Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification

We present a simple, sensitive, and cost-effective fluorescent assay of single-nucleotide polymorphism (SNP) with target-primed branched rolling circle amplification (TPBRCA). Designed padlock probe is circularized after perfect hybridization to mutant DNA. Then rolling circle amplification (RCA) reaction can be initiated from the mutant DNA that acts as primer and generates a long tandem single-stranded DNA (ssDNA) product. At the same time, the introduction of a reverse primer complementary to the target-primed RCA products leads to the branched RCA and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using SYBR Green I (SG) fluorescence dye. In contrast, the wild DNA contains a single mismatched base with the padlock probe and primes only a limited extension with the unligated padlock probe, generating weak background fluorescence with the addition of SG. Due to the excellent specificity and powerful amplification of TPBRCA reaction, the mutant DNA was distinctively differentiated from the wild DNA in a homogeneous and label-free manner. The assay is sensitive and specific enough to detect 5-amol (8.6-fM) mutant DNA strands. It was possible to accurately determine the mutant allele frequency as low as 1.0%.