一种用于优化PCR引物设计的短链DNA熔点分析方法

目前，PCR引物设计主要依赖于软件对引物熔点的模拟计算，而PCR退火条件的优化需进行不同条件下的扩增实验。为开发一种可高效、精确评价引物和确定退火条件的方法，本研究采用高分辨率熔解曲线（high resolution melting，HRM）测定技术直接分析短链DNA的熔点，用于引物优劣性的评价，并为退火条件的优化提供参考。本文用HRM法直接测定了非完全互补的双链DNA以及DNA发卡结构的熔点，结果显示：（1）与完全互补的双链DNA相比，较为稳定的单碱基错配A?G、G?G和T?G的熔点只降低2℃ ~ 3℃，部分双碱基错配的熔点只降低4℃ ~ 6℃，单碱基突出熔点只降低4℃~ 5℃。因此，如果采用的退火温度不当，部分错配的非目的模板可能会被扩增。（2）即使发卡结构的茎干区只有6 bp，当其环区碱基少于10 nt时，其熔点也可达到60℃以上。此外，环区的长度对发卡熔点也有较大影响。根据本研究结果发现，引物设计时应尽量避免模板引物结合区同其邻近的30 nt碱基有6 bp以上的互补部分。综上所述，本研究证明HRM熔点法是一种高效评价引物及确定退火温度的方法。

A Melting Temperature Measurement Method of Short Stranded DNA Duplex for PCR Primer Design

At present, the PCR primer design is mainly dependent on software simulation, and the annealing temperature needs to be optimized by amplification under various conditions. In order to provide an efficient and accurate method for measuring melting temperatures (Tms) of primers and determining annealing conditions, high resolution melting (HRM) was adopted as a technical means to analyze the binding ability of primers in this study. It was proved that HRM was capable to measure practical Tms of short DNA duplexes in solution, which could be applied to evaluate the performance of primers and optimize annealing temperatures. Moreover, Tms of some non-complementary DNA duplexes and some hairpins were measured by HRM. The results indicated that 1) compared with the Tm of complementary DNA duplex, Tms of more stable mismatches A?G, G?G and T?G were 2℃~3℃ lower, Tms of some double base mismatches were only 4℃~6℃ lower; Tms of single base bulges only reduced by 4℃~5℃. Thus, if the improper annealing temperature is used, some non-target DNA may be amplified. 2) Tms of DNA hairpins with 6 bp stems and loops shorter than 10 nt were nearly 60℃. In addition, it was also found that the loop length greatly affected the hairpin Tm. According to our result, the primer binding site of amplicon should not form a hairpin containing a stem longer than 6 bp and a loop shorter than 30 nt. In summary, HRM can be a powerful approach for the evaluation of primer and the optimization of annealing temperatures for PCR.