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Polymerase Chain Reaction

PCR. Polymerase Chain Reaction. PCR. Is a revolutionary method developed by Kary Mullis in1983. PCR is used in molecular biology to make many copies of small sections of DNA.

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Polymerase Chain Reaction

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  1. PCR Polymerase ChainReaction

  2. PCR • Is a revolutionary method developed by Kary Mullis in1983. • PCR is used in molecular biology to make many copies of small sections ofDNA. • It is based on the ability ofDNA polymeraseto synthesize new strand of DNA complementary to a templatestrand.

  3. Because DNA polymerase can only add a nucleotide onto a preexisting 3'-OHgroup, it needs a primer to which it can add the firstnucleotide. • This requirement makes it possible to delineate a specific region of template sequence that the researcher wantsto amplify. • At the end of the PCR reaction, the specific sequence will be accumulatedin billions of copies, calledamplicons.

  4. Kary B.Mullis The Nobel Prize in Chemistry1993 "for contributions to the developments of methods within DNA-based chemistry" "for his invention of the polymerasechain reaction (PCR)method”

  5. Taqpolymerase

  6. Enzymes are proteins that speed up reactions without being consumedthemselves. The most important enzyme in a PCR reaction is called taqpolymerase. A polymerase is an enzyme that attaches molecules together (i.enucleotides). Every cell that has DNA has its own polymerase that takes care of replication ofDNA. PCR uses a polymerase from a species of bacteria, Thermus aquaticus, which normally lives in hotsprings.

  7. Thermusaquaticus

  8. ThermalCycler • Is a laboratory apparatus used to amplify segments of DNA viaPCR). • The device has a thermal block with holes where tubes holding the reaction mixtures can beinserted. • The cycler then raises and lowers the temperature of the block in pre-programmedsteps.

  9. Materials ofPCR • target DNA • Taq DNApolymerase • 2Primers • ~20 nucleotides inlength • Forward andreverse • the fourDNTP’S • Adenine • Thymine • Cytosine • Guanine • cofactorMgCl2.

  10. Locate the TargetSequence • Scientists determine which GENEthey are interested instudying. • Locate Primers at the beginning andend ofgene.

  11. Step 1Denaturing • 60 seconds @94°C. • Hydrogen bonds between bases break, & backbones separate.

  12. Step 2Annealing • 60 seconds @60°C. • Forward and reverse primers bond to template DNA.

  13. TaqPolymerase • Once primers anneal, Taq Polymerase can attach tothem.

  14. Step 3Extension • 2 minute at72°C. • Taq Polymerase adds DNTP’s to complete the DNAcopy.

  15. The exponential amplification ofthe gene inPCR.

  16. Amplification

  17. Cycle3 • Obtain twocopies of targetDNA

  18. Cycle30 • 1,073,741,764 • targetcopies

  19. Is there a gene copied duringPCR and is it the right size?

  20. Applications ofPCR • A quick, reliable method for detecting all manner of mutations associated with geneticdisease. • Duchene musculardystrophy • Detect unwanted Geneticmaterial • Bacterial or viral infection(HIV). • Amplify degraded DNAsamples • Egyptianmummy • Termite inamber

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