Highly Specific And Sensitive Electrochemical Genotyping Via Gap Ligation Reaction And Surface Hybridization Detection

摘要

Single nucleotide polymorphisms (SNPs) are the most abundant and stable type of genetic variations found in human genome. Genotyping of such sequence variations is often diagnostic of particular genetic disorders and drug responses owing to their direct connections with transcriptional regulation or biological functions of many proteins. SNP genotyping is generally performed through a certain allele discrimination mechanism followed by detection of the allele-specific products. Typical allele discrimination mechanisms include allele-specific hybridization, allele-specific nucleotide incorporation, allele-specific cleavage, and allele-specific oligonucleotide ligation. Allele-specific hybridization requires double-stranded DNA hybrids resulting from single-base variations to show detectable differences in hybridization efficiency or thermal stability. Such a mechanism normally involves stringent control of the assay conditions, posing challenges in sequence-dependent probe design, and hybridization condition optimization. Enzyme-aided allele discrimination, such as allele-specific nucleotide incorporation, cleavage and ligation, provides a more selective and flexible arsenal for SNP genotyping. These techniques, though widely adapted to varying detection strategies from optical readouts to mass spectrometry and electronic measurements, still have not prevailed owing to relatively high cost, limited specificity, or inadequate sensitivity.