Odelberg S J, Weiss R B, Hata A, White R
Department of Human Genetics, Eccles Institute of Human Genetics, University of Utah, Salt Lake City 84112, USA.
Nucleic Acids Res. 1995 Jun 11;23(11):2049-57. doi: 10.1093/nar/23.11.2049.
Recombinant DNA molecules are often generated during the polymerase chain reaction (PCR) when partially homologous templates are available [e.g., see Pääbo et al. (1990) J. Biol. Chem. 265, 4718-4721]. It has been suggested that these recombinant molecules are a consequence of truncated extension products annealing to partially homologous templates on subsequent PCR cycles. However, we demonstrate here that recombinants can be generated during a single round of primer extension in the absence of subsequent heat denaturation, indicating that template-switching produces some of these recombinant molecules. Two types of template-switches were observed: (i) switches to pre-existing templates and (ii) switches to the complementary nascent strand. Recombination is reduced several fold when the complementary template strands are physically separated by attachment to streptavidin magnetic beads. This result supports the hypothesis that either the polymerase or at least one of the two extending strands switches templates during DNA synthesis and that interaction between the complementary template strands is necessary for efficient template-switching.
当存在部分同源模板时，重组DNA分子通常会在聚合酶链式反应（PCR）过程中产生[例如，见帕博等人（1990年）《生物化学杂志》265卷，4718 - 4721页]。有人提出，这些重组分子是截短的延伸产物在后续PCR循环中与部分同源模板退火的结果。然而，我们在此证明，在不存在后续热变性的情况下，单轮引物延伸过程中就能产生重组体，这表明模板转换产生了其中一些重组分子。观察到两种类型的模板转换：（i）转换到预先存在的模板，以及（ii）转换到互补的新生链。当互补模板链通过连接到链霉亲和素磁珠而物理分离时，重组率降低了几倍。这一结果支持了这样的假说，即在DNA合成过程中，要么是聚合酶，要么是两条延伸链中的至少一条会转换模板，并且互补模板链之间的相互作用对于有效的模板转换是必要的。
