Lec10_W11

Lec10_W11 - PCR • When we do PCR, we only amplify a...

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Unformatted text preview: PCR • When we do PCR, we only amplify a specific region in the template DNA – Template DNA may be millions of base pairs long – we only amplify a region of a few thousand basepairs • The region that is amplified is determined by the primers, which are incorporated into the ends of the PCR product Critical PCR parameters 1. Primer design – most important 1. Concentration of DNA template, nucleotides, divalent cations (especially Mg2+) 2. Error rate of the polymerase ( Taq, Vent exo, Pfu ) 1. Primer design • Critical variables are: – specificity – melting temperature ( Tm ) – depends on length and G/C content – complementary of primer sequences – 3’-end sequence Specificity of primers • The primer should only hybridize with the target sequence. – Primers that are longer than 15 base pairs are usually unique for just one sequence in a genome. (What are the chances of finding a specific 15 nucleotide sequence in a stretch of DNA? 1/4 15 !) – However, it is very important to check to confirm that your primer does not cross hybridize to other sequences – use Blast or similar program • Longer primers would be better in terms of specificity but not in terms of rapidity of annealing and ease of synthesis • Having 20-nucleotide long primers gives good specificity with shortest length and good Tm If primers are too short, they can bind in many places on target DNA – you would get multiple PCR products when you only want one lux A lux B Region in...
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This note was uploaded on 04/10/2011 for the course BIMM 101 taught by Professor Butler during the Spring '08 term at UCSD.

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Lec10_W11 - PCR • When we do PCR, we only amplify a...

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