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题名: 糖基化介导RCA与qPCR联用检测5-羟甲基胞嘧啶
作者: 刘迪
学位类别: 硕士
答辩日期: 2016-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 汪海林
关键词: 5-羟甲基胞嘧啶 ; 滚环扩增 ; 5-hydroxymethylcytosine, rolling circle amplification, qPCR ; qPCR
其他题名: Detection of 5-hydroxymethylcytosine with the combination of glucosylation-mediated RCA and qPCR
学位专业: 环境科学
中文摘要:     5-羟甲基胞嘧啶(5hmC)由5-甲基胞嘧啶(5mC)经Ten eleven tanslocation(Tet)双加氧酶催化氧化形成。作为一种重要的表观遗传修饰,5hmC在基因组中相对含量低,但其在多种生物过程发挥重要作用,如基因表达、细胞分化、胚胎发育及神经元功能等。此外,5hmC的修饰水平与癌症密切相关,多项研究显示癌症的发生伴随着5hmC整体水平的降低。因此,深入研究5hmC的生物学功能至关重要,而测定5hmC在基因组中的含量与分布是进一步探究5hmC的生物功能的基础。
    发展简单有效的方法测定5hmC是本课题的主要工作。据此,本研究使用恒温扩增酶phi29 DNA聚合酶,将糖基化介导的滚环扩增(RCA)反应与实时定量PCR(qPCR)相结合,实现特定序列中5hmC的定量检测。
    首先制备了含有不同类型胞嘧啶的双链DNA探针(C-/5mC-/5hmC-/5ghmC-dsDNA probe)及不同5ghmC修饰密度的系列探针(5ghmC probe D-1,D-2,D-3,D-4,D-5)。
RCA实时分析显示,5ghmC修饰使RCA反应的产物产量下降,即5ghmC修饰可阻碍phi29 DNA扩增酶的扩增效率。分别以各RCA反应的产物为模板进行qPCR扩增,得到各个反应的Ct值。以C相关的qPCR反应的Ct值为基准值,计算各个修饰参与qPCR反应的△Ct值。结果显示,C相关qPCR反应的△Ct值(-0.00±0.09)与5mC(-0.06±0.08)及5hmC(-0.02±0.03)相关的qPCR反应的△Ct值之间无统计学差异,但与5ghmC相关qPCR的△Ct值(1.59±0.03)存在显著性差异。5ghmC相关qPCR的Ct值的特异性增加使得该方法可检测5hmC修饰的存在。
使用5ghmC密度不同的探针进行RCA-qPCR反应,结果显示5ghmC密度与△Ct值线性关系良好:△Ct=0.0209*[5ghmC/1000 C]+0.111,R2=0.97,且当模板中5ghmC修饰密度低至7.3±0.4/1000 C时,该方法依旧能够检测到5ghmC。
    我们进一步将5ghmC-dsDNA probe与C-dsDNA probe按不同比例进行混合作为RCA-qPCR反应的模板。结果表明随着模板中5ghmC-dsDNA probe比例的增加,Ct值逐渐增大,且反应中5ghmC修饰的含量与相应qPCR的△Ct值呈线性相关(△Ct=0.0204*[5ghmC/1000C]+0.104,R2=0.98)。比例及密度实验得到的线性方程的相似性初步证明了qPCR反应△Ct值的主要决定因素为5ghmC修饰密度。
英文摘要:     5-hydroxymethylcytosine (5hmC) is converted from 5-methylcytosine (5mC) by Ten eleven translocation (Tet) proteins. As an important epigenetic mark, although with low content, 5hmC has been reported to participate in various biological processes, such as gene regulation and expression, cell proliferation differentiation, embryonic development and neuron functions. Meanwhile, ―loss of 5hmC‖ is an epigenetic hall-mark of many cancers, with diagnostic and prognostic implications. To elucidate the exact biological roles of this base modification, the development of new analytical technologies for detection of 5hmC contents and distribution, has become essential.
    Our research focuses on development of simple and efficient method to detect 5hmC. Taking advantage of phi29 DNA polymerase, we combined glucosylation mediated rolling circle amplification (RCA) with real time PCR (qPCR) for quantatitive detec-tion of 5hmC within specific fragments.
    We first synthesized double strand DNA probes with different cytosine types (C-/5mC-/5hmC-/5ghmC-dsDNA probe) and a batch of 5ghmC probes with various 5ghmC densities (5ghmC probe D-1, D-2, D-3, D-4, D-5).
    Real time RCA results showed that 5ghmC could lead to the decrease of RCA prod-ucts quantity, indicating the specific inhibition effect of 5ghmC on the replication ac-tivity of phi29 DNA polymerase. Then RCA products were used as templates in the following qPCR reactions. △Ct values of reactions with C-/5mC-/5hmC-/5ghmC-related templates were calculated by subtracting Ct values of C-related qPCR reaction from that of C-/5mC-/5hmC-/5ghmC-related qPCR reaction. The △Ct values of 5mC- (-0.06±0.08) and 5hmC-related qPCR (-0.02±0.03) showed no statistical difference to the value of C-related qPCR (-0.00±0.09), while △Ct value of 5ghmC-related qPCR (1.59±0.03) was statistically different from that of C-related qPCR reaction. The in-crease of Ct value of 5ghmC-related qPCR could be used for 5hmC detection.
    Using templates with different 5ghmC density to perform RCA-qPCR assay, a good linear relationship existed between 5ghmC contents and △Ct values: △Ct=0.111+0.0209*[5ghmC/1000 C], R2=0.97. Moreover, 5ghmC density as low as 7.3±0.4 per 1000 C could still be detected by our developed method.
    We further mixed 5ghmC-dsDNA probe and C-dsDNA probe at different molar ratios as templates of the RCA-qPCR assay. Results showed Ct values increased as the in-
crement of 5ghmC-dsDNA probe ratios. A positive correlation between △Ct values and 5ghmC modifications levels (5ghmC/1000 C) in initial RCA-qPCR templates could be observed (△Ct=0.0204*[5ghmC/1000 C]+0.104, R2=0.98). Basing on the similarity between these two linear equations, we could draw the preliminary conclu-sion that the sequence context had little influence on the performance of the RCA-qPCR method and 5ghmC content was the dominant factor that decidied the △Ct val-ues.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36894
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
刘迪. 糖基化介导RCA与qPCR联用检测5-羟甲基胞嘧啶[D]. 北京. 中国科学院研究生院. 2016.
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