Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.
Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.
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DOI：
10.1093/nar/gkt684
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发表时间：
2013-10
影响因子：
14.9
通讯作者：
Yang S
中科院分区：
文献类型：
--
作者：
Fraley SI;Hardick J;Masek BJ;Athamanolap P;Rothman RE;Gaydos CA;Carroll KC;Wakefield T;Wang TH;Yang S
基因异质性样品中核酸的综合分析对于临床和基础研究应用是重要的。通用数字高分辨率解链（U-dHRM）是一种基础广泛的PCR诊断和分析技术的新方法，可以克服由于污染核酸导致的灵敏度差和由于引物或探针杂交对单核苷酸变异的不准确性导致的特异性差的问题。U-dHRM方法使用宽基引物或连接的衔接子序列来通用地扩增异质样品中的所有核酸分子，所述异质样品已经被分区，如在数字PCR中。广泛的测定优化使得能够通过基于算法的解链曲线形状和Tm与已知序列特异性解链曲线的数据库的匹配来进行直接序列鉴定。我们表明，单分子检测和单核苷酸灵敏度是可能的。通过检测与多种微生物血液感染相关的细菌和与宿主对感染的反应相关的microRNA（miRNA），证明了U-dHRM的可行性和实用性。使用宽基16 S rRNA基因引物的U-dHRM证明了细菌病原体的通用单细胞检测，即使在存在大量污染细菌的情况下;在异质混合物中使用通用适应的Lethal-7 miRNA的U-dHRM展示了该方法的单拷贝灵敏度和单核苷酸特异性。
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.
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DOI：
10.1038/nrg3198
发表时间：
2012-04-18
期刊：
Nature reviews. Genetics
影响因子：
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作者：
Pritchard CC;Cheng HH;Tewari M
通讯作者：
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影响因子：
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作者：
Lee, Inhan;Ajay, Subramanian S.;Chen, Haiming;Maruyama, Atsushi;Wang, Nulang;McInnis, Melvin G.;Athey, Brian D.
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影响因子：
3.7
作者：
Dube S;Qin J;Ramakrishnan R
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