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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. Single-cell comparative genomic hybridization (SCOMP), described in this protocol, is a form of ligation-mediated PCR that was specifically designed for WGA of extremely limited sources of genomic DNA. The reaction volume is purposely kept to a minimum, and all buffers are optimized to eliminate the need to purify the reaction between steps. In addition, the entire reaction is performed in a single tube. This avoids initial template loss and reduces the risk of PCR contamination. SCOMP begins by converting the genome to a high-complexity representation with a fragment size of

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). GenomePlex WGA, described in this protocol, is a proprietary amplification technology based on nonenzymatic random fragmentation of genomic DNA. The protocol involves conversion of the genome into an in vitro molecular library of DNA fragments, followed by incubation at various temperatures to add adaptor sequences with specific PCR priming sites to both ends of every fragment. The fragment library can then be amplified several 1000-fold to generate milligram quantities of DNA starting with as little as 10-100 ng.

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.