A method to quantitatively detect H-ras point mutation based on electrochemiluminescence.
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-quantitative. 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.
Zhu
Debin
D
Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China. xingda@scnu.edu.cn
Xing
Da
D
Shen
Xingyan
X
Liu
Jinfeng
J
D002138:Calibration; D003062:Codon; D004252:DNA Mutational Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D016014:Linear Models; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011379:Prognosis; D012680:Sensitivity and Specificity; D001749:Urinary Bladder Neoplasms / Q000235:genetics
D003062:Codon; D004262:DNA Restriction Enzymes