指数线性聚合酶链式反应法制备焦磷酸测序反应的单链模板  

Preparation of Single-Stranded DNA Template for Pyrosequencing by Linear-after-the-Exponential-Polymerase Chain Reaction

作　　者：杨会勇[1] 奚涛[1] 梁超[1,2] 陈之遥[3] 徐定邦[3] 周国华[2,3] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009 [2]华东医学生物技术研究所,南京210002 [3]南京大学医学院,南京210093

出　　处：《分析化学》2009年第4期489-494,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.30470454);日本日立中中央研究所资助项目

摘　　要：建立了一种简单的焦磷酸测序用单链模板制备方法,以包含SNP6位点一段78bp序列为对象,采用非热启动Taq酶进行指数线性PCR扩增,通过加入甘油、BSA等PCR增强剂增加反应的效率和特异性,设计反应液A和B处理PCR产物中干扰焦测序的限制性引物、未完全反应的产物、焦磷酸和dNTPs等杂质,处理后1～2μLPCR产物就可直接用于焦测序检测。测定了BRCA1基因中5个乳腺癌相关的SNP位点,获得的图谱无非特异性信号,测得序列与参考序列一致,能够进行SNP分析,表明本方法可以制备高质量焦测序单链模板,且使焦测序的成本显著降低,操作更为简便,减少了操作过程中样本间的交叉污染,有利于焦测序样品预处理的自动化。In order to establish a simple method to prepare single-stranded DNA templates for pyrosequencing, the Linear-After-The-Exponential （LATE）-PCR technology based on Taq DNA polymerase without hot-start capacity was applied to amplify a 78-bp sequence（ containing the rs2207517 SNP site）, and the PCR-enhancing reagents（ glycerol and BSA） were used to increase the efficiency and specialization, much more reagents A and B were designed to eliminate the impurity（limited primers, PPi, dNTPs and so on） , and 1 -2 μL LATE-PCR products with simply treatment can be used in pyrosequencing directly. Then five SNPs related with human breast-cancers in the BRCAI gene were investigated, and the programs had no nonspecific signals and the resalts were consisted with the theoretic sequences. Moreover, the genotyping of the SNPs could also be distinguished easily. The results indicated this method can be used to prepare high quality single-stranded DNA templates for pyrosequencing and allows pyrosequencing be lower in cost, simpler in operation, and easier in automation, and the cross-contamination from sample preparation was also reduced.

关 键 词：焦测序 指数线性扩增 单链核酸模板的制备 单核苷酸多态性 

分 类 号：Q503[生物学—生物化学]