We have developed a new method for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The technique involves PCR amplification of genomic DNA with two tailed allele-specific primers that introduce priming sites for universal energy-transfer-labeled primers. The output of red and green light is conveniently scored using a fluorescence plate reader. The new method, which was validated on nine model SNPs, is well suited for high-throughput, automated genotyping because it requires only one reaction per SNP, it is performed in a single tube with no post-PCR handling, the same energy-transfer-labeled primers are used for all analyses, and the instrumentation is inexpensive. Possible applications include multiple-candidate gene analysis, genomewide scans, and medical diagnostics.

Background: Human hypertension is a complex, multifactorial disease with a heritability of more than 30–50%. A genetic screening test based on analysis of multiple single-nucleotide polymorphisms (SNPs) to assess the likelihood of developing hypertension would be helpful for disease management. Methods: Tailed allele-specific primers were designed to amplify by PCR six biallelic SNP loci [three in G protein-coupled receptor kinase type 4 (GRK4): R65L, A142V, and A486V; two in angiotensinogen: −6G→A and M235T; and one in aldosterone synthase: −344C→T] associated with essential hypertension. PCRs of SNP loci were coupled (via a common sequence of 21 nucleotide tails) to incorporate Universal Amplifluor™ primers labeled with fluorescein or sulforhodamine in a homogeneous format. Use of Amplifluors in SNP PCRs produced labeled amplicons, the fluorescence of which was quantified by a microplate reader and then analyzed via an Excel macro to provide genotypes for all six SNP loci. Unique restriction endonucleases were identified for five SNP loci that could independently confirm homogeneous PCR results when needed. Results: We developed six homogeneous PCR assays that were set up, performed, and fluorometrically analyzed in 96-well microplates. Allele frequencies were determined for six SNPs in 60 Italian hypertensive patients and a control group of 60 normotensive persons. A significant correlation (P = 0.034) between one SNP [GRK4 (A486V)] and the hypertensive patients was observed. Genotyping results for five of six SNPs were confirmed by digesting corresponding amplicons with locus-specific restriction endonucleases. Conclusions: We developed a simple and homogeneous fluorescent protocol that has been used to determine the SNP genotype for six loci in a population of hypertensive and normotensive persons. We also observed a significant association (P = 0.034) between one SNP (A486V) and an Italian population of mildly hypertensive patients.

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 costeffective with potential for use in industrial-scale genetic studies or in the clinical diagnostic setting.

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The Amplifluor method for single-nucleotide polymorphisms (SNP) genotyping provides homogeneous assays that utilize a pair of universal energy transfer-labeled primers. The main advantage of this single-step, loci-independent, low-cost method is that it can be readily adapted for new SNPs. The development of any new SNP assay requires only the design and synthesis of three conventional oligonucleotides. Furthermore, Amplifluor-based SNP assays require instrumentation found in most laboratories including a thermocycler and fluorescent plate-reader. Here, we provide detailed protocols for primer design, both manually and using AssayArchitect software. Protocols for SNP analysis are provided along with more than 100 examples for common polymorphisms. Specific cases including polymorphisms caused by the insertion/deletion of nucleotides, and dealing with the AT- and GC-rich sequences are addressed and discussed in detail.