title: Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene.
abstract: 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.
authors.affiliation: Department of Medicine, Beth Israel Hospital, Boston, MA 02215, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Van Orsouw
authors.forename: D
authors.initials: D
authors.lastname: Li
authors.forename: P
authors.initials: P
authors.lastname: van der Vlies
authors.forename: H
authors.initials: H
authors.lastname: Scheffer
authors.forename: C
authors.initials: C
authors.lastname: Eng
authors.forename: C H
authors.initials: CH
authors.lastname: Buys
authors.forename: F P
authors.initials: FP
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Vijg
mesh_terms: D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005091:Exons; D016147:Genes, Tumor Suppressor; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics*
chemical_list: D016160:Retinoblastoma Protein