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

Nathalie J. Van Orsouw, Daizong Li, Pieter Van Der Vlies, Hans Scheffer, Charis Eng, Charles H.C.M. Buys, Frederick P. Li, Jan Vijg

Research output: Contribution to journalArticle

27 Scopus citations


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

Original languageEnglish (US)
Pages (from-to)755-761
Number of pages7
JournalHuman molecular genetics
Issue number6
StatePublished - Jun 1 1996
Externally publishedYes


ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Genetics(clinical)

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