本文選題：表觀遺傳學(xué) + CpG島文庫　； 參考：《第三軍醫(yī)大學(xué)》2006年博士論文

【摘要】： 目的 1.建立分離和富集人類基因組CpG島(CpG islands, CGIs)的甲基化敏感性鏡像定位選擇(methylation- sensitive mirror orientation selection, MS-MOS)方法,并將MS-MOS產(chǎn)物制備成CGIs文庫。 2.建立抑制物控制性PCR(inhibitor-controlled PCR, IC-PCR)方法,并系統(tǒng)地探討內(nèi)對照處理(internal processing control, IPC)與目的DNA之間的相互作用關(guān)系,同時(shí),將IC-PCR應(yīng)用于比較分析多種不同DNA抽提方法降低或消除豬源性肝素粗制品(industrial crude porcine heparin, ICPH)中的PCR抑制物的能力。 3.分析不同核酸熒光染料在瓊脂糖凝膠電泳中的染色特性,找到一種比EB更安全的核酸熒光染色法。 方法 1. MS-MOS方法的建立和CGIs文庫的制備 1.1 MS-MOS分離和富集人類基因組CGIs 1.1.1樣本DNA(tester DNA, T-DNA)和驅(qū)動(dòng)DNA(driver DNA, D-DNA)的制備:男性基因組經(jīng)Mse I酶切后與H24接頭連接,一部分連接產(chǎn)物經(jīng)連接介導(dǎo)性PCR(ligation-medicated PCR, LM-PCR)制備成preT-DNA,另一部分連接產(chǎn)物則經(jīng)Hpa II/Hha I雙酶切后再經(jīng)LM-PCR制備成preD-DNA。preT-DNA和preD-DNA經(jīng)Mse I酶切分別制備成T-DNA和D-DNA。 1.1.2 CGIs的分離和富集:應(yīng)用鏡像定位選擇(mirror orientation selection, MOS)分離和富集只存在于T-DNA的非甲基化CGIs(nonmethylated CGIs, UM-CGIs),并以Spike消減為陽性對照,自身消減和反向消減為陰性對照。 1.2 CGIs文庫的制備與鑒定 正向消減的MS-MOS產(chǎn)物以TA克隆方式轉(zhuǎn)化至E.Coli XL1-Blue MRF’制備成CGIs文庫。通用引物PCR(universal primer PCR, UPPCR)鑒別CGIs文庫克隆是否存在
[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)
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 黃慶,郭穎,府偉靈;人類表觀基因組計(jì)劃[J];生命的化學(xué);2004年02期

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本文編號：1744956