Carcinogenesis, Vol 19, 979-984, Copyright © 1998 by Oxford University Press
RD Rines, NJ van Orsouw, I Sigalas, FP Li, C Eng and J Vijg
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.
ARTICLES
Comprehensive mutational scanning of the p53 coding region by two- dimensional gene scanning
Beth Israel Deaconess Medical Center, Harvard Institute of Medicine, Boston, MA 02115, USA.
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