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-quantitive. In this work, a protocol for quantitative detection of H-ras point mutation was developed. Electro...
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Veröffentlicht in: | Biochemical and biophysical research communications 2004-11, Vol.324 (2), p.964-969 |
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Sprache: | eng |
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Zusammenfassung: | 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 100fmol 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. |
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ISSN: | 0006-291X 1090-2104 |
DOI: | 10.1016/j.bbrc.2004.09.121 |