【摘 要】
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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
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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CGIs1.1.1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
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