title: 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.
authors.affiliation: Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 117871 Moscow, Russia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Shagin
authors.forename: Denis V
authors.initials: DV
authors.lastname: Rebrikov
authors.forename: Valery B
authors.initials: VB
authors.lastname: Kozhemyako
authors.forename: Ilia M
authors.initials: IM
authors.lastname: Altshuler
authors.forename: Alex S
authors.initials: AS
authors.lastname: Shcheglov
authors.forename: Pavel A
authors.initials: PA
authors.lastname: Zhulidov
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Bogdanova
authors.forename: Dmitry B
authors.initials: DB
authors.lastname: Staroverov
authors.forename: Valery A
authors.initials: VA
authors.lastname: Rasskazov
authors.forename: Sergey
authors.initials: S
authors.lastname: Lukyanov
mesh_terms: D000818:Animals; D033381:Anomura / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004720:Endonucleases / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D004720:Endonucleases