Amplification of DNA with double annealing temperature PCR

Abstract:

[Objective] Instead of standard PCR setting a single annealing temperature (S-T_m), we studied double annealing temperature PCR (D-T_m PCR) setting respective annealing temperature for forward and reverse primers from higher to lower. [Methods] A 4.3 kb pET20b-Xyn (Aspergillus niger xylanase gene) model DNA was amplified with Q5 DNA polymerase by using PxF61 and VPel as forward and reverse primers. The PCR procedure was:pre-denaturation at 98℃ for 3 min, and 30 cycles of denaturation at 98℃ for 30 s, annealing at T_m1 70℃ (PxF61) for 15 s and at T_m2 62℃ (VPel) for 15 s, extension at 72℃ for 130 s. [Results] The 4.3 kb target DNA band of D-T_m PCR was a little brighter, whereas non-specific DNA bands were two less than those of the S-T_m PCR (T_m=61℃). Twenty-five cycles of amplification created the brightest target DNA band in the D-T_m PCR. A 5.3 kb recombinant plasmid DNA was clearly amplified in the D-T_m PCR than the S-T_m PCR. [Conclusion] The D-T_m PCR amplified directly target DNA band without demanding for investigation of an optimal annealing temperature and for setting closer annealing temperatures between forward and reverse primers. Moreover, two respective annealing steps were clearly elucidated from theoretical viewpoint.