High sensitive approach for point mutation detection based on electrochemiluminescence.
An electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for point mutation detection has been developed. The target is amplified using a tris (bipyridine) ruthenium (TBR)-labeled forward and a biotinylated reverse primer. The amplification products are digested with specific restriction enzyme, then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. The established technique was further applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the system has a low detection limit of 100 fmol and a linear range of more than 3 orders of magnitude for H-ras amplicon; the two genotypes can be reliably discriminated. In summary, the mutant specific ECL-PCR method can be used to detect a point mutation that creates or destroys a restriction site in any gene. It is useful in single nucleotide polymorphism (SNP) and 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.
Xing
Da
D
Shen
Xingyan
X
Liu
Jinfeng
J
Chen
Qun
Q
D014408:Biomarkers, Tumor / Q000032:analysis; D002277:Carcinoma / Q000175:diagnosis / Q000235:genetics; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D005240:Feasibility Studies; D011905:Genes, ras / Q000235:genetics; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis / Q000235:genetics
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm