应用特异单引物法标记DNA探针

Labelling of DNA probes by specific sirigle-primer method

作者应用探针DNA上的一个片段作为DNA引物;以环化探针DNA或重组质粒DNA为模板,在DMA聚合酶的作用下,通过变性、退火和延伸过程,使探针DNA得到标记.3次实验结果表明,用过种单引物标法标记的DNA探针的放射比活性可达250×10^(14)cpm/g DNA,而用缺刻翻译法标记的DNA探针的放射比活性只有0.42×10^(13)cpm/g DNA,比单引物法要低5倍,打点杂交结果表明,用单引物法标记的探针能特异性地检测出70fg的靶基因序列,其敏感性比用缺刻翻译法标记的DNA探针要高两个数量级.

The present study has established a new method for . labelling of DNA probes A small fragment was used on probe DNA as a primer and a circularized probe of ptasmid containing probe sequence as a template, through repeated cycles of denaturation,anncalling and extcnsion. the DNA probes were labelled with high efficiency. Three experiments showed that th: radioactivity of DNA probe labelled by this, technic could reach 2.50×1014 com/g DNA, while that labelled by rick translation was only .0.42 × 1013 com,g DNA. Dot blot hybridization showed that probe labelled by singlc-primer method could specifically detect target sequence of 70 fg, two magnitudes higher in sensitivity than that labellec by nick translation.