Amplification of complex gene libraries by emulsion PCR

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. 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. 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).