一种用于优化PCR引物设计的短链DNA熔点分析方法
发布时间：2018-11-25 17:30
【摘要】：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AsIG、GsIG和TsIG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。
[Abstract]:At present, the design of PCR primer mainly depends on the simulation calculation of the melting point of the primer by software, but the optimization of PCR annealing conditions needs to carry out amplification experiments under different conditions. In order to develop an efficient and accurate method for evaluating primers and determining annealing conditions, the melting point of short-chain DNA was directly analyzed by high-resolution melting curve (high resolution melting,HRM, which was used to evaluate the advantages and disadvantages of primers. It also provides a reference for the optimization of annealing conditions. In this paper, the melting point of incomplete complementary double-stranded DNA and DNA card issuing structure has been directly determined by HRM method. The results show that: (1) compared with fully complementary double-stranded DNA, the melting point of stable single-base mismatch AsIG,GsIG and TsIG is only 2 鈩，
本文编号：2356895
[Abstract]:At present, the design of PCR primer mainly depends on the simulation calculation of the melting point of the primer by software, but the optimization of PCR annealing conditions needs to carry out amplification experiments under different conditions. In order to develop an efficient and accurate method for evaluating primers and determining annealing conditions, the melting point of short-chain DNA was directly analyzed by high-resolution melting curve (high resolution melting,HRM, which was used to evaluate the advantages and disadvantages of primers. It also provides a reference for the optimization of annealing conditions. In this paper, the melting point of incomplete complementary double-stranded DNA and DNA card issuing structure has been directly determined by HRM method. The results show that: (1) compared with fully complementary double-stranded DNA, the melting point of stable single-base mismatch AsIG,GsIG and TsIG is only 2 鈩，
本文编号：2356895
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