厦门大学学报（自然科学版）2016，Vol.55Issue(4)：485-494,10.DOI:10.6043/j.issn.0438-0479.201506017
Tm值差异型不对称PCR方法的建立及其在分子诊断中的应用
Establishment of the Tm Difference Asymmetric PCR Method and Its Application in Molecular Diagnosis
摘要
Abstract
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'-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.关键词
Tm值差异型不对称PCR/单链DNA模板/单核苷酸多态性/焦磷酸测序/禽流感病毒Key words
Tm difference asymmetric PCR (TDA-PCR)/single-stranded DNA template/single nucleotide polymorphism/pyrose-quencing/Avian influenza virus分类
生物科学引用本文复制引用
饶华春,滕继云,刁勇,宋沁馨,王立强,杨会勇,周国华..Tm值差异型不对称PCR方法的建立及其在分子诊断中的应用[J].厦门大学学报（自然科学版）,2016,55(4):485-494,10.基金项目
国家自然科学基金(31200979,81271691,82170734,30973591) （31200979,81271691,82170734,30973591）
国家国际科技合作项目(2011DFG33320) （2011DFG33320）
福建省自然科学基金(2016Y0063) （2016Y0063）
江苏省基础研究计划(自然科学基金)项目(BK20151445) （自然科学基金）
中国博士后科学基金(2012M512179,2013T6092) （2012M512179,2013T6092）
中国博士后科学基金特别资助(2013T60962) （2013T60962）
药物质量与安全预警教育部重点实验室项目(MKLDP2013MS01) （MKLDP2013MS01）
江苏省青蓝工程项目 （）
中央高校基本科研业务费专项(ZQN-PY319,2015ZD008) （ZQN-PY319,2015ZD008）
华侨大学高层次人才启动项目(09BS624) （09BS624）
