Whole-Genome Amplification by Adaptor-Ligation PCR of Randomly Sheared Genomic DNA (PRSG)

Abstract

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Ligation-mediated PCR techniques involve ligating an adaptor sequence onto a “representation” of DNA molecules, generated following enzymatic digestion, random shearing, or chemical cleavage. Adaptor-ligation PCR of randomly sheared genomic DNA (PRSG), described here, is based on ligation-mediated PCR and was designed to improve genome coverage. Rather than using enzymatically generated fragments, this method uses randomly fragmented DNA as the template. The process involves three steps: (1) the hydrodynamic shearing of genomic DNA to a 0.5-2-kb size range, (2) end filling and adaptor ligation, and (3) high-stringency PCR for faithful replication of the resulting fragments.