作者简介：  韩富天，男，1970年11月生，1994年09月师从中国科学院大连化学物理研究所林炳承教授，于1999年07月获得博士学位。

摘要

从开发能够进行高分辨基因分析的毛细管电泳无胶筛分体系入手，对以羟丙基甲基纤维素为基础的筛分介质进行研究，并予以改进和优化，对包括毛细管状态、温度、电场强度等影响DNA分离的各种因素进行了考察。开发出一系列全新的、以甘露醇添加剂为特征，具有超低粘度和高分辨率的毛细管电泳无胶筛分介质。根据对筛分机理的讨论和较为系统的应用实践，提出了对基因分析具有指导意义的毛细管电泳无胶筛分总体原则。以自行研制和开发的上述筛分介质为载体，对毛细管电泳在医学基因诊断和分子生物学的各个相关分支领城中的应用进行了系统的、有一定覆盖面的研究。结果表明，我们所发展的毛细管电泳无胶筛分介质不仅能够应用于基因突变的检测和基因的家系连锁分析，而且可以应用于分子生物学基础研究中实现对基因表达及剪接多样性的研究和基因的定量分析。鉴于毛细管电泳所具有的通过对体内新陈代谢产物的检测和定量进行疾病诊断的可能性，作为一个补充和应用实例，本论文使用毛细管区带电泳和激光诱导荧光检测，测定了正常人和癌症病人尿样中喋啶类化合物的含量。

    鉴于人类基因组计划（Human Genome Project, HGP）无可争辩的意义及毛细管电泳作为主流技术之一介入这一计划造成的实际影响，论文第一篇较为系统地介绍了基因及其分析在医学、法医学、分子生物学等领域的重要性，以及毛细管电泳在基因分析各个领域的应用价值。

    论文第二篇集中探讨了羟丙基甲基纤维素作为筛分介质的筛分性能以及各种因素，包括羟丙基甲基纤维素的浓度和分子量、电场强度、毛细管内壁的修饰、毛细管的温度、DNA嵌入试剂、甘露醇添加剂、尿素变性剂等等，对DNA片段分离的影响，探讨了在这一系统中DNA片段的分离机理。在国际上首次报导甘露醇添加剂对DNA片段分离的影响，并首次观察到DNA片段在非变性筛分介质分离中由于空间结构对迁移的影响而出现的峰倒转现象。发现不管是在常规粘度还是超低粘度筛分介质中，甘露醇添加剂的存在无一例外地提高了对DNA片段的分离能力，其中甘露醇对于超低粘度的筛分介质更是一种必要的添加剂。甘露醇特殊的分子结构使之优越于甘油或其它添加剂而具有重要的应用价值。鉴于DNA空间结构对迁移的影响能够导致PCR产物鉴定的错误，我们明确提出了尿素在毛细管电泳PCR产物检测中的重要性和必要性。

首次提出使用小分子量的高分子作为筛分介质分离DNA片段。以分子量为10，000g/mol的羟丙基甲基纤维素为基础，加以甘露醇改性剂，开发出一种具有超低粘度的能够对DNA片段进行高分辨分离的无胶筛分介质。其粘度仅为常规筛分介质的10%，对DNA分离的柱效高达1，800，000塔板数/米。这种超低粘度的筛分介质有望在具有重大应用前景的芯片毛细管电泳中得到广泛的使用，以克服后者由于微小尺寸所造成的尺寸效应带来的种种困难。此外，由于该筛分介质的粘度和水的粘度在同一数量级，因此克服了常规粘度筛分介质难以灌入毛细管以及会粘附于毛细管外壁从而给样品带来污染的困难。

论文第三篇集中探讨了利用上述自行研制的筛分介质在医学和分子生物学等领域中开展大规模基因分析的可能性。在医学领域主要表现为以基因突变检测，基因家系连锁分析，特定基因的PCR产物直接检测为基础的基因诊断。其中包括，结合多重PCR技术，对引起杜氏肌营养不良症的基因突变进行了检测，对三例由于基因缺失突变而导致的杜氏肌营养不良症进行了基因诊断；首次使用毛细管电泳技术，通过对与苯丙氨酸羟化酶基因连锁的短串联重复（PAH STR）位点多态性进行分析，对80例中国人的PAH STR等位基因多态性位点的分布情况作了调查，包括6个苯丙酮尿症家系的连锁分析，并对其中的一个家系进行了产前基因诊断；以结核杆菌、链球菌和解脲支原体等致病微生物为例，通过对病原体特异性PCR产物的直接检测对某些感染性疾病做出了早期诊断等。在对病原体检测的过程中，论文对平板电泳所做出的假阴性结果进行了校正。同时发现，毛细管电泳还可能用于病原体类型的鉴别。除此之外，通过对3-4个细胞的PCR产物的检测，进一步显示了毛细管电泳技术的灵敏度，为遗传性疾病的无创产前诊断、癌症的预后监测提供了技术储备。

在分子生物学基础研究领域，通过对特定基因的RT-PCR产物的分析，实现了对基因的表达和选择性剪接的直接分析。本篇使用毛细管电泳技术对引起脆性X综合症的FMR1基因的选择性剪接异构，及该基因在胎儿和成人不同组织中的表达情况进行了研究，揭示出其与克隆化技术相比在速度、灵敏度和成本等方面的优势。与此同时，针对医学、分子生物学中对PCR产物进行定量分析的要求，开发出－个快捷PCR产物定量方法。据此方法可以通过毛细管电泳一次运行对PCR产物进行准确定量，比传统的平板电泳-光密度扫描法和标准曲线法更为准确、迅速、可靠。

