基于环介导等温扩增（LAMP）反应高灵敏检测DNA甲基化

发布时间：2018-05-24 23:11

  本文选题：环介导等温扩增(LAMP) + DNA甲基化　； 参考：《陕西师范大学》2016年硕士论文

【摘要】：DNA甲基化最常见的是基因组DNA上CpG二核苷酸胞嘧啶的第五位碳原子的甲基化,它是基因转录调控的主要表观遗传机制,甲基化异常可能导致人类疾病,例如癌症的发生。先进的“第二代基因测序”技术能够在单碱基水平上,对基因组DNA甲基化进行分析,为我们了解基因组中CpG甲基化与癌症发生之间的关系提供了新的思路,因此,DNA甲基化已被越来越多地用作癌症诊断的生物标志物。用于常规的医学诊断中的DNA甲基化分析方法,要求能够简便、准确、灵敏的检测基因组中CpG甲基化,且不需要昂贵仪器设备。目前,基于PCR的分析方法被广泛地用来检测CpG甲基化,然而,这类方法在实际应用中存在着诸多挑战：第一,基于PCR的分析方法存在非特异性扩增,容易受假阳性信号干扰。其次,大多数以PCR为基础的方法需要耗时费力的实验操作和昂贵的实验仪器。许多新开发出来的方法不依赖于PCR扩增,然而这些方法灵敏度很低,无法满足基因组样品中CpG甲基化检测的要求。为了解决上述甲基化分析中存在的问题,在本论文中,利用甲基化敏感型限制性内切酶(HpaⅡ)特异性地区分甲基化和非甲基化DNA,我们建立了基于环介导等温扩增(LAMP)反应的DNA甲基化分析法。该方法首先用HpaⅡ处理目标DNA,在内切酶特异识别位点处,非甲基化目标DNA被切断,而甲基化DNA目标序列仍然保持完整。随后,利用LAMP反应快速扩增未被切割的甲基化DNA,能够获得高灵敏度的检测结果且几乎没有背景信号干扰。因此,与传统的甲基化分析方法相比,我们所提出的方法能够获得更高的灵敏度和选择性,可以检测浓度低至10 aM的甲基化DNA目标分子,在大量非甲基化DNA存在的样品中可检测到0.1%的甲基化DNA。另外,该方法在等温条件下进行,不需要任何DNA标记,用普通的荧光染料SG即可实现甲基化DNA的实时检测,因此,该方法还具有简单且成本低的优点。基于LAMP反应的甲基化法也可以很好地应用于基因组DNA中CpG甲基化分析,能检测到低至100 pg的甲基化基因组DNA。因此,该方法在疾病诊断研究和DNA甲基化的生物学功能研究领域具有很好的应用前景。
[Abstract]:The most common methylation of DNA is the fifth carbon atom of CpG dinucleotide cytosine on genomic DNA, which is the main epigenetic mechanism of gene transcription regulation. Abnormal methylation may lead to human diseases, such as cancer. Advanced "second generation gene sequencing" technology can analyze genomic DNA methylation at the single base level, which provides a new way to understand the relationship between CpG methylation and carcinogenesis in the genome. Therefore, DNA methylation has been increasingly used as a biomarker for cancer diagnosis. DNA methylation analysis for routine medical diagnosis requires simple, accurate and sensitive detection of CpG methylation in the genome, and does not require expensive instrumentation. At present, PCR based analysis method is widely used to detect CpG methylation. However, there are many challenges in the practical application of this method. Firstly, PCR based analysis method has non-specific amplification and is vulnerable to false positive signal interference. Secondly, most PCR-based methods require time-consuming and laborious experimental operations and expensive experimental instruments. Many newly developed methods do not rely on PCR amplification, however, these methods are not sensitive enough to meet the requirements of CpG methylation detection in genomic samples. In order to solve the above problems in methylation analysis, in this paper, Using methylation-sensitive restriction endonuclease (HPA 鈪，

本文编号：1930991