Detection of central single-nucleotide mismatches in short duplex DNAs on hyper-branched amine surfaces

摘要

High capability to distinguish single-nucleotide mismatches of genes using short oligonucleotide probes is essential in diagnostic methods for identification of point mutations and single nucleotide polymorphisms. To investigate the feasibility of using an aziri-dine-treated surface containing hyper-branched amine groups to discriminate single-nucleotide mismatches in a human gene, target probes for exons 5-8 of the p53 gene from liver cancer cells were hybridized with four types of surface-bound capture probes, one for perfect match and three for central single-nucleotide mismatches. The aziridine slide with high DNA-loading capacity exhibited greater ability to detect single-nucleotide mismatch than did the generic amine slide. When a T30 tether was linked to the capture probe, the mismatch discrimination capability increased when using a chemical cross-linker, but decreased when using UV irradiation for cross-linking. DNA duplexes had lower melting temperatures when the single-nucleotide mismatch was in the central region than when it was in the terminal region regardless of the type of mismatched nucleotide. Our results suggest that capture probes attached to the aziridine surface can effectively identify point mutations in a genomic sequence or and can estimate the affinity of gene-specific antisense oligonucleotide probes.