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.[Supplemental material is available online at . The sequence data from this study have been submitted to GenBank/EMBL/Date Bank under accession nos. . The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N.K. Yankovsky, A.V. Polyakov, and G.N. Rudenskaya.]
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机译:我们已经从堪察加蟹中鉴定出一种新型核酸酶,称为双链体特异性核酸酶(DSN)。与单链DNA相比,DSN在切割双链DNA和DNA-RNA杂交双链体中的DNA方面表现出强烈的偏好。而且,这种酶对短的,完美匹配的DNA双链体的切割速率基本上高于相同长度的非完美匹配的双链体的切割速率。因此,DSN可以区分DNA中的一个核苷酸变异。基于这种独特的特性,我们开发了一种用于单核苷酸多态性(SNP)检测的新颖检测方法,称为“双链体特异性核酸酶偏爱”(DSNP)。在这种创新的检测方法中,将扩增包含SNP位点的DNA区域,并将PCR产物与信号探针(FRET标记的短序列特异性寡核苷酸)和DSN混合。在孵育过程中,DSN仅切割DNA模板和信号探针之间完全匹配的双链体,以产生序列特异性荧光。 FRET标记的信号探针与DSN的特异性结合使用,提供了一种检测SNP的简单有效的方法。我们已采用DSNP分析法对纯合和杂合基因组DNA上的甲基四氢叶酸还原酶,凝血酶原和p53基因中的SNP进行分型。这项研究的序列数据已提交给GenBank / EMBL / Date Bank,登录号为。 。下列人员请按照论文中的指示提供试剂,样品或未发表的信息:扬科夫斯基波利亚科夫和G.N. Rudenskaya。]
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