Closed-Tube SNP Genotyping Without Labeled Probes: A Comparison Between Unlabeled Probe and Amplicon Melting

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Abstract

Two methods for closed-tube single nucleotide polymorphism (SNP) genotyping without labeled probes have become available: unlabeled probe and amplicon melting. Unlabeled probe and amplicon melting assays were compared using 5 SNPs: human platelet antigens 1, 2, 5, and 15 and a C>T variant located 13,910 base pairs (bp) upstream of the lactase gene. LCGreen Plus (Idaho Technology, Salt Lake City, UT) was used as the saturating DNA dye. Unlabeled probe data were readily interpretable and accurate for all amplicon lengths tested. Five targets that ranged in size from 42 to 72 bp were well resolved by amplicon melting on the LightScanner (Idaho Technology) or LightTyper (Roche, Indianapolis, IN) with no errors in genotyping. However, when larger amplicons (206 bp) were used and analyzed on lower resolution instruments (LightTyper and I-Cycler, Bio-Rad, Hercules, CA), the accuracy of amplicon genotyping was only 73% to 77%. When 2 temperature standards were used to bracket the amplicon of interest, the accuracy of amplicon genotyping of SNPs was increased to 100% (LightTyper) and 88% (I-Cycler).

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