Colorimetric detection of single nucleotide polymorphisms in the presence of 10-fold excess of a wild-type gene

Herein, we proposed a simple colorimetric assay for highly sensitive and specific detection of single nucleotide polymorphisms (SNPs). Briefly, SNP specific capture probes (CPs) were immobilized onto magnetic beads. The hybridization of a target SNP with the CPs and detection probes containing multiple DNAzyme sequences (DNAzyme-DPs) brought the target SNP and the DNAzyme-DPs onto the magnetic beads. Meanwhile, a mismatch-specific CEL II enzyme (Surveyor® nuclease) cleaved the imperfectly hybridized wild-type gene together with all other mismatched sequences off the magnetic beads, leaving only the perfectly matched SNP strands on the magnetic beads. Amplified colorimetric detection was carried out through the DNAzyme-catalyzed oxidation of 3,3’,5,5’-tetramethylbenzidine in the presence of H2O2. The excellent selectivity of Surveyor® nuclease toward all imperfectly-matched DNA duplexes produced an ultrahigh selectivity – one mutant in 1000 copies of the wild-type gene can be detected. In addition, the cumulative nature of the DNAzyme-amplified signal generation process produced a detection limit as low as 0.40 fM and a dynamic range from 1.0 to 200 fM. The simple protocol and its high sensitivity and selectivity allowed the proposed assay to be used in the detection of SNPs in genomic DNA samples.