Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene

With the rapid increase in the number of identified human disease genes,the development of accurate and cost-efficient mutation tests has becomeopportune. Here we present a combination of extensive PCR multiplexing andtwo-dimensional (2-D) DNA electrophoresis to screen for mutations in 26exons of the retinoblastoma (RB1) tumor suppressor gene. In 2-Delectrophoresis, fragments are separated according to size and base pairsequence in non-denaturing and denaturing gradient gels, respectively. Alltarget fragments, designed to have optimal melting characteristics, wereprepared in a two-step PCR (a 6-plex long-PCR pre- amplification and asubsequent 25-plex short-PCR) followed by heteroduplexing. The mixture ofPCR amplicons was then subjected to 2-D electrophoresis under a single setof experimental conditions. With this design, 35 previously identifiedmutations in 18 different exons were detected in 33 bilateralretinoblastoma patients. These results suggest that 2-D electrophoresis inthis format provides a generally applicable, practical and fast way todiagnose with high accuracy large genes for a broad spectrum of possibledisease-causing mutations.