COMPREHENSIVE MUTATIONAL SCANNING OF THE P53 CODING REGION BY 2-DIMENSIONAL GENE SCANNING

A comprehensive mutational scanning test for the p53 coding region bas ed on multiplex PCR and two-dimensional DNA electrophoresis was design ed and evaluated. In a 2-step multiplex PCR, the p53 coding region (ex ons 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 s tep two. The multiplex products were then separated, first on the basi s 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 desig ned using a recently developed computer program. The resulting two-dim ensional gene scanning (TDGS) test was evaluated by screening, in a bl inded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations, All mutations were cor rectly detected. This assay provides an accurate, cost-effective and n onradioactive method for simultaneous mutational scanning of all p53 c oding exons.