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

Hum Mol Genet. 1996 Jun;5(6):755-61. doi: 10.1093/hmg/5.6.755.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA Mutational Analysis*
  • Electrophoresis, Gel, Two-Dimensional / methods*
  • Exons*
  • Genes, Tumor Suppressor*
  • Humans
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Genetic
  • Polymorphism, Single-Stranded Conformational
  • Retinoblastoma / genetics*
  • Retinoblastoma / metabolism
  • Retinoblastoma Protein / genetics*


  • Retinoblastoma Protein