Phi29-based amplification of small genomes

Abstract

based on theisothermal reaction of the enzyme Phi29 and randomprimers, is one of the most popular methods currentlyused in genomic research [1–3]. However, there is onemajor drawback limiting applicability of this technique incertain Welds such as forensics, microbiology, and envi-ronmental monitoring. The WGA method is hardly appli-cable to small genomes and/or degraded DNA, especiallyif the number of genome copies is approaching 100–1000molecules. Here, we describe a simple procedure in whichWGA ampliWes 1fg of the linear pUC19 DNA su Ycientlyfor hundreds of downstream (i.e., post-WGA) PCR reac-tions. Thus, the ampliWcation of limited quantities ofother unknown small genomes or genomic fragmentsshould be possible as well.It was observed previously that self-priming of degener-ate 6-mers might compete with the priming of the templateDNA, especially if the quantity and/or length of the DNAtemplate are small [2–5]. However, resolving self-primingproblem did not remove performance limitations of thePhi29-based WGA procedure [5]. For example, in our labo-ratory, 300 copies of a single copy gene could be WGAampliWed in the context of human DNA but not as an iso-lated plasmid DNA. It seemed that the technical bottleneckwas due to a low hybridization of the degenerate 6-mers tothe template, suggesting a better WGA performance withan equivalent number of copies of longer templates orembedded within longer templates, thereby favoring ahigher number of priming events to occur. Therefore, wedecided to ligate a small template, such as pUC19 of2686bp, with a long carrier DNA, such as poly(dA–dT) orhuman DNA, before performing Phi29-based WGA.We compared WGA performance of serial dilutions of(i)