【作者】 胡华；

【导师】 史常旭；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 地中海贫血是世界上最常见的单基因遗传病之一,同时也是我国南方地区最常见的遗传病,重症地中海贫血终生需要输血且死亡率高,产前诊断是预防重症地中海贫血唯一有效的措施。孕妇外周血胎儿有核红细胞、游离胎儿DNA、mRNA的发现为无创伤性获得胎儿遗传物质提供了新的机会,促进了无创性产前诊断的发展,获取的这些微量胎儿遗传物质已渐渐用于胎儿性别、核型、胎儿Rh血型等方面的检测,另外从受精卵分裂中取得单个胎儿细胞进行全基因组扩增技术的发展也促进了胚胎移植前产前诊断,然而这些研究都难以推广应用于临床,它们都面临较严峻的问题:即样本量少,对检测技术方法的灵敏性提出了很高的要求。芯片毛细管电泳具有快速、方便、所需样本量少等优点,将其用于产前诊断有望提高解决目前基因检测速度慢、灵敏度低、检测所需样本量较多等问题,为无创性产前诊断提供新的策略或手段。目的:我们通过芯片毛细管电泳检测β-地中海贫血,为应用于临床地中海贫血无创性产前诊断、受精卵移植前诊断打下了基础,同时我们基于片段长度差异建立了芯片毛细管电泳快速检测基因突变的技术平台,为临床疾病相关基因突变的筛查和诊断提供了一种快速、灵敏的技术手段。本研究拟开展了如下工作:1、质粒样本构建:遗传病检测的研究中病例基因组样本一般不容易获得,它是制约实验顺利进行的重要因素,如何取得足量的研究样本是本实验首先需要解决的问题。构建β-地中海贫血点突变克隆,为突变基因样本做准备。其次,通过引物延伸反应建立稳定的β-地中海贫血检测条件,完成野生型和突变型引物单独检测,初步保证检测引物工作的特异性和灵敏性。2、建立多重PCR反应体系,通过具有较高分辨能力的DHPLC完成β-地中海贫血多位点同时检测,保证六个位点多重引物延伸反应的可行性;同时也为DHPLC与芯片毛细管电泳的区分度、灵敏度的比较奠定基础。3、构建芯片毛细管电泳变性胶检测点突变的技术平台,以完成β-地中海贫血多位点野生型和突变型共同检测的目标。最终,通过芯片毛细管电泳实现β-地中海贫血快速、微量、准确地检测,为产前诊断提供一种快速、灵敏的技术手段。方法:我们的研究内容分了以下三个部分:1、构建突变质粒模板,建立稳定的引物延伸反应检测体系(1)首先建立包括六个位点的β-珠蛋白基因正常序列克隆,然后通过定点突变的方法突变克隆中正常基因位点的碱基序列,分别构建含有β-珠蛋白基因突变位点的基因克隆,通过测序进行验证。(2)设计检测各位点的引物,不但要避免引物间二聚体形成,同时要使所有引物长度、退火温度等相近,以保证多重引物延伸反应的顺利进行。为提高引物延伸反应的特异性,采用三温循环反应程序,并对影响检测反应的各因素进行研究和优化,包括反应程序、引物浓度、引物序列、离子浓度、退火温度、人工错配引物等影响因素的研究,以提高检测反应的特异性和灵敏性,初步建立具有区分正常基因型和突变基因型能力的检测体系。(3)为了后续能在一个检测系统同时区分野生型和突变型扩增产物,我们在野生型检测引物5’端加上10个或20个非特异性的碱基序列,基于核酸片段长度差异在单管反应中可同时区分野生型和突变型,以此同时辨别六个位点的纯合子和杂合子基因型。2、多重引物延伸反应检测β-地中海贫血建立多重引物延伸反应体系,将六个位点的野生型、突变型引物同时在单管中反应,采用具有较高分辨能力的DHPLC检测多个位点的扩增产物,通过多重引物延伸实验完成β-地中海贫血六个位点同时检测。3、通过芯片毛细管电泳检测β-地中海贫血(1)构建双通道芯片毛细管电泳检测平台,包括毛细管电泳芯片、激光共聚焦检测装置、信号收集输出装置的搭建和优化;然后对检测胶的制作工艺、浓度等条件进行研究,提高其对长度差异核酸片段的区分能力;通过对核酸酶切产物、微卫星标准品的检测,评估系统的区分度和重复性。(2)通过多重引物延伸反应将β-地中海贫血六个位点的野生型和突变型引物同时检测每个待检样品。采用荧光Cy3和Cy5分别标记地中海贫血标准品和待检品,通过芯片毛细管电泳双通道同时检测,将待检样品与标准品进行对位比较,以辨别各样品的基因型,包括正常基因型、杂合子、突变纯合子的检测,通过临床病例样本完成β-地中海贫血芯片毛细管电泳检测平台的验证。(3)收集孕妇夫妻双方均为地中海贫血患者或携带者的家庭进行产前诊断,分别获取孕妇外周血浆、羊水或脐血样本,采用试剂盒及芯片毛细管电泳检测平台同时进行β-地中海贫血的产前诊断,分析和比较不同检测方法得到的胎儿基因型结果。结果1、通过定点突变方法成功构建了β-地中海贫血六个突变位点克隆,经测序完全正确。引物延伸反应体系的研究中,设计的引物工作较好,无明显引物二聚体产生。采用三温循环反应较传统单温循环反应具有较高的特异性,在三温循环中退火温度、循环次数对反应的扩增效率及特异性有明显的影响,在我们的研究中发现第二循环反应温度采用67度时适合所有位点检测引物的扩增,且扩增效率高、无非特异性扩增;同时引物延伸反应还受离子浓度、引物浓度、引物序列等因素的影响,对以上因素的调节有助于建立最佳的反应检测条件。在实验中为了降低单碱基差异检测的难度、提高检测引物的特异性和辨别力,我们在引物序列中引入人工错配碱基,发现其增大了野生型和突变型引物间的差异,解决了单碱基差异检测中非特异性扩增的难题。通过凝胶电泳的检测初步建立了具有较高稳定性、特异性的检测体系,以β-地中海贫血患者样本为检测模板上进行了验证,每个样品检测结果都符合试剂盒检测结果或测序结果。在引物加尾实验的研究中,我们发现加入10个或20个非特异的T碱基不但不会影响反应的退火温度,而且也没有造成引物二聚体,可较好的工作而不影响多重引物延伸反应。可用于下一步基于片段长度通过芯片毛细管检测的研究。2、通过多重引物延伸反应对β-地中海贫血六个位点同时扩增,采用DHPLC检测不同位点扩增产物,调节洗涤液的浓度及检测时间提高DHPLC检测的区分度,实现了六位点野生型或突变型同时进行检测。3、通过芯片毛细管电泳检测β-地中海贫血:构建了芯片毛细管电泳变性胶检测技术平台,在材料方面我们采用高分子为原材料可下降检测成本及实现再利用;检测装置为公司自产的激光共聚焦扫描仪改装而成,采用共聚焦光路,大大提高了检测底限,同时仪器还集成了632nm和532nm两种激光器以及各自的滤光片,因而能够实现Cy5、Cy3双通道同时采集;新的光路采用了激发光入射到芯片管道上,大部分的反射光经过反射镜的透射孔透射出去。其有两个优点,第一是反射镜上有一个透射孔,可以透过绝大部分的反射激光,降低背反射激光的影响;第二是采用共聚焦光路,使得非焦点位置的杂散光不能透过针孔,再一次降低了背景噪音。通过变性胶浓度以及制备程序等方面优化提高芯片毛细管电泳检测能力,采用3%的HPC胶时,检测区分能力较好,可区分100-200bp和300-400bp间相差4bp的不同长度核酸片段。在检测β-地中海贫血标准品的实验中,完成了对100-600bp间相差10bp不同长度片段的区分,并且建立的标准品和待检品双通道检测体系,其稳定性、重复性好,通过芯片毛细管电泳完成了临床病例样本多位点同时检测,可检测六个位点纯合子、杂合子基因型,芯片毛细管电泳检测所有样本的结果与试剂盒检测的结果完全一致。此检测系统平台仅需要0.5ng/μl的基因组样本,1μl的检测样本(0.04nM),检测时间只需要200s,实现了快速、微量检测的目标。将检测系统进行β-地中海贫血无创性产前诊断的研究,检测的结果与脐血或羊水基因检测结果一致。其所需样本量非常少、快速的优点为无创性产前诊断方法提供的一种新的选择。结论1、完成了从质粒构建、引物延伸反应到遗传病检测一套完整的方法体系,点突变质粒的构建为解决遗传病检测样本的不足提供了一种便捷的方法,引物延伸反应影响因素的全面研究为快速建立引物延伸反应条件奠定了基础,这些方法可用于其他相关遗传病检测的研究和应用。同时建立了β-地中海贫血多重引物延伸反应采用凝胶进行检测的方法,此方法简单、材料普通而便宜,可用于基层医院的筛查使用。2、建立了芯片毛细管电泳检测遗传病基因点突变的技术平台,完成了β-地中海贫血微量样品的快速检测,为外周血胎儿游离DNA及胚胎移植前遗传病诊断等微量标本要求的无创性产前诊断提供一种新的检测途径,同时也为其他遗传病的快速检测奠定了基础。3、我们将基于基因片段长度差异检测的方法进行了比较,包括琼脂糖凝胶电泳、HPLC、芯片毛细管电泳,显示了芯片毛细管电泳是一种快速、灵敏度高、区分能力强的基因突变检测工具。更多还原

