A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification
A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification
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DOI：
10.1016/j.mimet.2011.07.003
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发表时间：
2011-10-01
影响因子：
2.2
通讯作者：
Filion, Martin
中科院分区：
文献类型：
--
作者：
Gadkar, Vijay J.;Filion, Martin
基因组内已知序列侧翼区域的表征，称为基因组步移，是现代遗传分析的基石技术。在目前的工作中，我们已经开发了一种新的PCR依赖的，定向基因组步行协议的基础上独特的环化性质的一种新的DNA连接酶，CircLigase。在第一步中，使用磷酸化引物进行基于PCR的引物延伸，所述磷酸化引物被设计成从已知序列的边界延伸到侧翼区中。这种线性扩增导致单链（ss）DNA的产生，然后使用CircLigase将其环化。使用Phi 29 DNA聚合酶的超支化活性，然后通过滚环扩增使环状ssDNA线性化，产生大量的双链多聚DNA。使用反向PCR使用嵌套引物扩增侧翼序列。在琼脂糖凝胶上解析产物，鉴定、提取由于所用引物组合的嵌套位置而导致迁移率变化的条带，并克隆到质粒载体中进行测序。这一概念的经验证据是在假单胞菌LBUM 300中的两个抗微生物生物合成基因上产生的。使用hcnB和phlD基因作为起始点，成功地分离了约Ikb的侧翼序列。基因座特异性引物的使用确保了步移的方向性和特异性，减轻了通常在随机引发的步移方案中观察到的假扩增子的产生。所提出的基因组步移方案可以应用于任何微生物基因组，并且仅需要100-150 bp的先前序列信息。所提出的方法不需要费力地测试限制性酶或接头连接。这是第一次报告的一个成功的应用程序的新型连接酶，CircLigase基因组步行的目的。(C)2011爱思唯尔有限公司版权所有。
Characterization of regions flanking a known sequence within a genome, known as genome walking, is a cornerstone technique in modern genetic analysis. In the present work we have developed a new PCR-dependent, directional genome walking protocol based on the unique circularization property of a novel DNA ligase, CircLigase. In the first step, PCR based primer extension is performed using a phosphorylated primer, designed to extend from the boundary of the known sequence, into the flanking region. This linear amplification results in the generation of single-stranded (ss) DNA, which is then circularized using CircLigase. Using the hyperbranching activity of Phi29 DNA polymerase, the circular ssDNA is then linearized by rolling circle amplification, resulting in copious amounts of double stranded concatarneric DNA. Nested primers are used to amplify the flanking sequence using inverse PCR. The products are resolved on an agarose gel and the bands whose mobility change due to the nested location of the primer combination used are identified, extracted, and cloned into a plasmid vector for sequencing. Empirical proof for this concept was generated on two antimicrobial biosynthetic genes in Pseudomonas sp. LBUM300. Using the hcnB and phlD genes as starting points, ca 1 kb of flanking sequences were successfully isolated. The use of locus specific primers ensured both directionality and specificity of the walks, alleviating the generation of spurious amplicons, typically observed in randomly primed walking protocols. The presented genome walking protocol could be applied to any microbial genome and requires only 100-150 bp of prior sequence information. The proposed methodology does not entail laborious testing of restriction enzymes or adaptor ligation. This is the first report of a successful application of the novel ligase enzyme, CircLigase for genomic walking purposes. (C) 2011 Elsevier B.V. All rights reserved.