Journal
JOURNAL OF CLINICAL LABORATORY ANALYSIS
Volume 22, Issue 3, Pages 192-203Publisher
WILEY
DOI: 10.1002/jcla.20240
Keywords
fluorescence; single nucleotide polymorphism; genotype; RNase H; PCR
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Funding
- Intramural NIH HHS [ZIA HL006067-01] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [Z01HL001452] Funding Source: NIH RePORTER
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CataCleave probes are catalytically cleavable fluorescence probes having a chimeric deoxyribonucleic acid (DNA)ribonucleic acid (RNA)-DNA structure that can be used for real-time detection of single nucleotide polymorphisms (SNPs), insertions, and deletions. Fluorescent donor emission is normally quenched by Forster resonance energy transfer (FRET). Upon binding to the target, if the RNA/DNA hybrid is correctly base-paired, ribonuclease (RNase) H will cleave the RNA moiety and the probe fragments will dissociate. FRET is lost and the donor fluorescence signal is recovered. A single-base mismatch within the hybrid region causes probe cleavage to be significantly reduced. We designed CataCleave probes to detect SNPs located in the insulin-like growth factor 2 (IGF-2) gene and at position 702 within the NOD2/CARD15 gene. Probes were also designed to detect a six-basepair deletion in the amelogenin gene and a partially methylated target DNA. Discrimination between wild-type and SNP is demonstrated for both genes in homogeneous reactions under isothermal and temperature cycling conditions. These probes were also able to identify a multibase deletion and methylated DNA. Cleavage rates were proportional to target concentration. Probe length and position of fluorescent labels may also be modified for use in multiplexing high-throughput SNP assays. This represents a novel method for the detection of SNPs.
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