Combination of DNA Ligase Reaction and Gold Nanoparticle-Quenched Fluorescent Oligonucleotides: A Simple and Efficient Approach for Fluorescent Assaying of Single-Nucleotide Polymorphisms

摘要

A new fluorescent sensing approach for detection ofnsingle-nucleotide polymorphisms (SNPs) is proposednbased on the ligase reaction and gold nanoparticlen(AuNPs)-quenched fluorescent oligonucleotides. The designnexploits the strong fluorescence quenching of AuNPsnfor organic dyes and the difference in noncovalent interactionsnof the nanoparticles with single-stranded DNAn(ssDNA) and double-stranded DNA (dsDNA), where ss-nDNA can be adsorbed onto the surface of AuNPs whilendsDNA cannot be. In the assay, two half primer DNAnprobes, one being labeled with a dye and the other beingnphosphorylated, were first incubated with a target DNAntemplate. In the presence of DNA ligase, the two capturednssDNAs are linked for the perfectly matched DNA targetnto form a stable duplex, but the duplex could not benformed by the single-base mismatched DNA template.nAfter addition of AuNPs, the fluorescence of dye-taggednDNA probe will be efficiently quenched unless the perfectlynmatched DNA target is present. To demonstrate thenfeasibility of this design, the performance of SNP detectionnusing two different DNA ligases, T4 DNA ligase andnEscherichia coli DNA ligase, were investigated. In the casenof T4 DNA ligase, the signal enhancement of the dyetaggednDNA for perfectly matched DNA target is 4.6-foldnhigher than that for the single-base mismatched DNA.nWhile in the presence of E. coli DNA ligase, the valuenraises to be 30.2, suggesting excellent capability for SNPndiscrimination.