无胶筛分毛细管电泳检测基因突变的方法学研究及其在胃癌中的应用
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摘要
基因突变与肿瘤发生有着较高的相关性,所以探索研究新的、灵敏度高的检测基因突变的方法是相关领域专家共同关注的热点问题,也是交叉学科。目前几乎所有的基因突变检测都是建立于PCR基础之上,操作简便准确率高的基因分析技术限制性片段长度多态性(RFLP)常与其结合用于基因突变的检测,但结果往往需要借助电泳技术显示,而传统的平板凝胶电泳分辨率低、分析时间长,不能很好的用于各种长度DNA片段尤其是小片段的多态性分析。随着与高效毛细管电泳技术(CE)联合在分子生物学中的广泛应用,使得RFLP-CE具备了检测灵敏度高、分离度高和检测效率高的特点。建立高效的检测基因突变方法,可为临床肿瘤的准确诊断和预警提供更为科学的技术和方法。本论文以此为目的探索建立了一套适用于RFLP技术的毛细管电泳分析分离体系用于临床胃癌组织基因突变检测,系统考察了毛细管电泳体系中不同筛分介质分离不同大小和范围的DNA片段的能力。
本论文共分为三部分,第一部分毛细管电泳在基因突变及多态性分析中的研究背景,第二部分是毛细管无胶筛分电泳中不同筛分介质对DNA分离的方法学研究,第三部分是选择合适的毛细管电泳分离体系联合RFLP检测胃癌组织中p53基因和ras基因点突变,并探讨p53基因和ras基因点突变在胃癌发生的作用和地位。
第一部分研究背景介绍,主要综述了胃癌发生相关基因p53和ras基因及异常基因检测方法研究进展,毛细管电泳研究背景主要集中在筛分机理、筛分介质、毛细管涂层技术和其他一些影响电泳的因素以及毛细管电泳在基因突变及多态性分析方面的应用进展等。
第二部分毛细管无胶筛分电泳(NGS-CE)中不同筛分介质对DNA分离的方法学研究,目的是通过系统考察不同筛分介质分离不同大小和范围DNA片段的能力,建立一套适用不同大小的基因双链分离体系。首先应用甲基纤维素(MC)为筛分介质并结合本实验室成熟的毛细管共价涂层技术,考察了对pUC19 DNA/MspⅠ(HpaⅡ)DNAMarker、PBR322/BsuRI DNA Marker和FastRuler~(TM)DNA Ladder的分离情况,系统研究了MC浓度、温度、分离电压、筛分介质pH等因素对双链DNA分离的影响,结果表明MC是一种良好的筛分介质,能成功分离上述三种不同的DNA片段;其次,应用具有动态涂层能力的分子量相对较小的聚乙烯吡咯烷酮(PVP)、聚环氧乙烷(PEO)为筛分介质在空毛细管柱中考察了对pUC19 DNA/MspⅠ(HpaⅡ)DNA Marker、PBR322/BsuRI DNA Marker和FastRuler~(TM)DNA Ladder的分离情况,结果表明PVP分离pUC19 DNA/MspⅠ(HpaⅡ)DNA Marker、PBR322/BsuRI DNA Marker的效果不佳,而分离FastRuler~(TM)DNA Ladder能够获得很高的分离度;PEO能很好的分离pUC19DNA/MspⅠ(HpaⅡ)DNA Marker和FastRuler~(TM)DNA Ladder。建立的这一套双链DNA片段分离体系最终可以达到同时分离10bp～600bp的双链DNA,符合RFLP分析基因突变的要求。
第三部分应用已经建立的无胶筛分毛细管电泳分离体系与PCR-RFLP技术相结合用于胃癌组织p53基因175位、245位密码子和H-ras、K-ras基因12位密码子突变情况的同时检测。提取了59例胃癌患者及癌旁正常组织的基因组DNA,经测定DNA浓度适中,纯度较好。利用PCR扩增出相应的目的片段,使用UNIQ-10柱式DNA胶回收试剂盒纯化PCR产物,排除了PCR产物中离子和引物二聚体对后续研究的干扰,扩增的目的片段中,有的片段较小不能进行纯化。相应的限制性内切酶切割纯化的PCR产物,然后应用毛细管电泳分离体系(筛分介质为2.0%MC,pH8.0,分离电压7.5kV,电泳温度25℃)分析临床59例胃癌患者p53和ras基因扩增产物的限制性片段长度多态性。20min内检测了各个基因不同位点的突变情况。初步建立了快速、高分辨率诊断胃癌的方法。结果显示胃癌的p53基因点突变率为20.51%(8/39),ras基因点突变率为11.11%(5/45)。其中p53基因175位密码子有5例发生突变(5/39,12.82%),245位密码子有3例发生突变(3/39,7.69%),H-ras基因有1例发生突变(1/45,2.22%),K-ms基因有4例发生突变(4/45,8.89%),只有在2例中同时发现p53和ras基因发生突变。经统计学处理,p53基因突变和发生部位无明显相关性,ras基因的突变主要集中在胃体和胃底贲门。p53和ras基因在胃癌和胃癌组织不同病理分型中的突变率结果显示,p53基因突变在部分胃癌的发生中起着较重要的作用,ras基因突变可能在胃癌的发生中作用不明显。
Gene mutation is correlated to the pathogenesis of gastric cancer and its development. We should search for a new method to detect the gene mutation, which is a focal point to most experts and also is a crossing science. At present, the most gene mutation method is based on PCR technology. Restriction fragment length polymorphism which is characterized by simplicity, high veracity is often together with PCR to detect gene mutation, and it need electrophoresis for analysis of gene mutation. Slab-gel electrophoresis is characterized by low sensitivity, low automation and long time consuming. It can not be cosmically applied in clinical diagnosis. With the widespread application in the molecular biology of RFLP ether with CE, it becomes high sensitivity, high resolution and high efficiency.
