PCR介导重组的刺激与抑制
Stimulation and suppression of PCR-mediated recombination.
作者信息
Judo M S, Wedel A B, Wilson C
机构信息
Department of Biology and Center for the Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.
出版信息
Nucleic Acids Res. 1998 Apr 1;26(7):1819-25. doi: 10.1093/nar/26.7.1819.
Abstract
Recombination, or chimera formation, is known to occur between related template sequences present in a single PCR amplification. To characterize the conditions under which such recombinant amplification products form we monitored the exchange of sequence between two homologous templates carrying different restriction sites separated by 282 bp. Using a typical cycling program the rates of recombination between the two restriction sites were 1 and 7% using Taq and Vent polymerases respectively over 12 doublings. However, by using long elongation times and cycling only to the mid-point of the amplification recombination could be suppressed below visual detection with both polymerases. Conversely, cycling programs designed to promote incomplete primer elongation and subsequent template strand exchange stimulated recombination to >20%.
摘要
已知在单个PCR扩增中存在的相关模板序列之间会发生重组或嵌合体形成。为了表征此类重组扩增产物形成的条件，我们监测了携带由282 bp隔开的不同限制位点的两个同源模板之间的序列交换。使用典型的循环程序，在12次倍增过程中，使用Taq和Vent聚合酶时，两个限制位点之间的重组率分别为1%和7%。然而，通过使用较长的延伸时间并仅循环至扩增中点，可以将两种聚合酶的重组抑制到视觉检测以下。相反，旨在促进不完全引物延伸和随后模板链交换的循环程序会刺激重组至>20%。
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