Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 324, Issue 2, Pages 964-969Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2004.09.121
Keywords
point mutation; electrochemiluminescence; restriction endonuclease digestion; polymerase chain reaction; H-ras oncogene; bladder cancer
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Conventional methods for point mutation detection are usually multi-stage, laborious, and need to use radioactive isotopes or other hazardous materials, and the assay results are often semi-quantitive. In this work, a protocol for quantitative detection of H-ras point mutation was developed. Electrochemiluminescence (ECL) assay was coupled with restriction endonuclease digestion directly from PCR products. Only the wild-type amplicon containing the endonuclease's recognition site can be cut off, and thus cannot be detected by ECL assay. Using the PCR-ECL method, 30 bladder cancer samples were analyzed for possible point mutation at codon 12 of H-ras oncogene. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than three orders of magnitude. The point mutation was found in 14 (46.7%) out of 30 bladder cancer samples. The experiment results demonstrate that the PCR-ECL method is a feasible quantitative approach for point mutation detection due to its safety, high sensitivity, and simplicity. (C) 2004 Elsevier Inc. All rights reserved.
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