A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome
A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome
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一种新颖且简单的 PCR 步移方法，可快速获取微生物基因组中已知位点侧翼的长 DNA 序列
DOI：
10.1007/s12033-010-9332-z
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发表时间：
2011-03-01
影响因子：
2.6
通讯作者：
Ren, Chunhua
中科院分区：
文献类型：
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作者：
Luo, Peng;Su, Ting;Ren, Chunhua
在微生物基因组相关研究中，获取与已知DNA位点相邻的侧翼序列是一项重要任务。在这项研究中，我们开发了一种新的方法，包括两轮PCR，然后克隆和测序。首先在反应体系中加入特异性引物（SP）进行一次基因座特异性线性扩增，然后在冷却的反应体系中加入复合长引物（CLP），仅进行一个循环。来自第一轮PCR的扩增产物不经电泳直接纯化，稀释，并用作第二轮PCR的模板。其次，采用一条长特异引物（LSP）和一条长碱基固定引物（LFP）。纯化扩增子用于克隆和测序。长侧翼区特异性扩增的实现主要依赖于PCR程序的巧妙精确设置、CLP引物的结构设计、特异性线性扩增后CLP引物的添加、CLP与SP引物的浓度比、长引物的应用等。alginolyticus和V.胆汁。该方法为快速扩增微生物长未知DNA片段提供了一种稳健而简单的策略。
Acquisition of flanking sequence adjacent to a known DNA site is an important task in microbial genome-related research. In this study, we developed a new method containing two rounds of PCR followed by cloning and sequencing. Firstly, specific primer (SP) is added into the reaction system for primary locus-specific linear amplification, and then a complex long primer (CLP) is added into the cooled reaction system for only one cycle. Amplification products from the first round of PCR are directly purified without electrophoresis, diluted, and used as the templates of the second PCR. Secondly, one long specific primer (LSP) and one long base-fixed primer (LFP) are adopted. The amplicons are purified for cloning and sequencing. The achievement of specific amplification for long flanking region mainly depends on ingenious and precise settings of PCR programs, structure design of CLP primer, adding of CLP primer after specific linear amplification, concentration ratio of CLP and SP primer, applying long primers, etc. Through this method, we successfully performed the long PCR walkings (>1.5 Kb) onrpoBgene ofVibrio vulnificus, transposon-like gene ofV. alginolyticus, andstogene ofV. cholerae. The method provides a robust and simple strategy for rapid amplification of long unknown DNA fragments from microbes.