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PEP、DOP-PCR单细胞多基因位点检测研究
Genotyping analysis of multiple loci in single cell by primer extension preamplification and degenerate oligonucleotide primed-PCR
【作者】 袁淑慧;
【作者基本信息】 浙江大学 , 妇产科学, 2001, 硕士
【摘要】 植入前遗传学诊断(Preimplantation genetic Diagnosis,PGD)通过体外辅助受精、胚胎细胞遗传学分析,选择正常胚胎移植,能将人类遗传病控制推进到胚胎植入子宫内膜之前,避免了异常妊娠的发生,具明显的遗传优生学意义。但由于PGD的分析材料仅限于1-2个活检卵裂球细胞,单细胞多聚酶链反应(Polymerase chain reaction,PCR)和荧光原位杂交(Fluorescencein-situ hybridization,FISH)能提供的遗传信息非常有限,难以满足同时检测多个基因位点或重复检测的需要。 全基因组扩增(Whole genome amplification,WGA)技术通过扩增微量DNA模板甚至单个细胞的整个基因组,再将扩增产物分为多份,作为模板,进行后续分析,增加微量DNA分析的遗传信息量。应用于PGD,实现了单次胚胎活检,可分析多种不同基因病或多次重复检测同种基因病。WGA技术主要有扩增前引物延伸(Primer extension preamplification,PEP)、退变寡核苷酸引物PCR(Degenerate oligonucleotide primer-PCR,DOP-PCR)。PEP以随机组成 2001年浙江人学帧十学位论义 的 15碱基寡核昔酸作为引物,在低温下连接、延伸,随机扩增整个基因组 DNA。 DOP—PCR引物序列为 5’-CCGACTCGAGN’N’N’NNNATGTGG-3’,经低温引物 随机连接和常温特异引物连接延伸两步法扩增,按比例均匀扩增整个基因组 D「A。?2P和D0?-?[R的随机引物众多,单一循环的PCR条件无法使所有随机引 物同时处于良好的复性和延伸状态,其扩增全基因组DNA的完整性和扩增效率 尚存许多亟待探讨之处。 单细胞PEP用于检测基因位点的研究国外开展较早,迄今已有利用PEP 进行性别、神经节昔脂病GM。I型、口地中海贫血.、IU:ln血型、高度多态短串 联重复序列(short tandem repeat,STR)等基因位点的研究,但尚未见p地中 海贫血 CD17和 nt-28突变基因及连锁基因(An?rlyl?T)、18和 ZI号染色体特异短 串联重复序列 D18551、D21511和 D2151411位点的报道。国内研究才刚刚起步, 仅徐晨明和王敏分别进行单个淋巴细胞、单个胚胎细胞和单个胎儿有核红细胞 的杜氏肌营养不良症基因的检测。单细胞DOP-PCR全基因组扩增产物目前局 限于比较基因组杂交,检测全基因组或染色体组缺失和重复,应用于基因病的 检测报道甚少。若能在DOP-PCR全基因组扩增,CGH染色体非整倍体检测的 同时,利用其DOP-PCR产物,开展基因病的检测或筛查,将具有明显的临床 应用价值。 第一部分 单细胞PEP多基因位点检测的研究 目的:探索建立单细胞PEP全基因组扩增,后续巢式PCR 6地中海贫血 CD17和 nt-28突变基因及连锁基因(ATTTTTT)、18和 21号染色体短串联重复序 列 D18S51、D21Sll和 D2lS1411位点检测技术,考察单细胞陀P同步检测上述 基因位点以及囊性纤维病CF A F508及连锁基因(GATT)、杜氏肌营养不良症DMD 2 2001年浙江人学J袖十学位论义基冈外显子尸和48、Y染色体性别决定基因SRY的扩增准确率,分析单细胞陀P扩增全基【间mA的完整性。方法:显微操作获取单个人淋巴细胞和胚胎细胞,PEP全基回组扩增,后续特异性巢式PCR,检测上述川个基囚位点的单细胞扩增率(特异产物扩增出现的比率)、假阳性率(阴性管内山现扩增产物的比率)、假阴性率(川性管内未出现扩增产物的比率)。扩增准确率(单细胞利阴性、川性扩增产物的符合率)。结果:1.成功建立单细胞陀P全基回组扩增,后续巢式 PCR D地中海贫血 CD17 t[ nt-28突变基因及连锁基因 (ATI”TT)、18和 21号染色体短串联重复序列 D18551、D21511和 D2151411位点检测技术。2.成功进行单细胞PEP-巢式PCR,上述基因位点以及缀性纤维病CF八F508及连锁基因(GATTTT)、杜氏肌营养不良症皿D基因外显子17和48、Y染色体性别决定基因 SRY 10个位点的同步分析,单个淋巴细胞和单个胚胎细胞扩增准确率分别达97.17%(583/600)和 gi.58%(174/190)。3.单个淋巴扩增PEP后续巢式PCR扩增准确率高于单个胚胎细胞,假阳性率低于胚胎细胞,两者有显著性差异(P<0.05)。 第二部分 单细胞DOP-PCR多基因位点检测的研究 日的:探索建立单细胞DOP-PCR全基因组扩增,后续巢式PCR上述10个基冈位点的同步检测技术?
