SNP analysis using CataCleave probes

John J. Harvey, Steven R. Brant, Jay R. Knutson, Myun K. Han

Research output: Contribution to journalArticlepeer-review

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 languageEnglish (US)
Pages (from-to)192-203
Number of pages12
JournalJournal of Clinical Laboratory Analysis
Volume22
Issue number3
DOIs
StatePublished - 2008
Externally publishedYes

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)

Fingerprint

Dive into the research topics of 'SNP analysis using CataCleave probes'. Together they form a unique fingerprint.

Cite this