Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning.
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.
Beth Israel Deaconess Medical Center, Harvard Institute of Medicine, Boston, MA 02115, USA.
R D
RD
Rines
N J
NJ
van Orsouw
I
I
Sigalas
F P
FP
Li
C
C
Eng
J
J
Vijg
D001483:Base Sequence; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D016158:Genes, p53; D006801:Humans; D016864:Li-Fraumeni Syndrome / Q000235:genetics; D016133:Polymerase Chain Reaction
D017931:DNA Primers