Detection of Single-Nucleotide Polymorphisms by PCR with Universal Energy Transfer–Labeled Primers: Application to Folate- and Cobalamin-Related Genes

摘要

Many methods for detection of single-nucleotide polymorphisms (SNPs) are based on fluorescence resonance energy transfer (1). One of these, the Amplifluor SNP genotyping system, combines the use of universal energy transfer-labeled primers (uniprimers) (2) with allele-specific amplification (3). The uniprimer is a modification of the beacon concept (1)(4); it uses the beacon’s hairpin structure, but instead of acting as a probe, the hairpin is part of the primer, and when incorporated into the amplicon, it opens and starts to fluoresce (see Fig. S1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue9/) (2). The advantage of the technology is that one pair of uniprimers is used for any SNP. Several variants of the uniprimer system for SNP detection are available (5)(6)(7). We modified the method so that it became faster and less expensive and then used it to identify 3 polymorphisms related to homocysteine and B vitamins (8): transcobalamin 2 (TCN2) 776CG, methylenetetrahydrofolate reductase (MTHFR) 677CT, and reduced folate carrier 1 (SLC19A1) 80GA.For each SNP, 5 primers were used: 2 uniprimers, 2 allele-specific primers, and 1 common primer for the opposite direction. The uniprimers (Amplifluor?; Chemicon International) were labeled with either fluorescein or sulforhodamine and were provided by Flowgen. The other primers (Sigma) were designed by use of Amplifluor Assay-Architect (Serological Corporations) and Molecular Beacon Design software (Premier Biosoft International). The nonlabeled primers are listed in Table? S1 in the online Data Supplement.Purified DNA was obtained from EDTA-blood samples by use of the Wizard DNA Purification Kit (Promega Corporation). The genotype distribution was investigated in 500 EDTA-blood samples from Norwegian blood donors. For these samples, we used boiled blood supernatant as the DNA source (9). Briefly, 50 μL of whole blood was mixed with 250 …