本文選題：聚合酶鏈?zhǔn)椒磻?yīng) + 單細(xì)胞��； 參考：《江西醫(yī)學(xué)院》2005年碩士論文

【摘要】：目的:運(yùn)用巢式PCR擴(kuò)增牙釉質(zhì)基因(Amelogenin gene,AMEL)的特異性引物序列,比較四種不同的單細(xì)胞裂解方法對(duì)PCR擴(kuò)增率、診斷正確率、等位基因脫扣(allele dropout,ADO)率的影響,尋求最佳的裂解單細(xì)胞體系使之盡可能提高單細(xì)胞PCR的擴(kuò)增效率的同時(shí)降低等位基因的脫扣率并將之應(yīng)用到單卵裂球的分析,為胚胎植入前遺傳學(xué)診斷建立一種高效的細(xì)胞裂解體系,提高PGD中性別診斷和單基因疾病診斷的能力。 方法:以人外周血基因組DNA為陽(yáng)性對(duì)照,收集400份男性單個(gè)淋巴細(xì)胞和200份女性單個(gè)淋巴細(xì)胞隨機(jī)分成A、B、C、D四組,每組100份單個(gè)男性和50份單個(gè)女性淋巴細(xì)胞,分別放置于預(yù)先盛有純水、堿裂解液(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反應(yīng)管中,分別用液氮凍融法、堿裂解法、蛋白酶K裂解法I、蛋白酶K裂解法II進(jìn)行處理;隨后用巢式-聚合酶鏈?zhǔn)椒磻?yīng)擴(kuò)增牙釉質(zhì)基因在X、Y染色體上的兩對(duì)特異性同源序列,瓊脂糖凝膠電泳紫外透射儀下觀察結(jié)果,比較四種裂解單細(xì)胞的方法對(duì)PCR擴(kuò)增效率和等位基因脫扣率和性別診斷正確率的影響。收集單個(gè)卵裂球用蛋白酶K裂解法II進(jìn)行處理后用巢式-聚合酶鏈?zhǔn)椒磻?yīng)擴(kuò)增牙釉質(zhì)基因在X、Y染色體上的兩對(duì)特異性同源序列后用巢式-聚合酶鏈?zhǔn)椒磻?yīng)擴(kuò)增牙釉質(zhì)基因在X、Y染色體上的兩對(duì)特異性同源序列,未洗滌過(guò)細(xì)胞的生理鹽水為試劑空白對(duì)照;最后一次洗滌淋巴細(xì)胞后的生理鹽水為微滴陰性對(duì)照。結(jié)果:液氮凍融法、堿裂解法、蛋白酶K裂解法I、蛋白酶K裂解法II處理后的單個(gè)淋巴細(xì)胞擴(kuò)增率分別為:61.33%、86%、90%、95.33%,性別診斷正確率
[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.
【學(xué)位授予單位】：江西醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346

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