Multiplex detection of single-nucleotide variations using molecular beacons
Multiplex detection of single-nucleotide variations using molecular beacons
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DOI：
10.1016/s1050-3862(98)00018-7
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发表时间：
1999-02-01
期刊：
影响因子：
--
通讯作者：
Tyagi, S
中科院分区：
文献类型：
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作者：
Marras, SAE;Kramer, FR;Tyagi, S
我们证明，在DNA序列中的单核苷酸差异可以检测到在均相检测使用分子信标。在该方法中，在聚合酶链式反应中扩增序列变异位点周围的区域，并且通过观察四种不同颜色的分子信标中的哪一种与扩增产物结合来确定变体核苷酸的身份。每个分子信标与靶序列的一个变体完全互补，并且每个分子信标用不同的荧光团标记。为了证明这些测定的特异性，我们制备了四种模板DNA，它们仅在一个位置处的核苷酸的同一性上彼此不同。制备了四个扩增反应，每个反应包含所有四种分子信标，但每个反应仅用四种模板DNA中的一种启动。结果表明，在每个反应中，从与扩增的DNA完全互补的分子信标引起荧光响应，但从探针序列与靶序列不匹配的三个分子信标不引起荧光响应。在扩增过程中，每个试管中出现的荧光颜色表明变异位点存在哪种核苷酸。这些结果证明了分子信标的非凡特异性。此外，结果还说明了用不同颜色的荧光团标记分子信标的能力如何使简单的多重测定能够进行遗传分析。(C)1999 Elsevier Science B.V.保留所有权利。
We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surroundings the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the Variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly complementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, we prepared four template DNAs that only differed from one another by the identity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated with only one of the four template DNAs. The results show that in each reaction a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecular beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amplification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beacons. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assays to be carried out for genetic analysis. (C) 1999 Elsevier Science B.V. All rights reserved.