We should set up high efficiency method of gene mutation detection, which can provide scientific foundation of technology and method for clinical diagnosis and prewaming of gastric cancer and this is our purpose in this study. We have set up a system suitable for RFLP-CE and applied to the gene mutation detection of gastric cancer. We systematically studied the separation ability of different sieving matrixes to separate different size and range DNA standard fragments.
This dissertation has three parts. First part is the study background of capillary electrophoresis in gene mutation/polymorphism. That different sieving matrixes separate different DNA fragments in non-gel sieving capillary electrophoresis is studied in second part. The third part is about detection of point mutation in gastric cancer tissues with the suitable capillary electrophoresis system together with RFLP, and we discuss the effect and position of p53 and ras gene in the pathogenesis of gastric cancer.
The first part reviewed p53 and ras gene, which were relative to the pathogenesis of gastric cancer and the development of abnormal gene detection. CE technology development mainly concentrated to sieving mechanism, sieving matrixes, column coating and other effects, and the application of gene mutation/ polymorphism with CE.
In second part, the separation method of different DNA fragments using different sieving matrixes were investigated by NGS-CE. In order to set up a system of double-strands DNA separation, we systematically investigated the separation ability of different sieving matrixes separating different size and range DNA fragments. First, methyl cellulose was used to separate pUC19 DNA/Msp I (Hpa II) DNA Marker, PBR322/BsuRI DNA Marker and FastRuler~(TM) DNA Ladder with capillary column coating. The effects of concentration of methyl cellulose, running pH, running temperature and running voltage on dsDNA(< 600bp)analysis were investigated. Methyl cellulose is an excellent medium and it can separate different size and range DNA fragments; PVP and PEO, which are characterized by dynamic coating, relative smaller molecular weight were used to separate pUC19 DNA/Msp I (Hpa II) DNA Marker, PBR322/BsuRI DNA Marker and FastRuler~(TM) DNA Ladder in vacuous capillary column. pUC19 DNA/Msp I (Hpa II) DNA Marker and PBR322/BsuRI DNA Marker separation of PVP were not well, but FastRuler~(TM) DNA Ladder separation of PVP obtained high resolution. PEO can separate pUC19 DNA/Msp I (Hpa II) DNA Marker and FastRuler~(TM) DNA Ladder pretty well. The system we have set up can simultaneously separate dsDNA (10bp-600bp) and it is suitable for gene mutation analysis of RFLP.
In third part, the mutations of codon 175 and 245 in p53 and codons 12 in H-ras and K-ras were simultaneously detected by NGS-CE-RFLP. Genomes of gastric cancer patients were extracted. The extracted genomes DNA had better concentration and purification. The purpose DNA fragment were amplified by PCR. PCR product was purified by UNIQ-10 Column DNA Purification Kit to remove salts and primer-dimer. PCR products were cleaved by correct restrictive enzymes. The optimum separation condition was applied in analyzing RFLP of PCR product of p53 and ras gene. The optimum separation condition was 2.0% MC, running pH at 8.0, running voltage at 7.5 kV, running temperature at 25°C. Mutation detection was completed in about 20min.A fast, high resolving power method was initially established in clinical diagnosis of gastric cancer. We detected the mutation rates of p53 and ras gene in different gastric cancer tissue and disguss their effect and position in the pathogenesis of gastric cancer. p53 gene mutation was detected in 8 out of 39 gastric cancer cases (8/39, 20.51%), ras gene mutation was detected in 5 out of 45 gastric cases (5/45, 11.11%). 5 cases(5/39,12.82%) of mutation of p53 175 codon, 3 cases (3/39, 7.69 %)of mutation of p53 245 codon, 1 cases(l/45, 2.22%) of mutation of H-ras 12 codon and 4 cases(4/45, 8.89%)of mutation of K-ras 12 codon were detected. p53 and ras gene mutation were simultaneously detected in 2. The mutation of p53 has no obvious relationship to the pathogenesis areas of gastric cancer, ras gene mutation is mainly in gastric body and cardia. p53 gene mutation plays an important role in some of the gastric cancer patients, ras gene mutation plays no obvious role in the pathogenesis of gastric cancer.
