TEMPLATE-SWITCHING DURING DNA-SYNTHESIS BY THERMUS-AQUATICUS DNA-POLYMERASE-I
TEMPLATE-SWITCHING DURING DNA-SYNTHESIS BY THERMUS-AQUATICUS DNA-POLYMERASE-I
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DOI：
10.1093/nar/23.11.2049
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发表时间：
1995-06-11
影响因子：
14.9
通讯作者：
WHITE, R
中科院分区：
文献类型：
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作者：
ODELBERG, SJ;WEISS, RB;WHITE, R
当部分同源模板可用时，重组DNA分子通常在聚合酶链反应（PCR）中产生[例如，参见Paabo at al. (1990) J. Biol。化学学报，2004,18(2):418 - 421。有人认为，这些重组分子是在随后的PCR循环中将截断的延伸产物退火到部分同源模板的结果。然而，我们在这里证明，在没有后续热变性的情况下，重组分子可以在单轮引物延伸中产生，这表明模板切换产生了一些重组分子。观察到两种类型的模板切换：(i)切换到预先存在的模板和（ii）切换到互补的新生链。当互补模板链通过附着在链亲和素磁珠上物理分离时，重组减少了几倍。这一结果支持了一个假设，即在DNA合成过程中，聚合酶或至少两条延伸链中的一条会切换模板，而互补模板链之间的相互作用对于有效的模板切换是必要的。
Recombinant DNA molecules are often generated during the polymerase chain reaction (PCR) when partially homologous templates are available [e.g., see Paabo at 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.