Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye

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Abstract
Background: Homogeneous PCR methods for genotyping usually require fluorescently labeled oligonucleotide probes. Amplicon melting with the DNA dye LCGreen™ I was recently introduced as a closed-tube method of genotyping that does not require probes or real-time PCR. However, some single-nucleotide polymorphisms (SNPs) could not be completely genotyped without addition of a known genotype, and high-resolution melting techniques were necessary. Methods: A 3′-blocked, unlabeled oligonucleotide probe and the saturating dye, LCGreen I, were added to standard PCR reagents before amplification. After PCR, the samples were melted at 0.1–0.3 °C/s in high-resolution (HR-1™), high-throughput (LightTyper™), and rapid-cycle, real-time (LightCycler®) instruments, and fluorescence melting curves were recorded. Results: Derivative melting curves of the probe–target duplexes were characteristic of the genotype under the probe. With synthetic plasmid templates, all SNP base combinations could be genotyped. For human genomic DNA, the technique was demonstrated with mutations associated with cystic fibrosis, including SNPs (G542X, I506V, and F508C) and 3-bp deletions (F508del and I507del). Conclusions: Genotyping of SNPs and small deletions by melting analysis of an unlabeled probe in the presence of LCGreen I is simple and rapid. Only three unlabeled oligonucleotides (two primers and one probe), a saturating DNA dye, PCR, and a melting instrument are required. The method is closed-tube, does not require fluorescently labeled probes or real-time PCR, and can be completed in <10 min on any instrument capable of monitoring melting curves by fluorescence.