An ECL-PCR method for quantitative detection of point mutation

Debin Zhu; Da Xing; Xingyan Shen; Qun Chen; Jinfeng Liu

doi:10.1117/12.588909

4 April 2005 An ECL-PCR method for quantitative detection of point mutation

Debin Zhu, Da Xing, Xingyan Shen, Qun Chen, Jinfeng Liu

Proceedings Volume 5704, Genetically Engineered and Optical Probes for Biomedical Applications III; (2005) https://doi.org/10.1117/12.588909
Event: SPIE BiOS, 2005, San Jose, CA, United States

Abstract

A new method for identification of point mutations was proposed. Polymerase chain reaction (PCR) amplification of a sequence from genomic DNA was followed by digestion with a kind of restriction enzyme, which only cut the wild-type amplicon containing its recognition site. Reaction products were detected by electrochemiluminescence (ECL) assay after adsorption of the resulting DNA duplexes to the solid phase. One strand of PCR products carries biotin to be bound on a streptavidin-coated microbead for sample selection. Another strand carries Ru(bpy)₃²⁺ (TBR) to react with tripropylamine (TPA) to emit light for ECL detection. The method was applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than 3 orders of magnitude, thus, make quantitative analysis possible. The genotype can be clearly discriminated. Results of the study suggest that ECL-PCR is a feasible quantitative method for safe, sensitive and rapid detection of point mutation in human genes.

Citation Download Citation

Debin Zhu, Da Xing, Xingyan Shen, Qun Chen, and Jinfeng Liu "An ECL-PCR method for quantitative detection of point mutation", Proc. SPIE 5704, Genetically Engineered and Optical Probes for Biomedical Applications III, (4 April 2005); https://doi.org/10.1117/12.588909

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KEYWORDS

Electrodes

Blood

Calibration

Polymers

Magnetism

Quantitative analysis

Signal processing

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