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

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.

• Authors: R D Rines, N J van Orsouw, I Sigalas, F P Li, C Eng, J Vijg
• Journal: Carcinogenesis (Carcinogenesis) Vol. 19 Issue 6 Pg. 979-84 (Jun 1998) ISSN: 0143-3334 [Print] England
• PMID: 9667734 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA Primers

Topics

• Base Sequence
• DNA Primers
• Electrophoresis, Gel, Two-Dimensional (methods)
• Genes, p53
• Humans
• Li-Fraumeni Syndrome (genetics)
• Polymerase Chain Reaction