Screening for genomic rearrangements by multiplex PCR/liquid chromatography

Screening for large gene rearrangements has become established as an important part of molecular medicine; however, it is also challenging as these rearrangements range from an extra copy of a complete chromosome(s) to deletion or duplication of a single exon. In this chapter, we describe a versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography (MP/LC) assay. Multiple genomic fragments are amplified under semiquantitative conditions using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography, and quantitated by fluorescent detection using a postcolumn intercalation dye. The relative peak intensities for each target directly reflect DNA copy number. This technique can be used not only to screen intronic, exonic, and intergenic parts of the genome but also for transcript quantitation. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to DHPLC users, as it broadens the spectrum of available applications on a DHPLC system. The authors describe a detailed protocol for large rearrangement screening in the RB1 gene.