本论文共分为三部分,第一部分毛细管电泳在基因突变及多态性分析中的研究背景,第二部分是毛细管无胶筛分电泳中不同筛分介质对DNA分离的方法学研究,第三部分是选择合适的毛细管电泳分离体系联合RFLP检测胃癌组织中p53基因和ras基因点突变,并探讨p53基因和ras基因点突变在胃癌发生的作用和地位。
第一部分研究背景介绍,主要综述了胃癌发生相关基因p53和ras基因及异常基因检测方法研究进展,毛细管电泳研究背景主要集中在筛分机理、筛分介质、毛细管涂层技术和其他一些影响电泳的因素以及毛细管电泳在基因突变及多态性分析方面的应用进展等。
第二部分毛细管无胶筛分电泳(NGS-CE)中不同筛分介质对DNA分离的方法学研究,目的是通过系统考察不同筛分介质分离不同大小和范围DNA片段的能力,建立一套适用不同大小的基因双链分离体系。首先应用甲基纤维素(MC)为筛分介质并结合本实验室成熟的毛细管共价涂层技术,考察了对pUC19 DNA/MspⅠ(HpaⅡ)DNAMarker、PBR322/BsuRI DNA Marker和FastRuler~(TM)DNA Ladder的分离情况,系统研究了MC浓度、温度、分离电压、筛分介质pH等因素对双链DNA分离的影响,结果表明MC是一种良好的筛分介质,能成功分离上述三种不同的DNA片段;其次,应用具有动态涂层能力的分子量相对较小的聚乙烯吡咯烷酮(PVP)、聚环氧乙烷(PEO)为筛分介质在空毛细管柱中考察了对pUC19 DNA/MspⅠ(HpaⅡ)DNA Marker、PBR322/BsuRI DNA Marker和FastRuler~(TM)DNA Ladder的分离情况,结果表明PVP分离pUC19 DNA/MspⅠ(HpaⅡ)DNA Marker、PBR322/BsuRI DNA Marker的效果不佳,而分离FastRuler~(TM)DNA Ladder能够获得很高的分离度;PEO能很好的分离pUC19DNA/MspⅠ(HpaⅡ)DNA Marker和FastRuler~(TM)DNA Ladder。建立的这一套双链DNA片段分离体系最终可以达到同时分离10bp～600bp的双链DNA,符合RFLP分析基因突变的要求。
第三部分应用已经建立的无胶筛分毛细管电泳分离体系与PCR-RFLP技术相结合用于胃癌组织p53基因175位、245位密码子和H-ras、K-ras基因12位密码子突变情况的同时检测。提取了59例胃癌患者及癌旁正常组织的基因组DNA,经测定DNA浓度适中,纯度较好。利用PCR扩增出相应的目的片段,使用UNIQ-10柱式DNA胶回收试剂盒纯化PCR产物,排除了PCR产物中离子和引物二聚体对后续研究的干扰,扩增的目的片段中,有的片段较小不能进行纯化。相应的限制性内切酶切割纯化的PCR产物,然后应用毛细管电泳分离体系(筛分介质为2.0%MC,pH8.0,分离电压7.5kV,电泳温度25℃)分析临床59例胃癌患者p53和ras基因扩增产物的限制性片段长度多态性。20min内检测了各个基因不同位点的突变情况。初步建立了快速、高分辨率诊断胃癌的方法。结果显示胃癌的p53基因点突变率为20.51%(8/39),ras基因点突变率为11.11%(5/45)。其中p53基因175位密码子有5例发生突变(5/39,12.82%),245位密码子有3例发生突变(3/39,7.69%),H-ras基因有1例发生突变(1/45,2.22%),K-ms基因有4例发生突变(4/45,8.89%),只有在2例中同时发现p53和ras基因发生突变。经统计学处理,p53基因突变和发生部位无明显相关性,ras基因的突变主要集中在胃体和胃底贲门。p53和ras基因在胃癌和胃癌组织不同病理分型中的突变率结果显示,p53基因突变在部分胃癌的发生中起着较重要的作用,ras基因突变可能在胃癌的发生中作用不明显。
Gene mutation is correlated to the pathogenesis of gastric cancer and its development. We should search for a new method to detect the gene mutation, which is a focal point to most experts and also is a crossing science. At present, the most gene mutation method is based on PCR technology. Restriction fragment length polymorphism which is characterized by simplicity, high veracity is often together with PCR to detect gene mutation, and it need electrophoresis for analysis of gene mutation. Slab-gel electrophoresis is characterized by low sensitivity, low automation and long time consuming. It can not be cosmically applied in clinical diagnosis. With the widespread application in the molecular biology of RFLP ether with CE, it becomes high sensitivity, high resolution and high efficiency.
