Facile profiling of molecular heterogeneity by microfluidic digital melt.
Facile profiling of molecular heterogeneity by microfluidic digital melt.
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DOI：
10.1126/sciadv.aat6459
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发表时间：
2018-09
期刊：
影响因子：
13.6
通讯作者：
Wang TH
中科院分区：
文献类型：
--
作者：
O'Keefe CM;Pisanic TR 2nd;Zec H;Overman MJ;Herman JG;Wang TH
数字微流控技术能够分析肿瘤或细胞内的遗传和表观遗传差异。这项工作提出了一个数字微流控平台，称为HYPER-Melt（高密度分析和枚举的熔化）的高度并行化的复制复制的DNA分子分析。HYPER-Melt提供了一种简便的方法，通过在微芯片阵列中进行数字化来检测和评估数千个单个DNA分子的序列变异，通过检测由于序列依赖性变性引起的HRM荧光变化来扩增和询问单个模板分子。作为一个模型应用，HYPER-Melt在这里用于检测和评估经典肿瘤抑制基因启动子内DNA甲基化的分子间异质性。该平台的能力通过混合表观等位基因的系列稀释进行验证，证明检测限低至经典肿瘤抑制基因CDKN 2A（p14 ARF）的200万个未甲基化模板（0.00005%）中的1个甲基化变体。该平台的临床潜力是使用NDRG 4的数字检测来证明的，NDRG 4是一种在结直肠癌中通常甲基化的肿瘤抑制基因，在健康和结直肠癌患者的液体活检中。总的来说，该平台提供了信息的深度，使用的简单性和单分子灵敏度，这对于快速评估分子间变异有助于遗传和表观遗传异质性，从而在胚胎发生，致癌和罕见生物标志物检测中具有挑战性的应用是必要的。
Digital microfluidic technology enables profiling of genetic and epigenetic differences within tumors or cells. This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene, CDKN2A (p14ARF). The clinical potential of the platform is demonstrated using a digital assay for NDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.
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DOI：
10.1073/pnas.93.18.9821
发表时间：
1996-09-03
影响因子：
11.1
作者：
Herman, JG;Graff, JR;Baylin, SB
通讯作者：
Baylin, SB
影响因子：
3.6
作者：
Abou Tayoun, Ahmad N.;Burchard, Paul R.;Tsongalis, Gregory J.
通讯作者：
Tsongalis, Gregory J.
DOI：
10.1073/pnas.0507904102
发表时间：
2005-11-08
影响因子：
11.1
作者：
Diehl, F;Li, M;Vogelstein, B
通讯作者：
Vogelstein, B
DOI：
10.1016/j.cca.2013.07.023
发表时间：
2013-10-21
期刊：
Clinica chimica acta; international journal of clinical chemistry
影响因子：
--
作者：
Keeley B;Stark A;Pisanic TR 2nd;Kwak R;Zhang Y;Wrangle J;Baylin S;Herman J;Ahuja N;Brock MV;Wang TH
通讯作者：
Wang TH
影响因子：
3.4
作者：
Chen, Y;Müller, JD;Gratton, E
通讯作者：
Gratton, E