Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species
Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species
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DOI：
10.1016/j.mimet.2004.11.021
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发表时间：
2005-05-01
影响因子：
2.2
通讯作者：
Litthauer, D
中科院分区：
文献类型：
--
作者：
Nthangeni, MB;Ramagoma, F;Litthauer, D
自从聚合酶链式反应技术发明以来，克隆已知序列两侧DNA片段的适应性技术不断发展。我们描述了一种几乎无限数量的完美退火盒，它具有可变的粘性和钝端限制性内切酶识别位点，用于有效地限制和连接受限制的目标基因组DNA。该盒式磁带提供了一个200bp的序列，用于设计各种盒式磁带特异的引物。去磷酸化阻止了盒的自我连接，并在盒的目标基因组DNA连接位置产生了一个缺口，抑制了非特异性的PCR扩增。我们介绍了单链扩增聚合酶链式反应(SSA-PCR)技术，在第二轮常规套式聚合酶链式反应中，首先使用一个已知的唯一的位点特异性引物来丰富目标模板DNA链，从而产生显着的聚合酶链式反应产物特异性。已知的位点特异性引物与所使用的限制性内切酶的最近位置之间的距离决定了所获得的PCR产物的长度。我们使用这项技术向下游走到异丝裂解酶，并向上游走到地衣芽孢杆菌成熟的胞外脂肪酶基因两侧假设的保守基因。我们进一步证明了该技术在基于PCR的新基因勘探中作为一种经济有效的方法的潜力，通过设计检测芽孢杆菌物种中VII族细菌脂解基因同源的“通用”简并引物。利用盒式连接介导的聚合酶链式反应技术，克隆了地衣芽孢杆菌和短小芽孢杆菌脂解功能基因的全序列。(C)2004爱思唯尔B.V.保留所有权利。
Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky and blunt-end restriction enzynne recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing "universal" degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus. (C) 2004 Elsevier B.V. All rights reserved.