双退火温度PCR扩增DNA

Amplification of DNA with double annealing temperature PCR

摘要:

[目的]与设置单一退火温度的常规PCR（S-T_m PCR）不同，本研究探讨双退火温度PCR（D-T_m PCR）由高到低设置2条引物各自退火温度。[方法]以PxF61和VPel为正/反向引物，用Q5 DNA聚合酶扩增4.3 kb的模式DNA pET20b-Xyn（黑曲霉木聚糖酶基因）。PCR程序为：98℃预变性3 min，30次循环{98℃变性30 s，设置双退火[T_m1 70℃（PxF61）退火15 s、T_m2 62℃（VPel）退火15 s]，72℃延伸130 s}。[结果]与S-T_m PCR（61℃）相比，D-T_m PCR扩增4.3 kb的目的条带亮度更高，减少2条杂带；经25次循环目的DNA产物量最高。D-T_m PCR用于长片段引物扩增5.3 kb重组质粒DNA条带更明显。[结论]D-T_m PCR直接扩增目的条带，避免了探讨T_m的麻烦，不要求2条引物T_m相近，从理论上更加清晰地认识引物与各自模板分步退火过程。

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.