A Novel Method for SNP Detection Using a New Duplex-Specific Nuclease From Crab Hepatopancreas

Abstract

We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed "duplex-specific nuclease preference" (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA.

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

“…To further investigate the role of DDX1 in RNA clearance at DSBs, we designed an assay that uses duplexspecific nuclease (DSN) and RNase H to distinguish DNA-RNA from other nucleic acid structures. DSN specifically degrades DNA from DNA-DNA and DNA-RNA duplexes but has no effect on ssDNA or any form of RNA (58,59). DSN has been adapted to a variety of applications, including quantitative measurement of ssDNA overhangs in human telomeres (59).…”

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

“…DSN is an attractive candidate for the latter, since it preferentially cleaves DNA in a DNA-RNAduplex, leaving ssRNA and dsRNA virtually unchanged [15]. DSN acts as an endonuclease on DNA, cleaving within the phosphate backbone [16], although the direction of digestion is unknown. The enzyme's preference for .…”

“…To minimize these limitations, isothermal RNA amplification techniques have been developed [7][8][9][10][11][12][13][14], but these still remain dependent on nucleic acid replication and are therefore hindered by polymerase speed and fidelity. Recent RNA detection methods that have employed duplex specific nuclease (DSN) isolated from the Paralithodes camtschaticus crab [15,16], remain limited to microRNA [17] and are thus unsuitable for longer RNA templates like virus genomic or mRNA targets. DSN is an attractive candidate for the latter, since it preferentially cleaves DNA in a DNA-RNAduplex, leaving ssRNA and dsRNA virtually unchanged [15].…”

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

“…Various approaches have been reported in the literature for analyzing single-nucleotide polymorphisms. All of the approaches are basically grouped into four categories including allele-specific hybrization (18), allele-specific extension (13), allele-specific ligation (8) and enzyme cleavage-based methods (7,10,12,16,21,22,23).…”