Digital Polymerase Chain Reaction Paired with High-Speed Atomic Force Microscopy for Quantitation and Length Analysis of DNA Length Polymorphisms
Digital Polymerase Chain Reaction Paired with High-Speed Atomic Force Microscopy for Quantitation and Length Analysis of DNA Length Polymorphisms
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DOI：
10.1021/acsnano.0c05897
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发表时间：
2020-11-24
期刊：
影响因子：
17.1
通讯作者：
Reed, Jason
中科院分区：
文献类型：
--
作者：
Koebley, Sean R.;Mikheikin, Andrey;Reed, Jason
DNA长度多态性存在于许多严重疾病中，对其长度和丰度的评估通常对于准确诊断至关重要。然而，在大多数野生型背景下测量它们的长度和频率，由于它们的可变和重复性质，仍然具有挑战性。为了克服这些障碍，我们结合了两种强大的技术，数字聚合酶链反应（dPCR）和高速原子力显微镜（HSAFM），创造了一种简单，快速，灵活的方法来量化DNA长度多态性的大小和比例。在我们的方法中，来自每个dPCR分区的单个扩增子被直接成像和大小确定。我们关注的是位于FLT 3基因内的内部串联重复（ITDs），这与急性髓细胞白血病相关，通常表明预后不良。在对来自含有FLT 3-ITD的细胞系和临床样品的超过150万个HSAFM成像扩增子的分析中，dPCR-HSAFM返回了预期的变体长度和变体等位基因频率，低至5%的变体样品。dPCR-HSAFM作为一种高通量的单分子分辨方法，代表了HSAFM分析的一个进步，是诊断长度多态性的有力工具。
DNA length polymorphisms are found in many serious diseases, and assessment of their length and abundance is often critical for accurate diagnosis. However, measuring their length and frequency in a mostly wild-type background, as occurs in many situations, remains challenging due to their variable and repetitive nature. To overcome these hurdles, we combined two powerful techniques, digital polymerase chain reaction (dPCR) and high-speed atomic force microscopy (HSAFM), to create a simple, rapid, and flexible method for quantifying both the size and proportion of DNA length polymorphisms. In our approach, individual amplicons from each dPCR partition are imaged and sized directly. We focused on internal tandem duplications (ITDs) located within the FLT3 gene, which are associated with acute myeloid leukemia and often indicative of a poor prognosis. In an analysis of over 1.5 million HSAFM-imaged amplicons from cell line and clinical samples containing FLT3-ITDs, dPCR-HSAFM returned the expected variant length and variant allele frequency, down to 5% variant samples. As a high-throughput method with single-molecule resolution, dPCR-HSAFM thus represents an advance in HSAFM analysis and a powerful tool for the diagnosis of length polymorphisms.