What are the critical factors for amplification of long genomic targets?
What are the critical factors for amplification of long genomic targets?
1. Template quality
DNA integrity is critical for amplification of long targets. DNA damage—such as DNA breakage during DNA isolation or DNA depurination at elevated temperatures and low pH—results in a greater amount of partial products and decreased overall yield. DNA damage can also occur in acidic conditions; therefore, avoid using water for resuspending DNA templates. DNA is most stable at pH 7–8 or in buffered solutions.
2. PCR conditions
-Denaturation time should be kept to a minimum to decrease depurination events.
-Use touchdown PCR; start at a higher annealing temperature and reduce by two degrees per cycle for several cycles.
-Design primers with melting temperatures (Tm) above 68°C.
3. PCR polymerases
We offer several PCR polymerases optimized for long-range PCR. Takara LA Taq DNA polymerase, TaKaRa LA Taq Polymerase with GC Buffer, and PrimeSTAR GXL DNA Polymerase are recommended depending on the GC content and size of the target(s).