A method to quantitatively detect H-ras point mutation based on electrochemiluminescence

作者：Zhu, DB; Xing, D^*; Shen, XY; Liu, JF

来源：Biochemical and Biophysical Research Communications, 2004, 324(2): 964-969.

DOI：10.1016/j.bbrc.2004.09.121

• point mutation
• electrochemiluminescence
• restriction endonuclease digestion
• polymerase chain reaction
• H-ras oncogene
• bladder cancer

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.

• 出版日期2004-11-12
• 单位华南师范大学