Whole-Genome Amplification by Single-Cell Comparative Genomic Hybridization PCR (SCOMP).

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 <2 kb by digesting with the restriction enzyme MseI. This results in a smear in the range of 100-1500 bp. Following enzyme digestion, adaptors containing specific primer sequences (specific to the restriction enzyme used) are ligated onto the ends of the genomic DNA and amplified in a high-stringency PCR. This results in a smear of PCR products in the range of 100-1500 bp, which can be visualized by agarose gel electrophoresis.