Rapid and sensitive detection of point mutation by DNA ligase-based electrochemiluminescence assay.
The identification of single-base mutations in particular genes plays an increasingly important role in medical diagnosis and prognosis of genetic-based diseases. Here we report a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay, the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target; thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-alpha-1 cell line, PANC-1 cell line and blood cell in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China.
Huijuan
H
Zhou
Da
D
Xing
Debin
D
Zhu
Xiaoming
X
Zhou
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin; D002460:Cell Line; D056831:Coordination Complexes; D011088:DNA Ligases; D055664:Electrochemical Techniques; D016158:Genes, p53; D006801:Humans; D008163:Luminescent Measurements; D008722:Methods; D015335:Molecular Probes; D009942:Organometallic Compounds; D017354:Point Mutation; D012680:Sensitivity and Specificity; D019809:Streptavidin; D017975:Ruthenium Compounds
D015082:2,2'-Dipyridyl; D001710:Biotin; D056831:Coordination Complexes; D011088:DNA Ligases; D015335:Molecular Probes; D009942:Organometallic Compounds; D019809:Streptavidin; C547232:tris(2,2-bipyridine)-ruthenium(II); D017975:Ruthenium Compounds; C058901:tris(2,2'-bipyridine)ruthenium III