Ideal cycling conditions for longer PCR products

Amplification of PCR products longer than 3–4 kb is often compromised by nonspecific primer annealing, suboptimal cycling conditions, and secondary structures in the DNA template. Lengthy optimization is often necessary, by varying factors such as cycling conditions, primer and dNTP concentrations, and special additives.

While depurination is usually not a problem in standard PCR, it can significantly influence the amplification of longer PCR fragments. This is because longer templates are proportionally more depurinated than shorter ones. For this reason, very short denaturation steps of only 10 seconds give higher yields and no background smearing compared to denaturation steps of 30 seconds or 1 minute (which leads to PCR failure; see figure Effect of cycling conditions). Extensive depurination is also observed during the final extension step. Therefore, using a lower extension temperature of 68°C instead of 72°C dramatically improves yield of longer amplification products.

Ideal cycling conditions for longer PCR products are given in the table Cycling conditions for amplifying longer PCR products.

 Effect of cycling conditions
Cycling conditions for amplifying longer PCR products
Step Time/cycles Temperature
Initial activation step 2 min 95°C
3-step cycling    
Denaturation 10 s 94°C
Annealing 1 min 50–68°C*
Extension 1 min/kb  
Number of cycles 40 cycles 68°C
End of PCR cycling Indefinite 4°C

Secondary structures such as hairpin loops, which are often caused by GC-rich template stretches, interfere with efficient amplification of long PCR products. This problem can be overcome by adding reagents that modify the melting behavior of DNA to help resolve secondary structures at lower temperatures.

Taq DNA Polymerase introduces more errors into the PCR product while copying the template than do so-called proofreading DNA polymerases. Once a mismatch occurs during synthesis, Taq DNA polymerase will either extend the mismatched strand or fall off the template strand, leading to mutated or incomplete PCR products, respectively. Although this does not generally affect PCR efficiency when amplifying shorter PCR fragments, amplification of longer PCR products can be significantly impaired by mismatches introduced during DNA synthesis.

Proofreading DNA polymerases contain an inherent 3' to 5' exonuclease activity that removes base-pair mismatches. Adding a small amount of a proofreading DNA polymerase to the PCR mixture therefore significantly improves the amplification efficiency of longer PCR products.