A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR
A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR
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一种基于降落PCR和抑制PCR的未知侧翼序列扩增方法。
DOI：
10.1016/j.ab.2016.07.001
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发表时间：
2016-09-15
影响因子：
2.9
通讯作者：
Wang, Li
中科院分区：
文献类型：
--
作者：
Gao, Song;He, Dan;Wang, Li
热不对称交错PCR是获得侧翼序列最广泛使用的技术。但也有一定的局限性，如阳性率低，操作复杂等。本研究在抑制型PCR和降落型PCR的基础上，对该方法进行了改进。通过扩增获得的PCR片段在凝胶纯化后直接用于测序。用改进的方法扩增ATMT突变体侧翼序列的阳性率达到99%。此外，从DNA提取到侧翼序列分析的时间缩短到2天，每份样品约6美元。(C)2016 Elsevier Inc. All rights reserved.
Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample. (C) 2016 Elsevier Inc. All rights reserved.