Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay
Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay
复制标题
基于单碱基延伸反应的表面增强拉曼光谱用于 DNA 甲基化测定
DOI：
10.1016/j.bios.2011.11.014
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发表时间：
2012-01-15
影响因子：
12.6
通讯作者：
Zhang, Chun-yang
中科院分区：
文献类型：
--
作者：
Hu, Juan;Zhang, Chun-yang
DNA甲基化是遗传疾病、癌症进展和药物基因组学的关键诊断标记。目前，DNA甲基化检测技术已发展到多种程度，但大多数方法费时费力。本文基于单碱基延伸反应和表面增强拉曼光谱（Sers）技术，建立了一种简便、灵敏的DNA甲基化检测方法。在甲基化DNA存在下，金纳米粒子修饰的捕获探针可以通过单碱基延伸反应与菁5-脱氧核苷三磷酸（cy 5-dGTP）偶联，进一步加入金纳米粒子以增加局部电磁场后产生高Sers信号。而在未甲基化DNA存在下，金纳米粒子修饰的捕获探针由于存在错配碱基而不能与cy 5-dGTP偶联，没有观察到Sers信号。这种基于单碱基延伸反应Sers可以检测甲基化DNA，检测限为3 pM，甚至可以从混合物中区分低至1%的肿瘤抑制基因CDKN 2/p16/MTS 1（p16）甲基化水平。值得注意的是，与报道的基于金纳米颗粒的比色测定相比，该测定的灵敏度提高了5个数量级，并且与基于微阵列的甲基化敏感性单核苷酸引物延伸测定（Ms-SNuPE）相比提高了2个数量级。该方法有望进一步应用于肿瘤相关基因甲基化状态的检测。(C)2011 Elsevier B. V.保留所有权利。
DNA methylation is a key diagnostic maker for genetic disease, cancer progression and pharmcogenomics. So far various techniques have been developed for DNA methylation assay, but most of them are laborious and time-consuming. Here we develop a simple and highly sensitive DNA methylation assay based on single base extension reaction and surface enhanced Raman spectroscopy (SERS). In the presence of methylated DNA, gold nanoparticle-modified capture probe can couple with a cyanine 5-deoxyribonucleoside triphosphate (cy5-dGTP) through single base extension reaction, and generates a high SERS signal after further addition of gold nanoparticles to increase the local electromagnetic field. While in the presence of unmethylated DNA, gold nanoparticle-modified capture probe cannot couple with cy5-dGTP due to the presence of a mismatch base, and no SERS signal is observed. This single base extension reaction-based SERS can determine methylated DNA with a detection limit of 3 pM, and can even distinguish as low as 1% methylation level in tumor suppressor gene CDKN2/p16/MTS1 (p16) from the mixtures. Notably, the sensitivity of this assay has improved by 5 orders of magnitude as compared to reported gold nanoparticle-based colorimetric assay, and by 2 orders of magnitude as compared to microarray-based methylation-sensitive single nucleotide primer extension assay (Ms-SNuPE). This method might be further applied to detect the methylation status in tumor-linked genes for cancer diagnosis. (C) 2011 Elsevier B.V. All rights reserved.