A novel single nucleotide polymorphism detection of a double-stranded DNA target by a ribonucleotide-carrying molecular beacon and thermostable RNase HII
A novel single nucleotide polymorphism detection of a double-stranded DNA target by a ribonucleotide-carrying molecular beacon and thermostable RNase HII
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通过携带核糖核苷酸的分子信标和热稳定性 RNase HII 对双链 DNA 靶标进行新型单核苷酸多态性检测
DOI：
10.1016/j.ab.2009.10.042
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发表时间：
2010-03-01
影响因子：
2.9
通讯作者：
Liu, Jian-Hua
中科院分区：
文献类型：
--
作者：
Liu, Xi-Peng;Hou, Jing-Li;Liu, Jian-Hua
单核苷酸多态性（SNP）是最丰富的遗传变异形式。 SNP 是将序列变异与表型变化联系起来的重要标记。由于 SNP 在生命和医学科学中的重要性，人们投入了大量的精力来开发准确、快速且经济高效的 SNP 分析技术。在本文中，我们描述了一种新的 SNP 基因分型方法，该方法基于由嗜热栖热菌 RNase Hill (TthRNase HII) 切割含有 SNP 位点特异性嵌合 DNA-rN(1)-DNA 分子信标 (cMB) 的 DNA 异源双链体产生的差异荧光发射。我们构建了 cMB 的环序列，其中在中心位点包含单个 SNP 特异性核糖核苷酸。当 cMB 探针与目标双链 DNA (dsDNA) 杂交时，cMB/DNA 双链体的完美匹配可实现 TthRNase HII 的有效切割，而 SNP 导致的双链体错配会大大降低效率。切割效率通过信标的荧光发射增量差来测量。我们证明，相对于传统 DNA 测序，可以正确确定 10 个个体在一系列人类白细胞抗原 (HLA) 的 12 个 SNP 位点上的基因型。这种基于 TthRNase HII 的新颖方法为简单、准确的 SNP 分析提供了一个平台。 (C) 2009 Elsevier Inc. 保留所有权利。
Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation. SNPs are important markers that link sequence variations to phenotypic changes. Because of the importance of SNPs in the life and medical sciences, a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. In this article, we describe a novel method for SNP genotyping based on differential fluorescence emission due to cleavage by Thermus thermophilus RNase Hill (TthRNase HII) of DNA heteroduplexes containing an SNP site-specific chimeric DNA-rN(1)-DNA molecular beacon (cMB). We constructed a loop sequence for a cMB that contains a single SNP-specific ribonucleotide at the central site. When the cMB probe is hybridized to a target double-stranded DNA (dsDNA), a perfect match of the cMB/DNA duplex permits efficient cleavage with TthRNase HII, whereas a mismatch in the duplex due to an SNP greatly reduces efficiency. Cleavage efficiency is measured by the incremental difference of fluorescence emission of the beacon. We show that the genotypes of 10 individuals at 12 SNP sites across a series of human leukocyte antigen (HLA) can be determined correctly with respect to conventional DNA sequencing. This novel TthRNase HII-based method offers a platform for easy and accurate SNP analysis. (C) 2009 Elsevier Inc. All rights reserved.