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首页> 美国卫生研究院文献>Nucleic Acids Research >Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli
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Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli

机译：基于竞争性结合的光学DNA定位技术可快速鉴定细菌-多配体转移矩阵理论和在大肠杆菌中的实验应用

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摘要

We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50–160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.
机译：我们证明了一个单一的DNA分子光学作图分析能够解决其他菌株的特定大肠杆菌菌株。该测定基于荧光染料YOYO-1和AT特异性抗生素奈特罗普星的竞争性结合。通过在纳米流体通道中拉伸DNA分子可以看到光学图。我们优化实验条件以获得包含尽可能多信息的可再现条形码。我们实现了一种多配体转移矩阵方法，用于从已知DNA序列计算理论条形码。我们的方法将先前的理论方法扩展为将两种类型的配体竞争性结合到许多类型的配体，并引入了一种递归方法，该方法允许使用标准计算机浮点格式来计算长条形码。特定大肠杆菌菌株（CCUG 10979）的鉴定是基于使用发达理论将50–160个碱基对实验DNA片段映射到理论基因组上的。我们的识别协议引入了两种理论构造：最佳实验理论匹配的P值和信息得分阈值。所开发的方法提供了一种新颖的光学作图工具箱，用于鉴定细菌种类和菌株。该协议不需要细菌培养或DNA扩增，从而可以超快速地鉴定细菌病原体。

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