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PCR and qRT-PCR

PCR and qRT-PCR. May 3. PCR. The thermocycle analyzing the products essential components of the reaction optimization basic rules of primer design problems with contamination - false positives. Essential components of the reaction. template (DNA or RNA ->DNA)

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PCR and qRT-PCR

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  1. PCR and qRT-PCR May 3

  2. PCR • The thermocycle • analyzing the products • essential components of the reaction • optimization • basic rules of primer design • problems with contamination - false positives

  3. Essential components of the reaction • template (DNA or RNA ->DNA) • primers (define the amplicon) • dNTPs • divalent cations (Mg, Mn) • thermostable DNA polymerase • optimization of Mg conc (7-2)

  4. Basic rules for primer design • comparable length and melting temperature • Tm=2(A+T) + 4(G+C) • unique • no complementarity between 3’ ends (primer dimers) • perfect match with 3’ end (additions to 5’ end) • no extensive secondary structure

  5. Contamination! • strategies for eliminating contamination (or reducing effects) • physical separation of product analysis from reaction set up • dUTP and uracil-n-glycosylase • controls

  6. Real-time PCR • why end-point PCR is not always quantitative • cycle threshhold (Ct) • chemistry options • methods of quantitation • absolute • relative • primer design • single-step vs two-step PCR • analysis

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