A Robust and Sensitive Method For Detection of exonic deletions in the critical gatekeeper genes: VHL, APC, and RB1 By Multiplex Polymerase Chain Reaction (Mpcr) And High-Sensitivity High-Performance Liquid Chromatography (HS-HPLC). S. Lilleberg, J. Hempel, S. Edstrom, M. Nickerson. Translational & Clinical Res, Transgenomic, Omaha, NE.
   Reliable and sensitive screening for large gene rearrangements is a vital part of molecular genetic testing, however it can be technically challenging. A variety of robust methods can detect whole-gene deletions, but fail to detect more subtle rearrangements such as single exon deletions. Here we describe the implementation of a versatile and robust technique to assess exon copy number, which utilizes a multiplex polymerase chain reaction (mPCR) and high sensitivity (HS) post-column fluorescence detection of the amplicons after separation by nondenaturing high performance liquid chromatography (HPLC). The relative peak intensity for each target exon is associated with the exon copy number present in the sample. This novel approach was used to screen various patient samples for exon deletions in the VHL,APC and RB1 genes. The use of multiplex PCR coupled with HS-HPLC allows for analysis of small gene rearrangements in biological samples with limited source DNA (e.g. biopsy samples) on an automated platform without the use of fluorescent probes.