PCR and qRT-PCR May 3. PCR The thermocycle analyzing the products essential components of the reaction optimization basic rules of primer design problems.

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Presentation transcript:

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) primers (define the amplicon) dNTPs divalent cations (Mg, Mn) thermostable DNA polymerase optimization of Mg conc (7-2)

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

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

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