中文摘要：

多数分子诊断方法对靶基因的检测都是以单链DNA为基础的,如何获得大量和高质量的单链靶DNA分子一直是研究热点.以包含BRCA1基因上3个单核苷酸多态性（SNP）的DNA片段为研究对象,通过调整不对称扩增引物碱基序列组成（5′-端引入错配或加入锁核苷酸（locked nucleic acid,LNA））,使用相差10倍浓度来增加不对称引物间Tm值差异,改进扩增条件（添加增强剂的扩增缓冲液,不同退火温度的二阶循环扩增程序）后,进行不对称扩增来产生大量单链靶DNA产物.结果表明Tm差异型不对称PCR（Tm difference asymmetric PCR,TDA-PCR）可有效提高单链DNA分子产率,降低引物设计难度,也扩大了模板序列的适用范围.此外,还针对禽流感病毒H5N1的血凝素（haemagglutinin）HA基因保守序列进行不对称扩增,用获得的单链DNA为模板进行焦磷酸测序检测,所得结果与参考序列一致且无明显干扰信号.因此,采用Tm值差异型不对称PCR可使焦磷酸测序等分子诊断流程更为简便,成本更低,效率更高,具有很好的通用性和实用性.

英文摘要：

Detection of single-stranded DNA （ssDNA）is the basis of many molecular diagnostic methods,and how to obtain enough and high quality ssDNA has been one of the hot research topics.In this paper,several sections of DNA （containing one of the three SNPs of BRCA1 gene respectively）were as the research subj ects,we carefully optimized the sequences of primers （introducing mis-match bases at the 5＆#39;-end of the primers or introducing locked nucleic acid （LNA）into excess primers）and adj usted the concentra-tion of the primers （10 times difference between the excess primer and the limiting primer）to generate the difference of Tm between the excess primer and the limiting primer （3-8 ℃）,and the amplification program was also improved （some PCR enhancers were added into the reaction mixture and a two-stage amplification procedure with two annealing temperatures）,then the Tm difference asymmetric PCR （TDA-PCR）method was used and a large number of target ssDNA molecules were produced.The results showed that TDA-PCR could steadily increase the productivity of the ssDNA molecules,and it became easier than linear-after-the-exponen-tial-PCR （LATE-PCR）for the easier primer designing and the more target DNA producing.This assay amplified the conserved se-quence of the influenza HA gene using TDA-PCR,and pyrosequencing method was used to detect the conserved sequence.The pyro-grams obtained were of high quality and consistent with the reference sequence,indicating that TDA-PCR not only prepares ssDNA templates for pyrosequencing more easily and more efficiently,but also has good practicality and versatility.