Stimulation and suppression of PCR-mediated recombination

被引:202
作者
Judo, MSB
Wedel, AB [1 ]
Wilson, C
机构
[1] Univ Calif Santa Cruz, Dept Biol, Santa Cruz, CA 95064 USA
[2] Univ Calif Santa Cruz, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
关键词
D O I
10.1093/nar/26.7.1819
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Tag 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%.
引用
收藏
页码:1819 / 1825
页数:7
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