Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.
Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.
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DOI：
10.1186/s13568-017-0495-x
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发表时间：
2017-11-02
期刊：
影响因子：
3.7
通讯作者：
Chen J
中科院分区：
文献类型：
--
作者：
Jia X;Lin X;Chen J
目前的基因组步行方法非常耗时，而且许多方法产生的是非特异性扩增产物。为了扩增粘质沙雷氏菌FZSF02中与Tn5转座子插入位点相邻的侧翼序列，我们建立了一种基于Tail-PCR的基因组行走方法。该方法在指数扩增步骤之前增加了20个循环的线性扩增步骤，以提高目标序列的浓度。线性扩增和指数扩增的产物被稀释100倍，以降低引起非特异性扩增的模板浓度。该方法采用快速DNA聚合酶，扩增速度快，扩增程序快速扩增出较长的特异序列。利用这种线性指数型尾部聚合酶链式反应(LETAIL-PCR)，我们在3h内成功地从Tn5转座子插入突变株中获得了大于2kb的产物，该方法可广泛用于基因组步行研究中扩增与已知序列相邻的未知序列。本文的在线版本(10.1186/s13568-0170495-x)包含向授权用户提供的补充材料。
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. The online version of this article (10.1186/s13568-017-0495-x) contains supplementary material, which is available to authorized users.
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