Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning

R. David Rines, Nathalie J. Van Orsouw, Iakovos Sigalas, Frederick P. Li, Charis Eng, Jan Vijg

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.

Original languageEnglish (US)
Pages (from-to)979-984
Number of pages6
JournalCarcinogenesis
Volume19
Issue number6
DOIs
StatePublished - Jun 1998
Externally publishedYes

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Exons
Multiplex Polymerase Chain Reaction
Li-Fraumeni Syndrome
Genes
Polymerase Chain Reaction
Denaturing Gradient Gel Electrophoresis
Germ-Line Mutation
DNA
Freezing
Electrophoresis
Software
Gels
Costs and Cost Analysis
Mutation
polyacrylamide gels

ASJC Scopus subject areas

  • Cancer Research

Cite this

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning. / Rines, R. David; Van Orsouw, Nathalie J.; Sigalas, Iakovos; Li, Frederick P.; Eng, Charis; Vijg, Jan.

In: Carcinogenesis, Vol. 19, No. 6, 06.1998, p. 979-984.

Research output: Contribution to journalArticle

Rines, R. David ; Van Orsouw, Nathalie J. ; Sigalas, Iakovos ; Li, Frederick P. ; Eng, Charis ; Vijg, Jan. / Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning. In: Carcinogenesis. 1998 ; Vol. 19, No. 6. pp. 979-984.
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