Mutation detection and single-molecule counting using isothermal rolling-circle amplification
Mutation detection and single-molecule counting using isothermal rolling-circle amplification
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DOI：
10.1038/898
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发表时间：
1998-07-01
期刊：
影响因子：
30.8
通讯作者：
Ward, DC
中科院分区：
文献类型：
--
作者：
Lizardi, PM;Huang, XH;Ward, DC
DNA聚合酶驱动的滚环扩增（RCA）可以在等温条件下以线性或几何动力学复制环化的寡核苷酸探针。在存在两个引物的情况下，一个引物与DNA的+链杂交，另一个引物与DNA的-链杂交，DNA链置换的复杂模式在90分钟内产生每个环的10（9）个或更多个拷贝，使得能够检测人类基因组DNA中的点突变。使用单个引物，RCA在几分钟内产生数百个串联连接的共价闭合环拷贝。如果与基质结合，DNA产物保持结合在合成位点，在那里它可以被标记、浓缩和成像为点光源。共价结合在玻璃表面上的线性寡核苷酸探针可以产生RCA信号，其颜色指示靶标的结合状态，这取决于特异性靶向连接事件的结果。由于RCA允许使用颜色代码对数百万个单独的探针分子进行计数和分类，因此它特别适合于分析罕见的体细胞突变。RCA还显示出用于检测细胞学制备物中与单拷贝基因结合的挂锁探针的前景。
Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allel status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.