Zaphiropoulos P G
Department of Bioscience, Center for Nutrition and Toxicology, Karolinska Institute, Novum, 141 57 Huddinge, Sweden.
Nucleic Acids Res. 1998 Jun 15;26(12):2843-8. doi: 10.1093/nar/26.12.2843.
RT-PCR amplification of P450 2C6 from rat liver, using primers in opposite orientations of exon 6, resulted in PCR products containing segments of exons joined at non-consensus splice sites. Moreover, many of the PCR products identified were composed of not only a single region containing exonic segments joined at non-consensus splice sites but, instead, of several repeats of the non-canonically joined region. To investigate whether these PCR products represent pre-existing molecules or are generated during the amplification process, the liver cDNA template was replaced by a plasmid containing the P450 2C6 cDNA. Surprisingly, PCR products containing repeats of non-canonically joined exonic segments were again revealed. In some cases the position of this non-canonical joining was a sequence of one or two identical nucleotides; however, there were also a number of products lacking any nucleotide identity at the position of joining. DNA nicking and/or DNA damage is thought to favour recombination during PCR, probably by misalignment of incomplete DNA strands; however, the presence of multiple repeats of the recombined region in the PCR products identified suggests a certain repetitiveness of the underlying mechanism. It is therefore proposed that these products result from a template switching event that occurs several times during a single polymerization step, following a rolling circle model of DNA synthesis.
使用位于外显子6相反方向的引物，对大鼠肝脏中的细胞色素P450 2C6进行逆转录聚合酶链反应（RT-PCR）扩增，结果得到的PCR产物包含在外显子非共有剪接位点处连接的外显子片段。此外，鉴定出的许多PCR产物不仅由单个包含在外显子非共有剪接位点处连接的外显子片段的区域组成，而是由非经典连接区域的几个重复片段组成。为了研究这些PCR产物是代表预先存在的分子还是在扩增过程中产生的，将肝脏cDNA模板替换为含有细胞色素P450 2C6 cDNA的质粒。令人惊讶的是，再次发现了含有非经典连接外显子片段重复的PCR产物。在某些情况下，这种非经典连接的位置是一两个相同核苷酸的序列；然而，也有许多产物在连接位置缺乏任何核苷酸一致性。DNA切口和/或DNA损伤被认为有利于PCR过程中的重组，可能是由于不完全DNA链的错配；然而，鉴定出的PCR产物中重组区域的多个重复的存在表明了潜在机制的某种重复性。因此，有人提出这些产物是由在单个聚合步骤中多次发生的模板转换事件产生的，遵循DNA合成的滚环模型。
