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Polymerase Chain Reaction

Polymerase Chain Reaction. What is PCR? : Why “Polymerase”?. It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. What is PCR? : Why “Chain”?.

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Polymerase Chain Reaction

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  1. Polymerase Chain Reaction

  2. What is PCR? : Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

  3. What is PCR? : Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

  4. 1966, Thomas Brock discovers Thermus Aquaticus, 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

  5. 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

  6. What is PCR? : The “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

  7. The Reaction PCR tube THERMOCYCLER

  8. Denature (heat to 95oC) Lower temperature to 56oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

  9. Reaction Components • DNA template • Primers • Enzyme • dNTPs • Mg2+ • buffers

  10. 1- DNA template • DNA containing region to be sequenced • Size of target DNA to be amplified : up to 3 Kb

  11. 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

  12. Primers (ctnd) • 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 • Tm= .41(%G+C) + 16.6log(J+) + 81.5 where J+ is the concentration of monovalent ions

  13. 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

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

  15. http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdfhttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

  16. Standard thermocycle

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

  18. Molecular Identification: Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

  19. Classification of organisms Genotyping Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis Applications of PCR

  20. MOLECULARIDENTIFICATION:

  21. Molecular Identification: Detection Of Pathogens

  22. Summary blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Gel Analysis, Restriction Digestion, Sequencing

  23. Conclusion The speed and ease of use, sensitivity, specificity of PCR essential tool of molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications.

  24. 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)

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

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