本文選題：環(huán)介導(dǎo)等溫?cái)U(kuò)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(jiàn)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(jī)制,甲基化異�？赡軐�(dǎo)致人類(lèi)疾病,例如癌癥的發(fā)生。先進(jìn)的“第二代基因測(cè)序”技術(shù)能夠在單堿基水平上,對(duì)基因組DNA甲基化進(jìn)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(lái)越多地用作癌癥診斷的生物標(biāo)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(jiǎn)便、準(zhǔn)確、靈敏的檢測(cè)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(lái)檢測(cè)CpG甲基化,然而,這類(lèi)方法在實(shí)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(kuò)增,容易受假陽(yáng)性信號(hào)干擾。其次,大多數(shù)以PCR為基礎(chǔ)的方法需要耗時(shí)費(fèi)力的實(shí)驗(yàn)操作和昂貴的實(shí)驗(yàn)儀器。許多新開(kāi)發(fā)出來(lái)的方法不依賴(lài)于PCR擴(kuò)增,然而這些方法靈敏度很低,無(wú)法滿足基因組樣品中CpG甲基化檢測(cè)的要求。為了解決上述甲基化分析中存在的問(wèn)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?cái)U(kuò)增(LAMP)反應(yīng)的DNA甲基化分析法。該方法首先用HpaⅡ處理目標(biāo)DNA,在內(nèi)切酶特異識(shí)別位點(diǎn)處,非甲基化目標(biāo)DNA被切斷,而甲基化DNA目標(biāo)序列仍然保持完整。隨后,利用LAMP反應(yīng)快速擴(kuò)增未被切割的甲基化DNA,能夠獲得高靈敏度的檢測(cè)結(jié)果且?guī)缀鯖](méi)有背景信號(hào)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(cè)濃度低至10 aM的甲基化DNA目標(biāo)分子,在大量非甲基化DNA存在的樣品中可檢測(cè)到0.1%的甲基化DNA。另外,該方法在等溫條件下進(jìn)行,不需要任何DNA標(biāo)記,用普通的熒光染料SG即可實(shí)現(xiàn)甲基化DNA的實(shí)時(shí)檢測(cè),因此,該方法還具有簡(jiǎn)單且成本低的優(yōu)點(diǎn)。基于LAMP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(cè)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(hào)：1930991