Target DNA detection and quantitation on a single cell with single base resolution.
Target DNA detection and quantitation on a single cell with single base resolution.
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DOI：
10.1142/s2339547813500088
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发表时间：
2013-09
期刊：
影响因子：
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通讯作者：
Smolina IV
中科院分区：
文献类型：
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作者：
Konry T;Lerner A;Yarmush ML;Smolina IV
在本报告中，我们提出了一种以单碱基分辨率灵敏检测单细胞中短 DNA 位点的新方法。该方法结合了肽核酸 (PNA) 开启子作为标记探针，以及等温滚环扩增 (RCA) 和基于荧光的检测，所有这些都以细胞流形式进行。 Bis-PNA 提供单碱基分辨率，而 RCA 确保线性信号放大。我们应用这种方法使用流式细胞术系统检测癌细胞系中的癌病毒 DNA 插入片段。我们还展示了对微流体纳升液滴中单细胞内选定特征位点的定量检测。我们的结果显示了等温非变性条件下的单核苷酸多态性 (SNP) 辨别和拷贝数变异 (CNV) 检测。这种新方法非常适合许多需要在单细胞水平上进行单碱基分辨率超灵敏 DNA 表征的应用。
In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.