Received on March 22, 2008
Accepted on July 10, 2008

Molecular Diagnostics and Genetics

Snapback Primer Genotyping with Saturating DNA Dye and Melting Analysis

¹ Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah
² Idaho Technology, Salt Lake City, Utah
³ ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah
⁴ Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah and Idaho Technology, Salt Lake City, Utah and ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah

^* To whom correspondence should be addressed. E-mail: carl.wittwer{at}path.utah.edu.

BACKGROUND: DNA hairpins have been used in molecular analysis of PCR products as self-probing amplicons. Either physical separation or fluorescent oligonucleotides with covalent modifications were previously necessary.

METHODS: We performed asymmetric PCR for 40–45 cycles in the presence of the saturating DNA dye, LCGreen Plus, with 1 primer including a 5' tail complementary to its extension product, but without any special covalent modifications. Samples were amplified either on a carousel LightCycler for speed or on a 96/384 block cycler for throughput. In addition to full-length amplicon duplexes, single-stranded hairpins were formed by the primer tail "snapping back" and hybridizing to its extension product. High-resolution melting was performed on a HR-1 (for capillaries) or a LightScanner (for plates).

RESULTS: PCR products amplified with a snapback primer showed both hairpin melting at lower temperature and full-length amplicon melting at higher temperature. The hairpin melting temperature was linearly related to the stem length (6–28 bp) and inversely related to the log of the loop size (17–135 bases). We easily genotyped heterozygous and homozygous variants within the stem, and 100 blinded clinical samples previously typed for F5 1691G>A (Leiden) were completely concordant by snapback genotyping. We distinguished 7 genotypes in 2 regions of CFTR exon 10 with symmetric PCR using 2 snapback primers followed by product dilution to favor intramolecular hybridization.

CONCLUSIONS: Snapback primer genotyping with saturating dyes provides the specificity of a probe with only 2 primers that are free of special covalent labels in a closed-tube system.