Abstract
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 Förster 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.
Original language | English (US) |
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Pages (from-to) | 192-203 |
Number of pages | 12 |
Journal | Journal of Clinical Laboratory Analysis |
Volume | 22 |
Issue number | 3 |
DOIs | |
State | Published - 2008 |
Externally published | Yes |
Keywords
- Fluorescence
- Genotype
- PCR
- RNase H
- Single nucleotide polymorphism
ASJC Scopus subject areas
- Immunology and Allergy
- Hematology
- Public Health, Environmental and Occupational Health
- Clinical Biochemistry
- Medical Laboratory Technology
- Biochemistry, medical
- Microbiology (medical)