【Abstract】 Thalassemia is the commonest genetic disorder worldwide.There is high incidence rate in southern china.The homozygous state is associated with high morbidity and mortality,thus screening of at-risk pregnancies and prenatal testing are stongly advocated. Fetal cells and cell free fetal DNA in the maternal circulation are found before 10 years which can be used for noninvasive prenatal diagnosis on fetal sex,21 trimsome and RhD, but it was not applied widely,partly because of less sample and complex technology.The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted DNA,the analytical sensitivity,and the specificity.The discrimination of single-nucleotide difference between circulating DNA samples is technically challenging and demands the adoption of highly sensitive and specific analytical systems.Chip-based capillary electrophoresis can detect a little sample speedily and conveniently.This technique may be useed for prenatal diagnosis,preimplantation genetic diagnosis which sample is precious and less.It could resolve many problems which the gene detection was slow,tardiness,much sample to need which is not fit for prenatal diagnosis.Objectives:We construct the technique platform of detection the point mutation by the chip-based capillary electrophoresis.It can be used to detect other genetic diseases in clinic as a celerity and sensitive means.In our study we should do as follows:1) For sample preparation we should construct the clones of the mutation genes ofβ-thalassemia which is important for the experiment.The steady detection system of primer extension is to be accomplished.By the gel to be detected the wildness and mutation genotype of sample it was be proved that the primers can discriminate the different of mutation types sensitively and specially.2) Multiplex primer entension reaction:we amplify six loci through multiplex primer entension reaction and detect the six genotypes simultaneity by sensitive HPLC. 3) Detection theβ-thalassemia by chip-based capillary electrophoresis:we construct the technical platform to detect mutation by chip-based capillary electrophoresis and progress the ability to differentiate.In the end we should accomplish the rapid prenatal diagnosis ofβ-thalassemia by chip-based capillary electrophoresis.Mehods:our experiment involves three parts as follows:1) The construction of gene mutation clone and primer extension reactionWe construct the clone of nomalβ-globin gene and six clones ofβ-thalassemia mutation genes:CD28、CD17、CD26、CD41、CD71、IVS654 by site-directed mutagenesis technique which were be proved by sequencing.The primers were designed whose anneal temperature and length is close each other and primer dimmers don’t been produced so that the multiplex can be successful.We study the conditions which may influence the detecting system.Many factors have been studied include reaction of procedure,the concentration of primers,the sequence of primers,the conctration of Mg ion,artificial mismatch primer and so on.Detected by gel the primers were proved to be special and sensitive.In order to differentiate the wildness and mutation genotype we added ten bases of T at the location of primer 5’ end so that they can be differentiated by chip-based capillary electrophoresis.2) In the second part multiplex primer extention reaction were operated to amplify the six