ARTICLE · 1058809
混模板 PCR 中压低异源双链比的两条“杠杆”
1
背景：混模板 PCR 里的"幽灵双链"
The ratio of heteroduplexes to homoduplexes increases rapidly in the later PCR cycles. The most rapid increase is between the cycles 26 - 31.
As Thompson et al. (2002) hypothesized, heteroduplex formation could be caused by a low concentration of primers in the later PCR cycles.
它的危害是实质性的：克隆进 E. coli 后，宿主 MutHLS 错配修复会把异源双链"修"成嵌合序列；Thompson 等建模显示，假序列数随差异位点数与原始变种数指数级爆炸；在 DGGE / CDCE 上异源双链因构象差异被误判成"新变种"；直接测序则峰图混乱、碱基读错。（参考2）
2
核心发现：两个降异源双链比的杠杆
Thompson等（2002）提出，将扩增产物稀释10倍，随后进行三个循环的再扩增（“复原条件PCR”，reconditioning PCR）可以有效地从模板扩增中去除异源双链。
Thompson et al. (2002) proposed that a 10-fold dilution of the amplification product, followed by a three cycle re-amplification (“reconditioning”) could effectively remove heteroduplexes from template amplifications.
而在本文献中给出了两种方法：
2.1、补加 Taq 聚合酶
研究者尝试通过在第27个循环后添加2 U的Taq DNA聚合酶或2 U的Taq DNA聚合酶加8 pmol的每种引物（加入已含有Taq DNA聚合酶和8 pmol每种引物的标准PCR混合物中）来减少异源双链DNA的量，PCR在第28、29和30个循环后终止，通过Sau96I限制性消化检测异源双链DNA。
其观察到在添加Taq聚合酶后的第二个循环（第30个循环）异源双链与同源双链比率下降了30%。添加Taq聚合酶在两种情况下都增加了同源双链分子的绝对数量，因此降低了异源双链与同源双链的比率。
异源双链与同源双链比率的分析清楚地表明，与未添加额外Taq聚合酶的实验相比，添加Taq聚合酶降低了异源双链与同源双链的比率。
2.2、 减少 PCR 循环数
研究者观察到在少数PCR循环后异源双链与同源双链的比率较低，因此当PCR产物量不是很高时，复原条件PCR可能是消除异源双链形成的合适方法。
We have observed that the heteroduplex to homoduplex ratio is low after a few PCR cycles and that the ratio rapidly increases in the later PCR cycles.
Therefore, reconditioning PCR may be a suitable method to eliminate heteroduplex formation when the quantity of PCR products is not very high.
总之：发现表明异源双链的形成最可能取决于Taq聚合酶分子与PCR产物的比率。异源双链与同源双链的比率可以通过添加Taq聚合酶和减少PCR循环数来降低。然而，这种解决方案限制了PCR产量，因此通常不太有吸引力，但它避免了异源双链分子的形成。
Appropriate combination of both (a higher number of PCR cycles + the addition of Taq polymerase in the later PCR cycles) can be a method generally applicable to avoid, or at least decrease the formation of heteroduplexes.
1：Michu E, Mráčková M, Vyskot B, Žlůvová J. Reduction of heteroduplex formation in PCR amplification. Biologia Plantarum. 2010;54(1):173-176. DOI: 10.1007/s10535-010-0029-8;
2：Thompson JR, Marcelino LA, Polz MF. Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'. Nucleic Acids Research. 2002;30(9):e43. PMID: 11972349；
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