Detection DNA Point Mutation with Rolling-Circle Amplification Chip

We present a protocol with isothermal rolling-circle amplification (RCA) to detect DNA point mutation on chip. The basic principle of the method is an allele-specific oligonucleotide circularization mediated by special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and target DNA. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) Under isothermal condition. There are sequence regions to bind respectively with fluorescent probe, solid probe and Artificial template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probe and 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 and signal of fluorescence cannot be found. 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 situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.