point mutation simultaneously.The products of primer extention action were detected by HPLC.3) The third parts were to construct the technical platform to detect the different length PCR product by chip-based capillary electrophoresis.The condition of multiplex primer entension experiments were designed to detect sixβ-globin gene mutation loci simultaneously.We study the raw materials made of capillary electrophoresis chips.To improve the ability to differentiate we studied the concentration of denaturalization gel and the procedure.Through detection of STR standard sample we preliminarily identified the capility of chip-based capillary electrophoresis.We can detect the ladder and samples ofβ-thalassemia simultaneously by the two channels labeled with different fluorescence. Through comparing the ladder with sample we could discern the genotype of the detected sample.All sample including the clone and patient’s blood were detected by this system. The detection systems were validated by the previous methods.At last we processed prenatal dianosis for three families in which parent wereβ-thalassemia patients or carriers. The fetal samples were collected from the amniotic fluid,cord blood and maternal plasma fetal DNA.We compared the kit detection method and chip-based capillary electrophoresis.Results:We constructed the clones ofβ-thalassemia mutation gene by site-directed mutagenesis technique.The sequences of clones were accoding with publicized date completely at NCBI.The primers designed can work and no primer dimmer produced.In the procedure of multiplex primer extension action the three temperature cycle was better than single temperature cycle which primers can work well.In the second temperature cycle 67℃was fit to detect without any false positive results,moreover the primer extionsion were influenced by the concentration of iron and primer and the sequences of primers. Artifical mismatch primers were used on experiment to differentiate the wild and mutation primers and cut down the nonspecia produce.The ten bases T were added to 5’ end of the reverse loci specific primers to lengthen PCR product of wild primer and don’t influence anneal temperature and other primers.Through the preliminary detection of clinical sample the primers were be proved to be special and sensitive.We establish the steady system to detectβ-thalassemia by gel.The result of detection was according with sequencing of the patients’ sample.This method is easy,convenient,common material used,less cost to operate.So it may be useful for the convenient detection ofβ-thalassemia and reducing the cost of gene detection.The result was according with the sequencing and previous result.Through the multiplex primer extension six loci were amplificated and detected.The PCR products were detected by HPLC which can differentiate these six loci.We constructed the technique platform of chip-based capillary electrophoresis to detect the point mutation.We utilized the polymer substrates PMMA which is less expensive and easy to make.An improvement of this chip-based electrophoresis detector from conventional confocal setup was to use a hole reflecting mirror instead of a dichroic mirror, which could pass through both the exciting and reflected lasers while reflect most of the emitted fluorescences to the collecting pathway.This setup woμld significantly eliminate the impact of reflected lasers on the fluorescence detection.Another feature of this detector was that