High sensitive approach for point mutation detection based on electrochemiluminescence

Biosens Bioelectron. 2004 Oct 15;20(3):448-53. doi: 10.1016/j.bios.2004.02.029.

Abstract

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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / analysis
  • Carcinoma / diagnosis
  • Carcinoma / genetics
  • Cell Line, Tumor
  • DNA Mutational Analysis / instrumentation
  • DNA Mutational Analysis / methods*
  • DNA, Neoplasm / analysis*
  • DNA, Neoplasm / genetics*
  • Electrochemistry / instrumentation
  • Electrochemistry / methods*
  • Feasibility Studies
  • Genes, ras / genetics
  • Genetic Testing / methods
  • Humans
  • Luminescent Measurements / instrumentation
  • Luminescent Measurements / methods*
  • Lung Neoplasms / diagnosis
  • Lung Neoplasms / genetics
  • Point Mutation / genetics
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Single Nucleotide / genetics*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Urinary Bladder Neoplasms / diagnosis
  • Urinary Bladder Neoplasms / genetics

Substances

  • Biomarkers, Tumor
  • DNA, Neoplasm