Magnetic beads based rolling circle amplification–electrochemiluminescence assay for highly sensitive detection of point mutation

The identification of point mutations is particularly essential in the fields of medical diagnosis and prognosis of many pathogenic and genetic diseases. In this study, an rolling circle amplification (RCA) based electrochemiluminescence (ECL) assay for highly sensitive point mutation detection was developed. In the assay, an allele-discriminating padlock probe was designed for targeting the sequence in the p53 oncogene locus. A circular template generated by enzymatic ligation upon the recognition of a point mutation (CGT to CAT) on the oncogene could be amplified isothermally by Phi29 DNA polymerase. The elongated products, containing hundreds of copies of the circular DNA template sequence, were hybridized with Ru(bpy)32+ (TBR)-tagged probes and then captured onto streptavidin-coated paramagnetic beads. The resulting products were analyzed by magnetic bead based ECL platform. As low as 2 amol of mutated strands was detected by this assay, which could be attributed to the high amplification efficiency of Phi29 DNA polymerase and current magnetic bead based ECL detection platform. In addition, the positive mutation detection was achieved with a wild-type to mutant ratio of 10 000:1, due to the high fidelity of DNA ligase in differentiating mismatched bases at the ligation site. It is demonstrated that this proposed method provides a highly sensitive and specific approach for point mutation detection.