We should set up high efficiency method of gene mutation detection, which can provide scientific foundation of technology and method for clinical diagnosis and prewaming of gastric cancer and this is our purpose in this study. We have set up a system suitable for RFLP-CE and applied to the gene mutation detection of gastric cancer. We systematically studied the separation ability of different sieving matrixes to separate different size and range DNA standard fragments.
This dissertation has three parts. First part is the study background of capillary electrophoresis in gene mutation/polymorphism. That different sieving matrixes separate different DNA fragments in non-gel sieving capillary electrophoresis is studied in second part. The third part is about detection of point mutation in gastric cancer tissues with the suitable capillary electrophoresis system together with RFLP, and we discuss the effect and position of p53 and ras gene in the pathogenesis of gastric cancer.
The first part reviewed p53 and ras gene, which were relative to the pathogenesis of gastric cancer and the development of abnormal gene detection. CE technology development mainly concentrated to sieving mechanism, sieving matrixes, column coating and other effects, and the application of gene mutation/ polymorphism with CE.
In second part, the separation method of different DNA fragments using different sieving matrixes were investigated by NGS-CE. In order to set up a system of double-strands DNA separation, we systematically investigated the separation ability of different sieving matrixes separating different size and range DNA fragments. First, methyl cellulose was used to separate pUC19 DNA/Msp I (Hpa II) DNA Marker, PBR322/BsuRI DNA Marker and FastRuler~(TM) DNA Ladder with capillary column coating. The effects of concentration of methyl cellulose, running pH, running temperature and running voltage on dsDNA(< 600bp)analysis were investigated. Methyl cellulose is an excellent medium and it can separate different size and range DNA fragments; PVP and PEO, which are characterized by dynamic coating, relative smaller molecular weight were used to separate pUC19 DNA/Msp I (Hpa II) DNA Marker, PBR322/BsuRI DNA Marker and FastRuler~(TM) DNA Ladder in vacuous capillary column. pUC19 DNA/Msp I (Hpa II) DNA Marker and PBR322/BsuRI DNA Marker separation of PVP were not well, but FastRuler~(TM) DNA Ladder separation of PVP obtained high resolution. PEO can separate pUC19 DNA/Msp I (Hpa II) DNA Marker and FastRuler~(TM) DNA Ladder pretty well. The system we have set up can simultaneously separate dsDNA (10bp-600bp) and it is suitable for gene mutation analysis of RFLP.
In third part, the mutations of codon 175 and 245 in p53 and codons 12 in H-ras and K-ras were simultaneously detected by NGS-CE-RFLP. Genomes of gastric cancer patients were extracted. The extracted genomes DNA had better concentration and purification. The purpose DNA fragment were amplified by PCR. PCR product was purified by UNIQ-10 Column DNA Purification Kit to remove salts and primer-dimer. PCR products were cleaved by correct restrictive enzymes. The optimum separation condition was applied in analyzing RFLP of PCR product of p53 and ras gene. The optimum separation condition was 2.0% MC, running pH at 8.0, running voltage at 7.5 kV, running temperature at 25°C. Mutation detection was completed in about 20min.A fast, high resolving power method was initially established in clinical diagnosis of gastric cancer. We detected the mutation rates of p53 and ras gene in different gastric cancer tissue and disguss their effect and position in the pathogenesis of gastric cancer. p53 gene mutation was detected in 8 out of 39 gastric cancer cases (8/39, 20.51%), ras gene mutation was detected in 5 out of 45 gastric cases (5/45, 11.11%). 5 cases(5/39,12.82%) of mutation of p53 175 codon, 3 cases (3/39, 7.69 %)of mutation of p53 245 codon, 1 cases(l/45, 2.22%) of mutation of H-ras 12 codon and 4 cases(4/45, 8.89%)of mutation of K-ras 12 codon were detected. p53 and ras gene mutation were simultaneously detected in 2. The mutation of p53 has no obvious relationship to the pathogenesis areas of gastric cancer, ras gene mutation is mainly in gastric body and cardia. p53 gene mutation plays an important role in some of the gastric cancer patients, ras gene mutation plays no obvious role in the pathogenesis of gastric cancer.
引文
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