In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes
In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes
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DOI：
10.1038/nmeth723
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发表时间：
2004-12-01
期刊：
影响因子：
48
通讯作者：
Nilsson, M
中科院分区：
文献类型：
--
作者：
Larsson, C;Koch, J;Nilsson, M
需要研究单个分子的方法来揭示在分子群体水平上可能未被检测到的变异性、相互作用和机制。我们在这里描述了一系列集成的反应步骤，可以以出色的特异性检测单个核酸分子。寡核苷酸探针在与已经制备的靶序列杂交后被环化，使得可以从靶分子开始局部扩增反应。该过程导致锚定到靶分子的强的离散检测信号。我们使用的方法来观察分布，在人类细胞内和之间，个别正常和突变的线粒体基因组，不同的单核苷酸的位置。
Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after hybridization to target sequences that have been prepared so that localized amplification reactions can be initiated from the target molecules. The process results in strong, discrete detection signals anchored to the target molecules. We use the method to observe the distribution, within and among human cells, of individual normal and mutant mitochondrial genomes that differ at a single nucleotide position.