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Molecular techniques.pptx

Polymerase Chain Reaction

Shalini53
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Molecular techniques.pptx

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  1. Molecular techniques ZOOL 301(B) Animal Biotechnology Prepared by: Dr. Shalini Chouhan RGGDC, Shimla-4

  2. PCR PCR • Polymerase chain reaction Polymerase chain reaction • In vitro amplification of a DNA segment In vitro amplification of a DNA segment • Automated process (replication of DNA in lab Automated process (replication of DNA in lab conditions) conditions) • Forms millions of copies Forms millions of copies • In short time period In short time period

  3. Requirements for PCR Requirements for PCR • Target DNA Target DNA • Primer Primer • Polymerase Polymerase • dNTPs (dCTPs, dATPs, dTTPs, dGTPs) dNTPs (dCTPs, dATPs, dTTPs, dGTPs)

  4. Principle Principle • Denaturation Denaturation • Annealing Annealing • Extension Extension

  5. • PCR is a cyclic process PCR is a cyclic process • Involves 30-35 cycles Involves 30-35 cycles • Each cycle has 3 steps: denaturation, annealing and Each cycle has 3 steps: denaturation, annealing and extension. extension. • Each cycle lasts 3-5 minutes Each cycle lasts 3-5 minutes • Millions of copies obtained in 2.5 hrs Millions of copies obtained in 2.5 hrs

  6. Details of PCR 1. 2. 3. 4. 5. 6. 7. 8. Carried out in polypropylene tubes (called PCR tubes) Carried out in polypropylene tubes (called PCR tubes) Thermo-cycler (regulates temperature) Thermo-cycler (regulates temperature) ds DNA having target DNA ds DNA having target DNA Both strands act as template Both strands act as template Boarder sequences should be known Boarder sequences should be known Primers (12-14 nucleotides long) Primers (12-14 nucleotides long) Heat stable polymerase (Taq polymerase) Heat stable polymerase (Taq polymerase) dNTPs dNTPs

  7. References • Animal Biotechnology by Trueman Publications

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