發(fā)布時間：2018-04-16 22:24

  本文選題：聚合酶鏈式反應 + 單細胞　； 參考：《江西醫(yī)學院》2005年碩士論文

【摘要】：目的:運用巢式PCR擴增牙釉質基因(Amelogenin gene,AMEL)的特異性引物序列,比較四種不同的單細胞裂解方法對PCR擴增率、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胞體系使之盡可能提高單細胞PCR的擴增效率的同時降低等位基因的脫扣率并將之應用到單卵裂球的分析,為胚胎植入前遺傳學診斷建立一種高效的細胞裂解體系,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為陽性對照,收集400份男性單個淋巴細胞和200份女性單個淋巴細胞隨機分成A、B、C、D四組,每組100份單個男性和50份單個女性淋巴細胞,分別放置于預先盛有純水、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進行處理;隨后用巢式-聚合酶鏈式反應擴增牙釉質基因在X、Y染色體上的兩對特異性同源序列,瓊脂糖凝膠電泳紫外透射儀下觀察結果,比較四種裂解單細胞的方法對PCR擴增效率和等位基因脫扣率和性別診斷正確率的影響。收集單個卵裂球用蛋白酶K裂解法II進行處理后用巢式-聚合酶鏈式反應擴增牙釉質基因在X、Y染色體上的兩對特異性同源序列后用巢式-聚合酶鏈式反應擴增牙釉質基因在X、Y染色體上的兩對特異性同源序列,未洗滌過細胞的生理鹽水為試劑空白對照;最后一次洗滌淋巴細胞后的生理鹽水為微滴陰性對照。結果: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處理后的單個淋巴細胞擴增率分別為: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.
【學位授予單位】：江西醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

【參考文獻】

相關期刊論文 前9條

1 李崎,馬燕琳,潘乾,夏家輝,戴和平,夏昆;細胞裂解法對植入前診斷中聚合酶鏈反應擴增效率的影響[J];湖南醫(yī)科大學學報;2002年03期

2 陶冶,金帆,黃荷鳳,徐晨明,葉英輝;單細胞五重巢式PCR檢測DMD基因[J];中國計劃生育學雜志;2001年02期

3 徐晨明,金帆,黃荷鳳,陶冶,葉英輝;全基因組擴增在DMD植入前遺傳學診斷中的可行性研究[J];中國計劃生育學雜志;2001年05期

4 李汶,陸長富,盧光t;運用PCR對小鼠植入前胚胎進行性別診斷[J];生命科學研究;2001年01期

5 徐艷文,莊廣倫,周燦權,張敏芳,李莉琳;應用熒光原位雜交技術進行人類早期胚胎性染色體嵌合型的初步研究[J];中華婦產科雜志;1999年10期

6 焦?jié)尚?莊廣倫,周燦權,舒益民,李潔,梁曉燕,張敏芳,鄧明芬;引物延伸預擴增法應用于β-地中海貧血患者胚胎植入前遺傳學診斷——附一例報告[J];中華婦產科雜志;2003年03期

7 王陶然,陳漢平,盧運萍,馬庭元;用巢式PCR對孕婦外周血中的單個有核紅細胞進行胎兒性別診斷[J];中華醫(yī)學遺傳學雜志;2002年01期

8 焦?jié)尚?莊廣倫,周燦權,張敏芳,李莉琳;應用巢式PCR對單細胞進行性別診斷的初步研究[J];中華醫(yī)學遺傳學雜志;2003年01期

9 鐘昌高,李麓蕓,陸長富,林戈,傅俊江,羅克莉,盧光t;單細胞巢式PCR診斷軟骨發(fā)育不全[J];中華醫(yī)學遺傳學雜志;2003年03期

，

本文編號：1760850