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What is PCR?

What is PCR?. P olymerase C hain R eaction. Applications of PCR. DNA cloning for sequencing and gene manipulation DNA-based phylogeny and functional analysis of genes Diagnosis of hereditary diseases or somatic mutations

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What is PCR?

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  1. What is PCR? PolymeraseChainReaction

  2. Applications of PCR • DNA cloning for sequencing and gene manipulation • DNA-based phylogeny and functional analysis of genes • Diagnosis of hereditary diseases or somatic mutations • Identification of genetic fingerprints (used in forensic sciences and paternity testing) • Detection and diagnosis of infectious diseases

  3. PCR takes advantage of … Complementarity of DNA bases – and the hydrogen bonding of base pairs A T C G

  4. PCR takes advantage of … - the way that the hydrogen bonds will denature with heat, and reform upon cooling

  5. PCR takes advantage of … DNA polymerase, whic makes an identical copy of the DNA A heat stable DNA polymerase (Taq) from the heat stable bacterium Thermus aquaticus

  6. Melt (denature) double stranded DNA into single strands at 95°C Gene of interest 3’ 5’ 3’ 5’ Denature 3’ 5’ 3’ 5’

  7. Add DNA primers and anneal 3’ 5’ 3’ 5’ DNA primers usually 18-24 nucleotides in length 5’-3’ 3’-5’

  8. Add heat stable DNA polymerase and free dNTPs dATP, dGTP, dTTP, dCTP The first cycle: 3’ 5’ Taq Polymerase 3’ 5’

  9. Polymerase adds complementary nucleotides starting from annealing site • Results in double stranded DNA

  10. Next: Heat to Melt-Denature 3’ 5’ 3’ 5’

  11. Primers and Anneal 3’ 5’ 3’ 5’

  12. Taq and Extend 3’ 5’ 3’ 5’

  13. Thermal Cycles • Denature 94°C (melt to remove DNA-DNA, Primer-DNA, Primer- primer complexes) • Anneal ~ 65°C (primers bind to template DNA) • Extension 72°C (adds/synthesizes complementary bases)

  14. Amplification! One copy Eight copies

  15. Run DNA product (the PCR product=amplicon) out on agarose gel • Visualize with ethidium bromide • To verify results • Extract & sequence the DNA, (the PCR product = Amplicon) • Use Restriction enzymes to cut

  16. What do you want to copy or measure? • Extract DNA from cell (only two copies of each gene) • Do PCR to amplify the DNA (sequence/clone into plasmid or virus) • Extract mRNA from cell • Synthesize complementary DNA (cDNA) from mRNA (using reverse transciptase) • Then do PCR to amplify the cDNA • Q PCR = Quantitative PCR = Real Time PCR • Extract mRNA from cell • SynthsizecDNA from mRNA • DoqPCR to measure absolute or relative DNA levels

  17. Applications of PCR • DNA cloning for sequencing and gene manipulation • DNA-based phylogeny and functional analysis of genes • Diagnosis of hereditary diseases or somatic mutations • Identification of genetic fingerprints (used in forensic sciences and paternity testing) • Detection and diagnosis of infectious diseases

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