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Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites

文献类型： 外文期刊

作者： Jia, Xianbo 1 ; Lin, Xinjian 1 ; Chen, Jichen 1 ;

作者机构： 1.Fujian Acad Agr & Sci, Inst Soil & Fertilizer, Wusi Rd, Fuzhou 350001, Fujian, Peoples R China

2.Fujian Agr & Forestry Univ, Inst Appl Ecol, Fuzhou, Fujian, Peoples R China

关键词： LETAIL-PCR;Genome walking;Linear amplification;Tn5 transposon

期刊名称：AMB EXPRESS （ 影响因子：3.298； 五年影响因子：3.427 ）

ISSN： 2191-0855

年卷期： 2017 年 7 卷

页码：

收录情况： SCI

摘要： Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

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