Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface plasmon resonance biosensors
Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface plasmon resonance biosensors
复制标题
表面等离子共振生物传感器检测DNA甲基化中寡核苷酸和蛋白质的双重识别
DOI：
10.1016/j.bios.2010.08.007
复制
发表时间：
2010-10-15
影响因子：
12.6
通讯作者：
Lu, Shan
中科院分区：
文献类型：
--
作者：
Pan, Shiyang;Xu, Jian;Lu, Shan
DNA甲基化在维持细胞功能中起着至关重要的作用。越来越多的人类疾病被发现与异常DNA甲基化有关，特别是癌症。然而，目前用于DNA甲基化检测的技术复杂且耗时。将已被充分研究的肿瘤抑制基因--腺瘤性息肉病大肠杆菌（APC）基因的启动子用作检测靶DNA序列。通过寡核苷酸探针杂交和特异性蛋白结合实现了双识别机制。首先，互补的目标DNA被捕获的探针固定到表面等离子体共振（SPR）传感器芯片。然后，将重组甲基-CpG结合结构域（MBD）蛋白通过表面以识别并结合甲基化的CpG位点。结合导致折射率增加，并产生可检测的光学信号。用这种方法可以很容易地检测到5皮摩尔的甲基化APC启动子DNA。整个检测过程可在1 h内完成。本工作代表了第一个基于SPR的生物传感器技术，它实现了简单和特异性的DNA甲基化检测，避免了复杂的亚硫酸氢盐处理和甲基化敏感的限制性消化。它将提高我们特异性和快速检测DNA甲基化的能力，促进我们对DNA甲基化在基因调控和疾病中作用的理解。(C)出版社：Elsevier B. V.
DNA methylation plays an essential role in maintenance of cellular function. A growing number of human diseases have been found to be associated with aberrant DNA methylation, especially cancer. However, current technologies used in DNA methylation detection are complicated and time consuming. A promotor of the Adenomatous polyposis coli (APC) gene, a well-studied tumor suppressor gene, was used as the detection target DNA sequence. The double recognition mechanism was realized with oligonucleotide probe hybridization and specific protein binding. First, complementary target DNA was captured by the probe immobilized onto a surface plasmon resonance (SPR) sensor chip. Then, the recombinant methyl-CpG binding domain (MBD) protein was passed over the surface to recognize and bind to methylated CpG sites. Binding resulted in an increase in the refractive index, and a detectable optical signal was generated. Five picomoles of methylated APC promotor DNA could be easily detected with this method. The entire detection could be completed within 1 h. This work represents the first SPR based biosensor technology, which achieves simple and specific DNA methylation detection and avoids complicated bisulfite treatment and methylation-sensitive restriction digestion. It will improve our ability to detect DNA methylation specifically and rapidly, and promote our understanding of the role of DNA methylation in gene regulation and diseases. (C) 2010 Published by Elsevier B.V.