A new approach to SNP genotyping with fluorescently labeled mononucleotides.

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3' end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.