Saitoh Kenji, Chen Wei-Jen
Tohoku National Fisheries Research Institute, Shinhama, Shiogama 985-0001 Japan.
Gene. 2008 Oct 15;423(1):92-5. doi: 10.1016/j.gene.2008.06.025. Epub 2008 Jul 2.
Occurrence of chimeric sequences and related artifacts in PCR cloning procedures gives us risks of over-estimation of haplotypes or alleles. Recombination among haplotypes occurs through template switching during PCR cycles or through random repair of mismatch sites on heteroduplex DNA by the host cell. To eliminate the chimeric cloning artifacts, we tested two alternative protocols using T7 endonuclease I cleavage of mismatch sites and re-extension of nascent strands. Though T7 endonuclease I effectively eliminated chimeric clones in some cases, it produced many short fragments. Protocol with single re-extension of PCR products successfully recovered non-recombinant clones with fewer short fragments. In spite of the improvement of allelic recovery through these two protocols, there were still a few recombinants that remained in both reaction mixtures, and thus interpretation of the results for haplotype diversity in a PCR-amplified DNA population should be cautionary. Because re-extension in a diluted reaction mixture is quick, inexpensive and effective, it is advisable to use this procedure for recovery of chromosomal alleles with PCR cloning.
聚合酶链式反应（PCR）克隆过程中嵌合序列及相关假象的出现，使我们面临单倍型或等位基因估计过高的风险。单倍型之间的重组通过PCR循环中的模板切换或宿主细胞对异源双链DNA错配位点的随机修复发生。为了消除嵌合克隆假象，我们测试了两种替代方案，即使用T7核酸内切酶I切割错配位点和新生链的重新延伸。尽管在某些情况下T7核酸内切酶I有效地消除了嵌合克隆，但它产生了许多短片段。PCR产物单次重新延伸的方案成功回收了非重组克隆，且短片段较少。尽管通过这两种方案提高了等位基因的回收率，但两种反应混合物中仍残留一些重组体，因此在PCR扩增的DNA群体中对单倍型多样性结果的解释应谨慎。由于在稀释的反应混合物中重新延伸快速、廉价且有效，因此建议使用此程序通过PCR克隆回收染色体等位基因。
