Labeled Primers for Mutation Scanning: Making Diagnostic Use of the Nucleobase Quenching Effect

[摘要] The advent of the PCR has revolutionized many molecular biological techniques, including single-nucleotide polymorphism (SNP) detection. Interest in the detection and discovery of human polymorphisms increased further with the complete sequencing of the human genome. The main applications for SNP detection include diagnosis of genetic disease and identification of functional mutations in genes that are of pharmacogenetic importance. The results from testing for somatic mutations remain with a person for a lifetime, and retesting is performed only rarely. To be able to deliver a molecular diagnosis of the highest quality, we must have a sound understanding of the different principles used for the detection of polymorphic DNA.The clinical laboratory today is equipped with a plethora of assays for the detection of SNPs, and new detection methods continue to be published. Molecular diagnostic assays often make use of the real-time monitoring of hybridization for the detection of base mutations. A common assay set-up uses a fluorescence resonance energy transfer (FRET) probe combination. A 3′-dye-labeled detection probe together with a 5′-dye-labeled and 3′-phosphorylated anchor probe interact by FRET in the hybridized state. In the unhybridized state, no FRET occurs, making this technique applicable to homogeneous single-tube assays. Variations of this theme include the use of exonuclease probes and molecular beacons. Initially it was recommended to avoid attaching the dye to a terminal guanosine residue because quenching was observed (1), but for molecular beacons, a design modification was suggested. Instead of the second dye or quencher, a string of guanosine bases was opposed …

[学科分类] 过敏症与临床免疫学