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Polymerase Chain Reaction. Polymease Chain Reaction Salwa Hassan Teama. Polymerase Chain Reaction Salwa Hassan Teama. Roche. Polymerase Chain Reaction. Invented by Kary Mullis and his colleagues in the 1983. Nobel prize 1993. PCR machine otherwise called Thermocycler.

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  1. Polymerase Chain Reaction Polymease Chain Reaction Salwa Hassan Teama Polymerase Chain Reaction Salwa Hassan Teama Roche

  2. Polymerase Chain Reaction • Invented by Kary Mullis and his colleagues in the 1983. • Nobel prize 1993.

  3. PCR machine otherwise called Thermocycler. -1983—Kary Mullis, a scientist working for the Cetus Corporation was driving along US Route 101 in northern California when he came up with the idea for the polymerase chain reaction. -1985—the polymerase chain reaction was introduced to the scientific community at a conference in October .Cetus rewarded Kary Mullis with a $10,000 bonus for his invention -Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million.

  4. NEW AUTOMATED PCR OLD PCR

  5. PCR Reaction Components • Template: previously isolated and purified. • Two primers: to flank the target sequence. • Four deoxynucleosides triphosphate (dNTPs) : to provide energy and nucleosides for the synthesis of DNA. • Buffer system containing magnesium. • DNA polymerase.

  6. Thermal Cycling Profile for Standard PCR • Initial Denaturation • Each cycle includes three successive steps: • Denaturation • Annealing • Extention

  7. PCR Phases Three phases: • Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. • Linear: The reaction components are being consumed, the reaction is slowing, and products are starting to degrade. • Plateau:The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.

  8. Results

  9. Variants of PCR • Reverse transcriptase-PCR • Nested-PCR • Hot-start PCR • Quantitative PCR • Multiplex-PCR • Mutagenesis PCR • Allele specific PCR • Inverse PCR • Asymetric PCR • In Situ PCR • RFLP-PCR • Alu-PCR • Colony PCR • DD-PCR • Degenerative PCR • Long-PCR • PCR-ELISA • PCR-SSCP • OC-PCR • RACE • RAPD • Rep-PCR • Touchdown • Assembly PCR &

  10. Polymerase Chain Reaction: Uses The polymerase chain reaction (PCR) is a technique widely used in: Molecular biology, Microbiology, Genetics, Diagnostics clinical laboratories, Forensic science, Environmental science, Many other applications………………………………………

  11. Protocol • DNA template: 5 microliter • F. Primer: 2 microliter • R. Primer: 2 microliter • DNTP,s: 2microliter • Taq. Polymerase: 2 microliter • Buffer: 1 microliter • dH2O: 1 microliter Total Master Mix= 15 microliter

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