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 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)755-761
Number of pages7
JournalHuman Molecular Genetics
Volume5
Issue number6
DOIs
StatePublished - Jun 1996
Externally publishedYes

Fingerprint

Electrophoresis
Polymerase Chain Reaction
Mutation
Retinoblastoma
Genes
Exons
Tumor Suppressor Genes
Base Pairing
Freezing
Gels
Costs and Cost Analysis
DNA

ASJC Scopus subject areas

  • Genetics

Cite this

Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis : Application to the RB1 gene. / Van Orsouw, Nathalie J.; Li, Daizong; Van Der Vlies, Pieter; Scheffer, Hans; Eng, Charis; Buys, Charles H C M; Li, Frederick P.; Vijg, Jan.

In: Human Molecular Genetics, Vol. 5, No. 6, 06.1996, p. 755-761.

Research output: Contribution to journalArticle

Van Orsouw, Nathalie J. ; Li, Daizong ; Van Der Vlies, Pieter ; Scheffer, Hans ; Eng, Charis ; Buys, Charles H C M ; Li, Frederick P. ; Vijg, Jan. / Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis : Application to the RB1 gene. In: Human Molecular Genetics. 1996 ; Vol. 5, No. 6. pp. 755-761.
@article{77e7c3fbb5814ed285a94fd6a03c5d24,
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.",
author = "{Van Orsouw}, {Nathalie J.} and Daizong Li and {Van Der Vlies}, Pieter and Hans Scheffer and Charis Eng and Buys, {Charles H C M} and Li, {Frederick P.} and Jan Vijg",
year = "1996",
month = "6",
doi = "10.1093/hmg/5.6.755",
language = "English (US)",
volume = "5",
pages = "755--761",
journal = "Human Molecular Genetics",
issn = "0964-6906",
publisher = "Oxford University Press",
number = "6",

}

TY - JOUR

T1 - Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis

T2 - Application to the RB1 gene

AU - Van Orsouw, Nathalie J.

AU - Li, Daizong

AU - Van Der Vlies, Pieter

AU - Scheffer, Hans

AU - Eng, Charis

AU - Buys, Charles H C M

AU - Li, Frederick P.

AU - Vijg, Jan

PY - 1996/6

Y1 - 1996/6

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0029943489&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029943489&partnerID=8YFLogxK

U2 - 10.1093/hmg/5.6.755

DO - 10.1093/hmg/5.6.755

M3 - Article

C2 - 8776589

AN - SCOPUS:0029943489

VL - 5

SP - 755

EP - 761

JO - Human Molecular Genetics

JF - Human Molecular Genetics

SN - 0964-6906

IS - 6

ER -