Long PCR Yanfei Yang

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 2 nd structure: Q- solution, 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 anneal- extension, 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)