不同的DNA制备方法对单细胞PCR扩增率和ADO率影响的比较研究

发布时间：2018-04-16 22:24

本文选题：聚合酶链式反应 + 单细胞　；参考：《江西医学院》2005年硕士论文

【摘要】：目的:运用巢式PCR扩增牙釉质基因(Amelogenin gene,AMEL)的特异性引物序列,比较四种不同的单细胞裂解方法对PCR扩增率、诊断正确率、等位基因脱扣(allele dropout,ADO)率的影响,寻求最佳的裂解单细胞体系使之尽可能提高单细胞PCR的扩增效率的同时降低等位基因的脱扣率并将之应用到单卵裂球的分析,为胚胎植入前遗传学诊断建立一种高效的细胞裂解体系,提高PGD中性别诊断和单基因疾病诊断的能力。方法:以人外周血基因组DNA为阳性对照,收集400份男性单个淋巴细胞和200份女性单个淋巴细胞随机分成A、B、C、D四组,每组100份单个男性和50份单个女性淋巴细胞,分别放置于预先盛有纯水、碱裂解液(KOH/DTT)、蛋白酶K裂解液I(PK/SDS)和蛋白酶K裂解液II(PK/Tween-20)的PCR反应管中,分别用液氮冻融法、碱裂解法、蛋白酶K裂解法I、蛋白酶K裂解法II进行处理;随后用巢式-聚合酶链式反应扩增牙釉质基因在X、Y染色体上的两对特异性同源序列,琼脂糖凝胶电泳紫外透射仪下观察结果,比较四种裂解单细胞的方法对PCR扩增效率和等位基因脱扣率和性别诊断正确率的影响。收集单个卵裂球用蛋白酶K裂解法II进行处理后用巢式-聚合酶链式反应扩增牙釉质基因在X、Y染色体上的两对特异性同源序列后用巢式-聚合酶链式反应扩增牙釉质基因在X、Y染色体上的两对特异性同源序列,未洗涤过细胞的生理盐水为试剂空白对照;最后一次洗涤淋巴细胞后的生理盐水为微滴阴性对照。结果:液氮冻融法、碱裂解法、蛋白酶K裂解法I、蛋白酶K裂解法II处理后的单个淋巴细胞扩增率分别为:61.33%、86%、90%、95.33%,性别诊断正确率
[Abstract]:Objective: to amplify the specific primer sequence of enamel gene Amelogenin gene Amelogenin gene (Amelogenin gene AMEL) by nested PCR, and compare the effects of four different single cell lysis methods on PCR amplification rate, diagnostic accuracy rate and allele dropout ADO rate.To find the best single cell system to improve the amplification efficiency of single cell PCR and to reduce the allelic release rate and to apply it to the analysis of single cleavage cells.To establish an efficient cell lysis system for preimplantation genetic diagnosis, and to improve the ability of sex diagnosis and single gene disease diagnosis in PGD.Methods: using human peripheral blood genomic DNA as a positive control, 400 male and 200 female single lymphocytes were randomly divided into four groups, 100 single male and 50 female lymphocytes in each group.The PCR reaction tubes containing pure water, alkaline lytic solution (Koh / DTT), protease K (IPKK / SDSs) and protease K lytic liquid (IIK / Tween-20) were treated with liquid nitrogen freeze-thawing method, alkaline lytic method, protease K lysis method and protease K lysis method II, respectively.Then, two pairs of specific homologous sequences of enamel gene were amplified by nested polymerase chain reaction on XY chromosome. The results were observed by agarose gel electrophoresis and UV transmission instrument.The effects of four methods of single cell cleavage on PCR amplification efficiency, allelic tripping rate and sex diagnosis accuracy were compared.Two pairs of specific homologous sequences of enamel gene on XNY chromosome were amplified by nested polymerase chain reaction after treatment with protease K cleavage method II, and then the teeth were amplified by nested polymerase chain reaction.Two pairs of specific homologous sequences of enamel gene on XMY chromosome.The normal saline of unwashed cells was used as the reagent blank control, and the saline after the last washing of lymphocytes was a microdrop negative control.Results: the amplification rates of single lymphocytes treated by liquid nitrogen freeze-thawing method, alkaline lysis method, protease K lysis method I and protease K lysis method II were: 1: 61.338610% and 95.33%, respectively. The correct rate of sex diagnosis was correct.
【学位授予单位】：江西医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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