A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas.
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.
Shagin
Dmitry A
DA
Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 117871 Moscow, Russia.
Rebrikov
Denis V
DV
Kozhemyako
Valery B
VB
Altshuler
Ilia M
IM
Shcheglov
Alex S
AS
Zhulidov
Pavel A
PA
Bogdanova
Ekaterina A
EA
Staroverov
Dmitry B
DB
Rasskazov
Valery A
VA
Lukyanov
Sergey
S
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
D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D004720:Endonucleases