title: Closed-tube SNP genotyping without labeled probes/a comparison between unlabeled probe and amplicon melting.
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 13910 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).
authors.affiliation: Institute for Clinical and Experimental Pathology, ARUP, Salt Lake City, UT 84108-1221, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Liew
authors.forename: Michael
authors.initials: M
authors.lastname: Seipp
authors.forename: Jacob
authors.initials: J
authors.lastname: Durtschi
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Margraf
authors.forename: Shale
authors.initials: S
authors.lastname: Dames
authors.forename: Maria
authors.initials: M
authors.lastname: Erali
authors.forename: Karl
authors.initials: K
authors.lastname: Voelkerding
authors.forename: Carl
authors.initials: C
authors.lastname: Wittwer
mesh_terms: D004247:DNA / Q000097:blood / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D006801:Humans; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D044366:Transition Temperature
chemical_list: D015345:Oligonucleotide Probes; D004247:DNA