利用线性PCR高效合成32P标记的单链DNA探针；该方法在链特异性DNA修复分析中的应用。

Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair.

作者信息

Ruven H J, Seelen C M, Lohman P H, Mullenders L H, van Zeeland A A

机构信息

MGC Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, The Netherlands.

出版信息

Mutat Res. 1994 Sep;315(2):189-95. doi: 10.1016/0921-8777(94)90018-3.

DOI:10.1016/0921-8777(94)90018-3

PMID:7521000

Abstract

We have developed a rapid method to synthesize radioactively labeled single-stranded DNA probes suitable for strand-specific analysis of single copy genes on Southern blot. Linear PCR with 10 microCi alpha 32P-dATP (3000 Ci/mmol) as the only dATP source enabled us to generate strand-specific DNA probes with high specific activity. The probes synthesized by this method have higher specific activities and the same strand specificity compared to the end-labeled single-stranded DNA probes obtained from single-stranded M13mp18/19 vectors. Application of the method for strand-specific analysis of ultraviolet-induced DNA lesions in defined DNA sequences significantly improved the hybridization signal.

摘要

我们开发了一种快速方法来合成放射性标记的单链DNA探针，适用于在Southern印迹上对单拷贝基因进行链特异性分析。以10微居里的α-32P-dATP（3000居里/毫摩尔）作为唯一的dATP来源进行线性PCR，使我们能够生成具有高比活性的链特异性DNA探针。与从单链M13mp18/19载体获得的末端标记单链DNA探针相比，通过该方法合成的探针具有更高的比活性和相同的链特异性。将该方法应用于对特定DNA序列中紫外线诱导的DNA损伤进行链特异性分析，显著提高了杂交信号。