Journal Article

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

R D Rines, N J van Orsouw, I Sigalas, F P Li, C Eng and J Vijg

in Carcinogenesis

Volume 19, issue 6, pages 979-984
Published in print June 1998 | ISSN: 0143-3334
Published online June 1998 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/19.6.979
Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning.

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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.

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Subjects: Clinical Cytogenetics and Molecular Genetics

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