Unlabeled Hairpin DNA Probe for Electrochemical Detection of Single-Nucleotide Mismatches Based on MutS-DNA Interactions
Unlabeled Hairpin DNA Probe for Electrochemical Detection of Single-Nucleotide Mismatches Based on MutS-DNA Interactions
复制标题
用于基于 MutS-DNA 相互作用的单核苷酸错配电化学检测的未标记发夹 DNA 探针
DOI：
10.1021/ac901371n
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发表时间：
2009-10-15
影响因子：
7.4
通讯作者：
Kraatz, Heinz-Bernhard
中科院分区：
文献类型：
--
作者：
Gong, He;Zhong, Tianying;Kraatz, Heinz-Bernhard
该论文描述了一种用于检测单核苷酸错配的无标记测定法，其中利用未标记的发夹 DNA 探针和 MutS 蛋白缀合物（His6-MutS-连接肽-链霉亲和素结合肽 (HMLS)）通过电化学阻抗谱 (EIS) 检测错配。我们针对八个单核苷酸错配演示了这种方法。目标链与发夹 DNA 探针杂交后，茎环结构打开，形成双链 DNA。在含有单核苷酸错配的双链体中，错配存在于溶剂暴露侧，从而实现更有效的 HMLS 识别和结合。结合事件通过 EIS 进行评估，并借助兰德尔斯等效电路进行分析。蛋白质与双链 DNA 结合之前和之后电荷转移电阻 Delta R-CT 的差异允许明确检测所有八个单核苷酸错配。 Delta R-CT 可以用低至 100 pM 的目标链浓度来区分 C-A 错配。
The paper described a label-free assay for the detection of single-nucleotide mismatches in which an unlabeled hairpin DNA probe and a MutS protein conjugate (His6-MutS-linker peptide-streptavidin binding peptide (HMLS)) are exploited for the detection of mismatches by electrochemical impedance spectroscopy (EIS). We demonstrate this method for eight single-nucleotide mismatches. Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened forming a duplex DNA. In duplexes containing a single nucleotide mismatch, the mismatch is present at the solvent exposed side, enabling more effective HMLS recognition and binding. The binding event is evaluated by EIS and analyzed with the help of Randles' equivalent circuits. The differences in the charge transfer resistance Delta R-CT before and after protein binding to the duplex DNA allows the unequivocal detection of all eight single-nucleotide mismatches. Delta R-CT allows the discrimination of a C-A mismatch with the concentration of the target strand as low as 100 pM.