NOVEL PCR TECHNIQUE USING ALU-SPECIFIC PRIMERS TO IDENTIFY UNKNOWN FLANKING SEQUENCES FROM THE HUMAN GENOME
NOVEL PCR TECHNIQUE USING ALU-SPECIFIC PRIMERS TO IDENTIFY UNKNOWN FLANKING SEQUENCES FROM THE HUMAN GENOME
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DOI：
10.1006/geno.1995.9004
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发表时间：
1995-09-20
期刊：
影响因子：
4.4
通讯作者：
PATERLINI, P
中科院分区：
文献类型：
--
作者：
MINAMI, M;POUSSIN, K;PATERLINI, P
与已知序列相邻的新细胞DNA序列的快速和可重复性鉴定是目前可用的程序难以实现的。在这里，我们描述了一种基于聚合酶链反应（PCR）的新方法，使用已知序列特异性引物和另一种针对人类Alu重复序列的引物。为了避免在Alu序列之间产生不希望的扩增，引物用dUTPs构建，在初始扩增10个周期后用尿嘧啶DNA糖基化酶处理破坏。只有需要的片段，然后用特定的引物进一步扩增到已知区域和在alu特异性引物中引入的标签序列。利用这一方案，我们已经成功地从三种肝癌组织的人类基因组中鉴定了整合乙型肝炎病毒DNA的细胞序列。该方法可以实现直接特异性扩增，而无需像以前基于pcr的协议所要求的任何连接或非特异性退火步骤。这种快速而直接的方法将成为研究病毒整合位点的有力工具，但也广泛适用于人类基因组的其他研究。(C) 1995学术出版社，Inc。
The rapid and reproducible identification of new cellular DNA sequences adjacent to known sequences is difficult to achieve with the currently available procedures. Here we describe a novel approach based on the polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesirable amplifications between Alu sequences, primers are constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 10 initial cycles of amplification. Only desirable fragments are then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-specific primer. Using this protocol, we have successfully identified cellular sequences flanking integrated hepatitis B virus DNA from the human genome of three hepatoma tissues. The method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based protocols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicable to other studies of the human genome. (C) 1995 Academic Press, Inc.