Amplification of complex gene libraries by emulsion PCR
Amplification of complex gene libraries by emulsion PCR
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DOI：
10.1038/nmeth896
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发表时间：
2006-07-01
期刊：
影响因子：
48
通讯作者：
Griffiths, Andrew D.
中科院分区：
文献类型：
--
作者：
Williams, Richard;Peisajovich, Sergio G.;Griffiths, Andrew D.
PCR对基因组文库、cDNA文库和其他复杂基因组合的有效扩增存在以下两种现象：一是短片段倾向于优先扩增，而不是大片段；其次，通过DNA同源区域之间的重组产生人工片段(1)。在这种情况下，当引物在一个PCR周期中部分延伸到一个模板上，并在随后的周期中进一步延伸到另一个模板上时，就会发生重组。因此，产生嵌合分子，然后优先扩增图1中所述的短分子。已经提出了各种PCR方案来最大限度地减少这些问题，其中大多数依赖于高模板浓度和低PCR循环数（2,3）。然而，很明显，如果可用的模板DNA很少，这种方法是不可行的。在这里，我们描述了一种扩增复杂DNA混合物的方案，基于油包水（w/o）乳液中基因的区隔化。模板片段在乳剂的微小水滴中分离，并在分离中通过PCR扩增（图1）。这种方法减轻了上述问题，同时允许使用少量的模板DNA和大量的PCR循环。框1描述了使用表面活性剂ABIL EM 90生成非常稳定的乳液用于乳液PCR的替代方法（图2）。
The efficient amplification of genomic libraries, cDNA libraries and other complex mixtures of genes by PCR is impeded by two phenomena: firstly, short fragments tend to be amplified in preference to larger ones; and, secondly, artifactual fragments are generated by recombination between homologous regions of DNA(1). Recombination in this case occurs when a primer is partially extended on one template during one cycle of PCR and further extended on another template during a later cycle. Thus, chimeric molecules are generated, the short ones of which are then preferentially amplified as described in Figure 1. A variety of PCR protocols have been proposed to minimize these problems, most of which rely on high template concentrations and low numbers of PCR cycles(2,3). Clearly, however, such an approach is not viable if little template DNA is available. Here we describe a protocol for amplifying complex DNA mixtures, based on the compartmentalization of genes in a water-in-oil (w/o) emulsion. Template fragments are segregated in the minute aqueous droplets of the emulsion and amplified by PCR in isolation (Fig. 1). This approach alleviates the problems described above while enabling the use of small amounts of template DNA and high numbers of PCR cycles. Box 1 describes an alternative method for generating very stable emulsions for emulsion PCR using the surfactant ABIL EM 90 (Fig. 2).