Abstract
The chief attribute of the fluorogenic 5′ nuclease assay is that it is completely homogeneous. After mixing the sample and reaction components, the assay is run in a closed tube format with no postpolymerase chain reaction (PCR) processing steps. Results are obtained by simply measuring the fluorescence of the completed reactions. By eliminating post-PCR processing, allelic discrimination with fluorogenic probes reduces the time of analysis, eliminates the labor and supply costs of post-PCR steps, reduces the risk of crossover contamination, and minimizes sources of error. The assay has the sensitivity of PCR so that a minimum amount of genomic DNA is required. The use of endpoint fluorescence measurements maximizes throughput. Using a single ABI Prism® 7900HT Sequence Detection System and 24-h operation, it is possible to generate up to 250,000 SNP results per day. TaqMan® MGB probes and the entire operating system outlined below make it possible to quickly apply the 5′ nuclease assay to any allelic discrimination application where high throughput is of paramount concern.
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