本文選題：環(huán)介導(dǎo)等溫擴增(LAMP) + DNA甲基化��； 參考：《陜西師范大學(xué)》2016年碩士論文

【摘要】：DNA甲基化最常見的是基因組DNA上CpG二核苷酸胞嘧啶的第五位碳原子的甲基化,它是基因轉(zhuǎn)錄調(diào)控的主要表觀遺傳機制,甲基化異常可能導(dǎo)致人類疾病,例如癌癥的發(fā)生。先進的“第二代基因測序”技術(shù)能夠在單堿基水平上,對基因組DNA甲基化進行分析,為我們了解基因組中CpG甲基化與癌癥發(fā)生之間的關(guān)系提供了新的思路,因此,DNA甲基化已被越來越多地用作癌癥診斷的生物標志物。用于常規(guī)的醫(yī)學(xué)診斷中的DNA甲基化分析方法,要求能夠簡便、準確、靈敏的檢測基因組中CpG甲基化,且不需要昂貴儀器設(shè)備。目前,基于PCR的分析方法被廣泛地用來檢測CpG甲基化,然而,這類方法在實際應(yīng)用中存在著諸多挑戰(zhàn)：第一,基于PCR的分析方法存在非特異性擴增,容易受假陽性信號干擾。其次,大多數(shù)以PCR為基礎(chǔ)的方法需要耗時費力的實驗操作和昂貴的實驗儀器。許多新開發(fā)出來的方法不依賴于PCR擴增,然而這些方法靈敏度很低,無法滿足基因組樣品中CpG甲基化檢測的要求。為了解決上述甲基化分析中存在的問題,在本論文中,利用甲基化敏感型限制性內(nèi)切酶(HpaⅡ)特異性地區(qū)分甲基化和非甲基化DNA,我們建立了基于環(huán)介導(dǎo)等溫擴增(LAMP)反應(yīng)的DNA甲基化分析法。該方法首先用HpaⅡ處理目標DNA,在內(nèi)切酶特異識別位點處,非甲基化目標DNA被切斷,而甲基化DNA目標序列仍然保持完整。隨后,利用LAMP反應(yīng)快速擴增未被切割的甲基化DNA,能夠獲得高靈敏度的檢測結(jié)果且?guī)缀鯖]有背景信號干擾。因此,與傳統(tǒng)的甲基化分析方法相比,我們所提出的方法能夠獲得更高的靈敏度和選擇性,可以檢測濃度低至10 aM的甲基化DNA目標分子,在大量非甲基化DNA存在的樣品中可檢測到0.1%的甲基化DNA。另外,該方法在等溫條件下進行,不需要任何DNA標記,用普通的熒光染料SG即可實現(xiàn)甲基化DNA的實時檢測,因此,該方法還具有簡單且成本低的優(yōu)點�；贚AMP反應(yīng)的甲基化法也可以很好地應(yīng)用于基因組DNA中CpG甲基化分析,能檢測到低至100 pg的甲基化基因組DNA。因此,該方法在疾病診斷研究和DNA甲基化的生物學(xué)功能研究領(lǐng)域具有很好的應(yīng)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，

本文編號：1930991