Label free detection of 5'hydroxymethylcytosine within CpG islands using optical sensors.
Label free detection of 5'hydroxymethylcytosine within CpG islands using optical sensors.
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DOI：
10.1016/j.bios.2014.10.041
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发表时间：
2015-03-15
影响因子：
12.6
通讯作者：
Armani, Andrea M.
中科院分区：
文献类型：
--
作者：
Hawk, Rasheeda M.;Armani, Andrea M.
在基因变异与不同患病概率之间的关联方面，已经进行了重要的研究。最近，很明显，其他DNA修饰，如在胞嘧啶中添加甲基或羟甲基，也可以发挥作用。虽然这些修改不会改变序列，但它们会对功能产生负面影响。因此，能够读取遗传密码并识别这些修饰是至关重要的。目前，羟甲基胞嘧啶(5‘HMC)及其两个密切相关的变异体-胞嘧啶(C)和5’甲基胞嘧啶(5‘MC)的检测依赖于核苷酸修饰步骤的组合，随后是聚合酶链式反应和基因测序。然而，这种方法并不理想，因为聚合酶链式反应过程中固有的转录错误可能被误解为相对C：5‘MC：5’HMC浓度的波动。因此，一种不依赖于PCR或核苷酸修饰的替代方法是可取的。一种方法是基于无标签光学谐振腔传感器。在本工作中，环形共振腔传感器被抗体功能化，以实现无标记检测和实时区分C，5‘MC和5’HMC，而不需要PCR。具体地说，使用环氧化物化学将5‘HMC抗体共价连接到空洞表面。随后，为了彻底表征传感器平台，在从PM到NM的浓度范围内执行C、5‘MC和5’HMC的检测。在低(Pm)浓度下，羟甲基化胞嘧啶产生的信号明显大于结构相似的表观遗传标记；从而证明了该平台的适用性。
Significant research has been invested in correlating genetic variations with different disease probabilities. Recently, it has become apparent that other DNA modifications, such as the addition of a methyl or hydroxymethyl group to cytosine, can also play a role. While these modifications do not change the sequence, they can negatively impact the function. Therefore, it is critical to be able to both read the genetic code and identify these modifications. Currently, the detection of hydroxymethylated cytosine (5′hmC) and the two closely related variants, cytosine (C) and 5′methylcytosine (5′mC), relies on a combination of nucleotide modification steps, followed by PCR and gene sequencing. However, this approach is not ideal because transcription errors which are inherent to the PCR process can be misinterpreted as fluctuations in the relative C:5′mC:5′hmC concentrations. As such, an alternative method which does not rely on PCR or nucleotide modification is desirable. One approach is based on label-free optical resonant cavity sensors. In the present work, toroidal resonant cavity sensors are functionalized with antibodies to enable label-free detection and discrimination between C, 5′mC, and 5′hmC in real-time without PCR. Specifically, epoxide chemistry is used to covalently attach the 5′hmC antibody to the surface of the cavity. Subsequently, to thoroughly characterize the sensor platform, detection of C, 5′mC, and 5′hmC is performed over a concentration range from pM to nM. At low (pM) concentrations, the hydroxymethylated cytosine produces a significantly larger signal than the structurally similar epigenetic markers; thus demonstrating the applicability of this platform.
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