【Abstract】 Preimplantation genetic Diagnosis (PGD) allows selective transfer of unaffected embryos using artificial reproductive techniques and single cell genetic analysis, enables genetic diagnosis to be offered before implantation, thus avoiding the need for termination of pregnancies in couples at risk for having children with genetic and chromosomal disorders. Because PGD are performed on single cell, genetic information is limited by either polymerase chain reaction (PCR) or fluorescent in-situ hybridization (FISH). Therefore, it is almost impossible to detect multiple genetic loci simultaneously or confirm the same locus repeatedly.Whole genomic amplification (WGA) can amplify all genomic sequence from minute DNA samples even from a single cell, provide sufficient DNA templates for a number of subsequent PCR amplification. Implication of WGA for PGD, the number of loci studied can be substantially increased and the genotyping can be confirmed.WGA has various strategies such as primer extension preamplification (PEP) and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). In PEP a mixture 15-base random oligonucleotides to prime Taq DNA synthesis frequently throughout the genome. DOP-PCR uses a primer 5 ’ -CCGACTCGAGNNNNNNATGTGG-3’ , two step PCR strategy consists of several low temperature annealing and extention to tagged many binding sites in the genome , and followed by more specific at the fragments tagged with the above primer sequence and this process is accomplished by increasing the annealing temperature, allows an unselected amplification of any source of DNA. A mixture random oligonucleotides and the uniform PCR temperature cycling perform in both PEP and DOP-PCR, in this condition, the efficiency of every primer annealing and extention is different. The coverage and yield of WGA may be different.It is reported that PEP from a single cell has implicated for ZFX/ZFY, Tay Sachs Disease , Cystic Fibrosis , Hemophilia A, Duchenne muscular dystrophy , RliD blood type and some short tandem repeat (STR). DOP-PCR from a single cell has been used mainly for cytogenetic studies by subsequent comparative genomic hybridization(CGH ) , is seldom for genetic studies by sequent PCR. Combining cytogenetics by CGH with genetics by PCR, DOP-PCR has the potential to diagnosis simultaneously genetic and chromosomal disorders.I Genotypic analysis of multiple loci in single cell by PEP protocols Objective To establish a technique of PEP from single cell followed by nest PCR for the detection of CD17 and nt-28 for P -thalassemia and ATTTT repeat linked with P -thalassemia, STR D18S51, D21S11 and D21S1411 genes. Thesimultaneous detection using PEP-nest PCR for the above loci with CFAF508 for cystic fibrosis and linked GATT repeat, DMD17 and 48 for Duchenne muscular dystrophy, SRY, sex-determination gene of chromosome Y for was carried out for examination of the coverage of the techniques. Methods Single lymphocyte in normal males or female and single blastomere from couple with no family history of genetic disorders were obtained. Using 15-base random primers, the entire genome of a single cell was amplified by primer extension preamplification (PEP). The 10 specific genetic loci were amplified from a small aliquot of the PEP reaction by nest PCR. Results: 1. PEP from single cell followed by nest PCR for the detection of CD17 and nt-28 for P -thalassemia and ATTTT repeat linked with P -thalassemia, STR D18S51, D21S11 and D21S1411 genes was successfully established. 2. The simultaneous detection using PEP-nest PCR for the above loci with CFAF508 for cystic fibrosis and linked GATT repeat, DMD17 and 48 for Duchenne muscular dystrophy, SRY, sex-determination gene of chromosome Y for was carried out, and the coverage of the genome from single lymphocytes is 97.17 %(583/600), single blastomere is 91.58% (174/190). ㏕he coverage of single blastomere is significantly lower than that of the single lymphocyte, P<0.05.II Genotypic analysis of multiple loci in single cell b
- 【网络出版投稿人】 浙江大学 【网络出版年期】2002年 01期
- 【分类号】R710.2;R394.3
- 【下载频次】180