Miniaturized sealed-tube allele-specific PCR

DNA diagnostics has been progressively moving from expensive, low,throughput, multi-step methods towards inexpensive, robust, and high,throughput methods. Here we describe the further validation and refinement of a recently described novel genotyping method that has the latter characteristics. An evolved form of allele,specific PCR, the method generates a fluorescent signal through the use of universal labeled primers, which can be quantified directly from microplates using standard plate readers. We have applied the method successfully to a test set of 12 novel single nucleotide polymorphisms (SNPs) on a panel of 47 individuals using low reaction volumes. We demonstrate that the method is extremely accurate, robust, and can be optimized in a simple and predictable manner. By conducting the assay in closed tube format, the potential for contamination is reduced to a minimum. By virtue of its simplicity, the method is versatile and cost, effective with potential for use in industrial, scale genetic studies or in the clinical diagnostic setting.