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Long PCR Yanfei Yang 2008.8.14 Compromise of longer PCR (>3,4kb) • • • • Nonspecific primer annealing Suboptimal cycling conditions Secondary structures in the DNA template Dupurination: longer templates are proportional more depurinated • Mismatches introduced during DNA synthesis Keep DNA quality and avoid non-specific primer binding Primer Primer design: 20-30bp, high specificity, high Tm ~62-70’C, avoid primer hairpin and 3’ complementarity. Primer concentration: concentration from 0.1 to 1.0 mM: too low, poor yield; too high, non-specific bands. Lower concentrations for highly complex DNA(such as human genomic DNA) or high concentrations of template DNA; higher concentrations for low complexity templates (plasmid DNA) or low template DNA. Template • Should be good quality, intact (free of nicks). Chose proper methods fully purify template samples. Puregene DNA Isolation Kit, QIAGEN Genomic tips, phenol-extraction, Megapore dialysis… • Store genomic DNA at 4’C to avoid introducing nicks during freeze-thaw. Buffer Cosolvents to stabilize enzyme, lower melting behavior of DNA, resolve 2nd structure: Qsolution, DMSO, glycerol, betaine. Alkaline tricine(PH8.7) to protect protect DNA from being nicked at high T in acidic conditions. Magnesium concentration: excess, non-specific reactions; scarce, less products. Varying the concentration in 0.5 mM increments throughout a range of 1.5 to 4 mM to determine optimal magnesium concentration Enzyme • Mismatch occurs during synthesis, Taq DNA polymerase will extend or fall off the template strand, leading to mutated or incomplete PCR products. • Amplification of longer PCR products can be significantly impaired by mismatches introduced during DNA synthesis. • Adding a small amount (1/20) of proof-reading DNA Polymerase to the PCR mixture corrects mismatch, therefore significantly improves the amplification efficiency of longer PCR products. Hot start: eliminate non-specific reactions, suppresses adverse effects of the 3’ to 5’ exonuclease activity on the primers. Enzyme amount: too high, non-specific reactions 2.5 units of TaKaRa LA Taq /50 ml Cycling conditions Longer templates are proportional more depurinated, so to protect template, use shorter denaturation t(10s) and lower extension T(68’C). Denaturation: need short time and low temperature, too short time/low temperature, diffuse smearing upon electrophoresis/poor amplification efficiency; too long time/high temperature, no identifiable product. Annealing and extension: 45-68’C. Aneal T too low, non-specific reactions; extension time too short, DNA synthesis can’t be competed, but too long causes diffusely smeared electrophoresis bands. Cycle numbers: 25 to 30 cycles over cycling, diffuse smear in electrophoresis. Two step PCR: combined annealextension, 68’C 30s~1min/1kb. If below 68’C, a longer time period is required. Shuttle PCR (Autosegment Extension ): a significant increase in amplification efficiency for long PCR. References • A new protocol for highly efficient amplification of long PCR products (Susan Kobsch, Katja Decker, and Dirk Löffert QIAGEN GmbH, Hilden, Germany) • LA PCR protocol (Alam lab) • XL PCR amplification of long targets from genomic DNA. (Lori A. Kolmodin, Methods in molecular biology, 2002)