SNP analysis using CataCleave probes.
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.
Harvey
John J
JJ
Excimus Biotech, Inc., 8510 Corridor Road, Savage, MD 20763, USA. harvey@excimus.com
Brant
Steven R
SR
Knutson
Jay R
JR
Han
Myun K
MK
D015342:DNA Probes; D005838:Genotype; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015347:RNA Probes; D016914:Ribonuclease H / Q000378:metabolism; D017422:Sequence Analysis, DNA
D015342:DNA Probes; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D009841:Oligonucleotides; D015347:RNA Probes; D007335:Insulin-Like Growth Factor II; D016914:Ribonuclease H