Walker F, Bedel C, Boucher O, Dauge M C, Vissuzaine C, Potet F
Service d'Anatomie Pathologique, Hôpital Bichat, Paris.
Ann Pathol. 1995;15(6):459-65.
In situ polymerase chain reaction is a recent technique which combines the sensitivity of PCR reaction to intracellular localization of genomic sequences with the same specificity as in situ hybridization. This reaction is based on the in situ annealing and polymerisation of oligonucleotides complementary to nucleotides located at each side of the target DNA sequence to amplify. We describe the Hot Start PCR (DNA) and the Hot Start PCR after reverse transcription step (RNA). It allows to amplify some nucleic sequences to a high level, becoming easier to detect. The vizualisation can be realized by direct in situ PCR, the product obtained being directly identifiable by incorporation of labeled nucleotides or primers, or preferentially by indirect in situ PCR. In this case, the amplification is followed by in situ hybridization with labeled probes. This last procedure is more specific. Numerous controls are essential at each step of the technique for validating results.
原位聚合酶链反应是一种最新的技术,它将聚合酶链反应对基因组序列细胞内定位的敏感性与原位杂交相同的特异性相结合。该反应基于与位于待扩增靶DNA序列两侧的核苷酸互补的寡核苷酸的原位退火和聚合。我们描述了热启动聚合酶链反应(用于DNA)以及逆转录步骤后的热启动聚合酶链反应(用于RNA)。它能够将一些核酸序列大量扩增,从而更易于检测。可视化可通过直接原位聚合酶链反应实现,通过掺入标记的核苷酸或引物直接鉴定所获得的产物,或者更优选通过间接原位聚合酶链反应实现。在这种情况下,扩增之后是用标记探针进行原位杂交。最后这个程序更具特异性。在该技术的每个步骤都必须进行大量对照以验证结果。