lasers were focused onto a very small spot(diameter of 5μm) by the objective. Two advantages could be resulted from this feature.One is that with the smaller focused spot,higher intensity of laser could be obtained,causing higher exciting efficiency of fluorescence.The other advantage is that the focused spot is much smaller than the width of separation channel(60μm),thus avoiding the illumination of rough side walls,which would cause scattering of lasers.Both of the two advantages would increase the sensitivity. The microvolumetr characteristics of chip-based capillary electrophoresis provide obvious advantages over slab-gel electrophoresis and capillary electrophoresis for biomedical and clinical applications.We sudied and optimizied the concentrations of denaturalization gels. The 3%LPA gel was used to detect with the best results which could differentiate the discrepancy of 4bp between the 100bp to 200 and 300-400bp and 10bp between 100-600bp. We detected size ladders and samples simultaneously by a dual-channel detection system which labeled respectively with Cy3 and Cy5 fluorescence so as to improve the detection precision.The work exploited the efficient separation of amplified DNA and differentiated the wide and mutation genotype by PCR products length.The genotype including homozygous wild type,heterozygous variant,and homozygous variant can be known.Sensitivity study show the detection system of the chip-based capillary electrophoresis could detect 0.5ng genomic DNA sample(0.5ng/μl).Chip-based capillary electrophoresis allowed the analysis time for the genotypes to be decreased to 200 sec.The high sensitivity is important for prenatal diagnosis in which the samples are always precious and a little, especially in early pregnancies.We utilized the chip-based capillary electrophoresis and developed rapid assays for prenatal diagnosis ofβ-thalassemia.We processed the prenatal diagnosis on three cases by the amniotic or a umbilical blood sample and maternal plasma fetal DNA.The results of chip-based capillary electrophoresis are according with that of the previous method and sequencing.Fetal genotypes were predicted correctly in all cases studied.The microvolume characteristics of CE provide obvious advantages for biomedical and clinical applications.Conclusion:1) we finished the experiments form the construction,primer extention reaction and detection.The clones of mutation gene were a suitable method for preparation the mutation samples.The study of factors inflenced the primer extension reacton would be based for quickly constrcting the reaction condition.We construct a suit of method to study and detect the point mutation which can be used to other diseases related to gene mutation. moreover we constructed the convenient method to detectβ-thalassemia by gel which can be used at common hospital.2) We constructed the platform of chip-based capillary electrophoresis to detect the point mutation and processed prenatal diagnosis ofβ-thalassemia quickly by maternal plasma fetal DNA.This detection system of chip-based capillary electrophoresis could be used to noninvasive prenatal diagnosis ofβ-thalassemia. It woμld facilitate prenatal diagnosis of genetic disorder rapidly and sensitively.3) we compared the three methods including agarose gel,HPLC and chip-based capillary electrophoresis.The results show chip-based capillary electrophoresis is a quick,sensitive detection instrument with stronger discrimination power.更多还原