通过校对PCR检测已知突变。
Detection of known mutation by proof-reading PCR.
作者信息
Bi W, Stambrook P J
机构信息
Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati, College of Medicine, PO Box 670521, Cincinnati, OH 45267-0521, USA.
出版信息
Nucleic Acids Res. 1998 Jun 15;26(12):3073-5. doi: 10.1093/nar/26.12.3073.
Abstract
Proof-reading PCR (PR-PCR) is designed to detect known mutations within genomic DNA. It differs from standard PCR approaches in that one of the two primers has its 3' end aligned with a putative mutation site, and has its 3'-OH replaced by a blocking group. Distinguishing a mutant gene from wild-type depends upon preferential removal of the blocked 3' terminal nucleotide by the polymerase proof-reading activity when it is mismatched with the template. Preferential removal of the blocked nucleotide allows subsequent extension and selective amplification, and provides the basis for distinguishing mutant from normal genes. This method has been used here to detect a transition mutation within the P53 gene of HaCaT cells with verification by direct sequencing of the selectively amplified DNA.
摘要
校对PCR（PR-PCR）旨在检测基因组DNA中的已知突变。它与标准PCR方法的不同之处在于，两条引物中的一条引物的3'端与假定的突变位点对齐，并且其3'-OH被一个封闭基团取代。区分突变基因和野生型基因取决于当聚合酶校对活性与模板不匹配时，优先去除封闭的3'末端核苷酸。优先去除封闭的核苷酸允许随后的延伸和选择性扩增，并为区分突变基因和正常基因提供了基础。本文使用该方法检测HaCaT细胞P53基因内的一个转换突变，并通过对选择性扩增的DNA进行直接测序来验证。
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