文章基本信息

标题：Randomly broken fragment PCR with 5′ end-directed adaptor for genome walking
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作者：Wentao Xu ; Ying Shang ; Pengyu Zhu 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2013
卷号：3
DOI：10.1038/srep03465
出版社：Springer Nature
摘要：Many genome walking methods based on polymerase chain reaction (PCR) are available, including those with and without restriction enzyme modification. Nevertheless, these methods suffer from low reproducibility, inefficiency, and non-specificity. Here, we present a traceable and efficient PCR strategy: randomly broken fragment PCR with 5′ end-directed adaptor for genome walking. The genome is first fragmented randomly. After blunting ends, the fragments are ligated to the 5′ end-directed adaptors. Semi-nested PCR is then performed. Thus, we can obtain an unknown sequence by cloning the fragments of interest, followed by sequencing. This method effectively bypasses the above-mentioned obstacles and offers the advances: 1) genome fragmentation without using restriction enzymes; 2) enhancement of primer specificity and the prevention of self-ligation between the adaptors by employing a 5′ end-directed adaptor. All of the steps in this new method are straightforward, and the unknown sequence can be definitively obtained by merely applying the method once.