We present a protocol to detect point mutation on a chip using isothermal rolling-circle amplification (RCA). The basic principle of the technique is an allele-specific oligonucleotide circularization mediated by DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and target DNA allow ligase to covalently circularize the probe. Mismatches around the ligation site prevent probe circularization. Under isothermal condition, the circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA). There are four adjacent sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probe and solid probe which immobilized on a glass slide composing a regular microarray pattern. The signal of fluorescence can be monitored by a scanner in the presence of nucleic acids templates, whereas the probe cannot be circularized, ssDNA cannot be generated and signal of fluorescence cannot be monitored. The stringency discrimination of the molecular templates are up to 102-103 folds between matched and mismatched sequences. The development of C-probe-based technologies offers a promising prospect for molecular diagnosis, situ detection, microarray, immunoassay, single nucleotide polymorphism, and whole genome amplification.
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