    在论文的第四篇，作为对基因分析的一个补充，以喋啶类化合物的分析为例，探讨了毛细管电泳在人体新陈代谢产物分析中的应用，揭示了喋啶类化合物组群作为肿瘤标记物的可能性。喋啶是人体新陈代谢过程中重要的因子之一，应用毛细管电泳-激光诱导荧光检测可以测定尿液中喋啶类化合物的含量。通过对健康人和癌症病人尿液中喋啶类化合物组群分析发现，在罹患癌症的病人的尿液中，喋啶类化合物的含量和正常人相比会有明显升高。这一结果提示喋啶类化合物可能是一种重要的癌症标记物，对喋啶类化合物量的测定可望提供一种癌症普查、早期诊断、预后跟踪以及疗效确定的简便方法。

Abstract

A series of sieving matrices, based on hydroxypropylmethylcellulose and modified by mannitol, were developed for DNA separation and analysis by capillary electrophoresis (CE). The many factors which influence the separation of DNA fragments were also investigated. The sieving mechanism of the DNA fragments in the sieving buffers were discussed, and the basic principles for DNA separation were presented. Some applications were carried out based on the sieving matrices we developed, and it was found that those sieving matrices can be used in clinical or molecular biology institutes for mutation detection, family linkage analysis and PCR product detection and characterization. Due to the advantages of CE in the area of biochemical analysis, it can also be used to monitor the levels of some metabolic products. As a supplement and example, capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF) was employed to determine the pteridine levels in urines from normal persons and cancer patients.

 In view of the significance of the Human Genome Project (HGP) and the importance of CE as a main technique in the implement of this project, in the first part of this thesis, we have introduced the basic knowledge of gene analysis, and its application in clinical diagnosis, forensic identification, and molecular biology sciences. The significance of CE in the area of gene analysis was also presented.

In the second part of this thesis, we have explored the possibility of separation of some DNA fragments such as PBR322/Hae III and PGEM- 3Zf(+)/Hae III. The factors that influence the migration and separation of the DNA fragments including capillary, temperature, electric field and additives were investigated. The migration mechanism of the DNA fragments in these sieving matrices was also discussed. For the first time in the world, we have employed mannitol as a buffer modifier to enhance the separation ability of HPMC-based sieving matrices. It was found that the separation of the DNA fragments can be improved by adding mannitol to the sieving buffer. This is true not only for the buffer with a conventional viscosity but also for the buffer with an ultra-low viscosity. Mannitol is proved to be an indispensable additive to the latter one. The special structure of mannitol enables itself to be a more valuable additive than glycerol. We are also among the very first who have observed a peak reversion when two co-amplified PCR products migrated together, which implies the influence of DNA conformation on migration is not neglectible. This influence will cause trouble in the identification of some PCR products. It is proposed in this thesis that a denaturant, such as urea, must be present in the sieving matrix in order to eliminate the conformation influence when doing analysis of PCR products.

A sieving buffer with an ultra-low viscosity was developed based on a HPMC polymer with a low molecular weight (1,000,000 g/mol). The viscosity of this sieving matrix is only about 10% of the viscosity of the conventional sieving matrices. The separation ability is satisfactory, and the highest efficiency (l,800,000 plates/m) was obtained in this sieving matrix. Ultra-low viscosity is one of the main advantages because this sieving matrix can be used in a microfabricated electrophorus device, for which conventional sieving matrices are not suitable. In addition, this sieving matrix enables an easy capillary fill-in and pump-out and also prevents the samples from being contaminated by the viscous buffer sticked on the capillary wall.

The third part of this dissertation mainly talked about the applications of the above sieving matrices on gene diagnosis of some inherited disease and infectious disease, and on molecular biology researches. First of all, mutation analysis of Duchenne muscular dystrophy (DMD) gene was carried out by CE coupled with multi-PCR technique. Three patients who carried deletion mutations of DMD gene were diagnosed. The short tandem repeat (STR) alleles of the PAH gene were separated and characterized, and the polymorphism of the PAN STR alleles in 80 Chinese people including 6 phenylketonuria (PKU) family was analyzed. Family linkage analysis of some PKU families were carried out, and prenatal diagnosis of PKU was applied to one of the PKU family. Early diagnosis is important for the early treatment of infectious disease. Early diagnosis was done by direct detection of the PCR products amplified from several kinds of pathogens, such as Mycobacterium Tuberculosis, S streptococcus and Bacteroids Ureolyticus, etc. Our experiments showed that CE is a fast, convenient technique for such kind of diagnosis. The more important is that CE can correct the false negative result obtained by slab gel electrophoresis, and can identify different subtypes of pathogens, Detection of trace amount of PCR product is very important in practical works. PCR product amplified from 3-4 cells can be detected successfully in our study, which implies that this technique may be a latent method for non-invasive prenatal diagnosis, early detection of infectious disease, and prognosis of cancers.

Investigation of gene expression and gene alternative splicing by CE was earned out in this study by analyzing the RT-PCR products of the target gene, FMR1 gene, a gene leading to fragile X syndrome. The results showed that CE is a more advantageous technique over traditional techniques such as colonization method. At the same time, a method for fast quantitation of PCR products was developed. A more precise result for quantitation will be obtained within a single run.

Except the application of CE in gene analysis, it also has abundant applications on the determination of some small molecules, of which most of them are metabolic products or intermediate products. As an example, we examine the pteridine levels in human urine by CE-LIF. The results showed a very good correlation between pteridine levels and tumor disease. There was a significant enhancement in some kinds of pteridines excreted in urine by patients carrying malignant tumors. This may indicate that pteridines may be important tumor markers, and this CE-LIF method can be used in clinical laboratories either for cancer  monitoring or pre-cancer screening.