A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions
A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions
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DOI：
10.2144/000112680
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发表时间：
2008-02-01
期刊：
影响因子：
2.7
通讯作者：
Ceci, Luigi R.
中科院分区：
文献类型：
--
作者：
Leoni, Claudia;Gallerani, Raffaele;Ceci, Luigi R.
在许多基因组规模的研究中，经常需要确定与已知区域相邻的核苷酸序列。为了实现这一目标并克服筛选基因组文库的耗时方法，已经开发了基于PCR技术的各种方法。通常，这些方案依赖于特定的要求和策略，例如存在合适的核苷酸限制性位点和连接特定的单链或双链接头，从而在一定程度上限制了它们的应用。在本文中，我们提出了一种替代的基于PCR的协议，包括四个主要步骤：（i）延伸的序列特异性引物;（ii）3 '-加尾的延伸单链DNA;（ii i）PCR;和（iv）巢式PCR扩增。这种方法，这似乎是一个有效的替代其他PCR为基础的协议，被用来识别序列侧翼的cDNA编码区的Lhcb1.1基因（一个成员的多基因家族编码的捕光蛋白Lhcb 1）在菠菜基因组中。
Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill this aim and overcome the time-consuming approach of screening genomic libraries. Usually these protocols rely on specific requirements and strategies, such as the presence of suitable nucleotide restriction sites and ligation of specific single- or double-strand linkers, thus limiting their application to a certain extent. In this paper we present an alternative PCR-based protocol, consisting of four main steps: (i) extension of a sequence-specific primer; (ii) 3 '-tailing of extended single-strand DNA; (ii i) PCR; and (iv) nested PCR amplifications. This method, which appears to be a valid alternative to the other PCR-based protocols, was used for the identification of sequences flanking the cDNA encoding region of the Lhcb1.1 gene (one member of the multigene family coding for the light harvesting protein Lhcb1) in the spinach genome.