High-Throughput Genotyping With Energy Transfer-Labeled Primers

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 thermocyler and fluorescent plate-reader. Here, we provide detailed protocols for primer design, both manually and using AssayArchitectTM 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.