Detection and Quantitation of DNA Mutations Based on Gold Magnetic Nanocomposites and Single Primer Extension Assay
Detection and Quantitation of DNA Mutations Based on Gold Magnetic Nanocomposites and Single Primer Extension Assay
复制标题
基于金磁性纳米复合材料和单引物延伸分析的 DNA 突变检测和定量
DOI：
10.1166/nnl.2019.2878
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发表时间：
2019-02
影响因子：
--
通讯作者：
Zeng Xin
中科院分区：
文献类型：
--
作者：
Liu Ming;Yang Haowen;Jiang Hongrong;He Nongyue;Zeng Xin
利用 Fe3O4 @SiO2 @Au（金涂层磁性纳米复合材料，GMNP）和单引物延伸测定（SPEA）开发了一种 DNA 突变检测和定量的新方法。在该方案中，单个荧光标记引物与含有野生型突变位点的靶DNA特异性延伸，随后与链霉亲和素修饰的GMNP捕获的生物素标记探针杂交，然后检测荧光信号。为了测试该方法的可行性，我们尝试在最佳条件下对已知突变体 L858R 和 T790M 的表皮生长因子受体 (EGFR) 合成的 DNA 片段进行定量。通过计算目标基因和参考基因之间的荧光强度比，对合成的目标DNA进行相对定量。由于使用具有低荧光背景的 GMNP，结果显示出出色的读数，并且具有良好的线性相关性，目标浓度范围为 1.25 至 20 nM。这种方法有潜力开发一种简单、快速、特殊且低成本的技术，用于对真实样本进行 DNA 突变定量。
A new method for DNA mutations detection and quantification was developed by using Fe3O4 @SiO2 @Au (Gold-coated magnetic nanocomposites, GMNPs) and single primer extension assay (SPEA). In this scheme, the single fluorescent-labeled primers were extended specifically with target DNA containing mutation sites from wild types, and subsequently hybridized with biotinlabeled probes captured by streptavidin-modified GMNPs, followed by detection of fluorescence signals. To test the feasibility of the method, we attempted to quantify the synthesized DNA segments from epidermal growth factor receptor (EGFR) with known mutant L858R and T790M under optimum conditions. The synthesized target DNA was relatively quantified by calculating the ratio of fluorescence intensity between the target and reference genes. Results exhibited an excellent readout due to the use of GMNPs with low fluorescence background, and a fine linear correlation, with target concentration ranging from 1.25 to 20 nM. This approach has potential in development of a simple, rapid, special and low-cost technique for DNA mutation quantification from real samples.