人类基因组CpG岛文库的构建和抑制物控制性PCR的建立与应用

发布时间：2018-04-13 14:31

本文选题：表观遗传学 + CpG岛文库　；参考：《第三军医大学》2006年博士论文

【摘要】： 目的 1.建立分离和富集人类基因组CpG岛(CpG islands, CGIs)的甲基化敏感性镜像定位选择(methylation- sensitive mirror orientation selection, MS-MOS)方法,并将MS-MOS产物制备成CGIs文库。 2.建立抑制物控制性PCR(inhibitor-controlled PCR, IC-PCR)方法,并系统地探讨内对照处理(internal processing control, IPC)与目的DNA之间的相互作用关系,同时,将IC-PCR应用于比较分析多种不同DNA抽提方法降低或消除猪源性肝素粗制品(industrial crude porcine heparin, ICPH)中的PCR抑制物的能力。 3.分析不同核酸荧光染料在琼脂糖凝胶电泳中的染色特性,找到一种比EB更安全的核酸荧光染色法。方法 1. MS-MOS方法的建立和CGIs文库的制备 1.1 MS-MOS分离和富集人类基因组CGIs 1.1.1样本DNA(tester DNA, T-DNA)和驱动DNA(driver DNA, D-DNA)的制备:男性基因组经Mse I酶切后与H24接头连接,一部分连接产物经连接介导性PCR(ligation-medicated PCR, LM-PCR)制备成preT-DNA,另一部分连接产物则经Hpa II/Hha I双酶切后再经LM-PCR制备成preD-DNA。preT-DNA和preD-DNA经Mse I酶切分别制备成T-DNA和D-DNA。 1.1.2 CGIs的分离和富集:应用镜像定位选择(mirror orientation selection, MOS)分离和富集只存在于T-DNA的非甲基化CGIs(nonmethylated CGIs, UM-CGIs),并以Spike消减为阳性对照,自身消减和反向消减为阴性对照。 1.2 CGIs文库的制备与鉴定正向消减的MS-MOS产物以TA克隆方式转化至E.Coli XL1-Blue MRF’制备成CGIs文库。通用引物PCR(universal primer PCR, UPPCR)鉴别CGIs文库克隆是否存在
[Abstract]:Purpose1.A methylation-sensitive mirror orientation selection (MS-MOS) method for the isolation and enrichment of human genomic CpG island CpG islandsCGIsa was established, and the MS-MOS product was prepared into CGIs library.2.To establish an inhibitor controlled PCR(inhibitor-controlled PCR (IC-PCR) method, and to explore the interaction between internal control processing (IPCs) and objective DNA.IC-PCR was applied to compare and analyze the ability of different extraction methods of DNA to reduce or eliminate PCR inhibitor in crude porcine heparin (ICPH), a crude product of porcine heparin.3.The staining characteristics of different nucleic acid fluorescent dyes in agarose gel electrophoresis were analyzed and a more safe method of nucleic acid fluorescence staining than EB was found.Method1.Establishment of MS-MOS method and preparation of CGIs Library1.1 isolation and enrichment of human genomic CGIs by MS-MOS1.1.1 preparation of DNA(tester DNA (T-DNA) and driving DNA(driver DNA (D-DNA): the male genome was digested by Mse I and connected to the H24 junction.Some of the ligation products were prepared into preT-DNA by ligation-mediated PCR(ligation-medicated PCR (LM-PCR). The other part was digested by Hpa II/Hha I and then LM-PCR was used to prepare preD-DNA.preT-DNA and preD-DNA into T-DNA and D-DNA by Mse I digestion, respectively.1.1.2 Separation and enrichment of CGIs: mirror orientation selection (moss) was used to isolate and enrich T-DNA only in non-methylated CGIs(nonmethylated CGIs. UM-CGIsa, and Spike subtractive as positive control, self-subtractive and reverse subtractive as negative control.Preparation and Identification of 1.2 CGIs LibraryThe positive subtractive MS-MOS product was transformed into E.Coli XL1-Blue MRF'by TA clone to form CGIs library.Identification of CGIs library clones by universal primer PCR(universal primer PCR (UPPCR)
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

【参考文献】