EFFICIENT SYNTHESIS OF P-32 LABELED SINGLE-STRANDED-DNA PROBES USING LINEAR PCR - APPLICATION OF THE METHOD FOR ANALYSIS OF STRAND-SPECIFIC DNA-REPAIR
EFFICIENT SYNTHESIS OF P-32 LABELED SINGLE-STRANDED-DNA PROBES USING LINEAR PCR - APPLICATION OF THE METHOD FOR ANALYSIS OF STRAND-SPECIFIC DNA-REPAIR
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DOI：
10.1016/0921-8777(94)90018-3
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发表时间：
1994-09-01
期刊：
影响因子：
--
通讯作者：
VANZEELAND, AA
中科院分区：
文献类型：
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作者：
RUVEN, HJT;SEELEN, CMJ;VANZEELAND, AA
我们开发了一种快速合成放射性标记单链DNA探针的方法，适用于Southern印迹上单拷贝基因的链特异性分析。线性PCR与10 μ Ci α（32）P-dATP（3000 Ci/mmol）作为唯一的dATP源，使我们能够产生链特异性DNA探针具有高比活性。该方法合成的探针比M13 mp 18/19单链载体末端标记的单链DNA探针具有更高的特异性活性和相同的链特异性。应用该方法对紫外线诱导的DNA损伤进行链特异性分析，显著提高了杂交信号。
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 mu Ci alpha(32)P-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.