbefoundon10.000positions inthegenome. OfeachCA-repeat anindividualgets onecopyofthe fatherandone copyofthemother. Thenumberof primersetsinthe PCRdetermines thespecificityof theidentification.

9. PCR • PCR, polymerase chain reaction, is an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence • Before PCR, DNA of interest could only be amplified by over-expression in cells and this with limited yield

10. 1966, Thomas Brock discovers ThermusAquaticus, a thermostable bacteria in the hot springs of Yellowstone National Park • 1983, Kary Mullis postulated the concept of PCR ( Nobel Prize in 1993) • 1985, Saiki publishes the first application of PCR ( beta-Globin ) • 1985, Cetus Corp. Scientists isolate Thermostable Taq Polymerase (from T.Aquaticus), which revolutionized PCR

11. Reaction Components • DNA template • Primers • Enzyme • dNTPs • Mg2+ • Buffers

12. 1- DNA template • DNA containing region to be sequenced • Size of target DNA to be amplified : up to 3 Kb

13. 2- Primers • 2 sets of primers • Generally 20-30 nucleotides long • Synthetically produced • complimentary to the 3’ ends of target DNA • not complimentary to each other

14. Primers • Not containing inverted repeat sequences to avoid formation of internal structures • 40-60% GC content preferred for better annealing • Tm of primers can be calculated to determine annealing T0

15. 3-Enzyme • Usually Taq Polymerase or anyone of the natural or Recombinant thermostable polymerases • Stable at T0 up to 950 C • High processivity • Taq Pol has 5’-3’ exo only, no proofreading

16. The PCR Cycle Comprised of 3 steps: 1. Denaturation of DNA at 950C - 2. Primer hybridization ( annealing) at 40-500C 3. DNA synthesis ( Primer extension) at 720C

22. Standard thermocycle

23. RT-PCR • Reverse Transcriptase PCR • Uses RNA as the initial template • RNA-directed DNA polymerase (rTh) • Yields ds cDNA

26. rTth DNA polymerase is a thermostable DNA polymerase derived from the thermophilic bacteria Thermus thermophilus (Tth) HB8. The enzyme has a reverse transcriptase activity in addition to a 5’→3’ polymerase activity and a double strand specific 5’→ 3’ exonuclease activity in the presence of Mn2+ ions.

28. Detection of amplification products • Gel electrophoresis • Sequencing of amplified fragment • Southern blot • etc...

29. Applications • Genome mapping and gene function determination • Biodiversity studies ( e.g. evolution studies) • Diagnostics ( prenatal testing of genetic diseases, early detection of cancer, viral infections...) • Detection of drug resistance genes • Forensic (DNA fingerprinting)

30. Advantages • Automated, fast, reliable (reproducible) results • Contained :(less chances of contamination) • High output • Sensitive • Broad uses • Defined, easy to follow protocols

31. In Conclusion PCRissensitiveandversatile diagnostictool: 1)todetecthereditarydiseases 2)todetectinfections 3)tomonitordevelopmentofadisease 4) toidentifyfathersorothercriminals PCRisextremelyusefulifone. 1)knowsthesequenceofagene. 2)carefullyselectsprimers 3)avoidscontamination

32. Instrumentation

33. Real Time PCR

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