【作者】 朱伟锋；

【导师】 万福生；

【作者基本信息】 南昌大学医学院 ， 内科学， 2012， 博士

【摘要】 随着人类基因组计划的完成,越来越多的研究开始关注基因多态性在医学方面的应用。要分析基因多态性,首先要得到DNA。口腔黏膜上皮细胞是分子流行病学、法医学和遗传学研究中重要的DNA来源。目前有多种方法可以用于口腔黏膜上皮细胞DNA的提取,各有其优缺点。还没有一种方法能满足安全、快速、简便、经济、可靠的要求。盐析法是全血基因组DNA提取常用的方法之一,具有简便快捷、经济安全、稳定可靠的特点。如果盐析法能用于口腔黏膜上皮细胞DNA的提取,可能会是一种较好的口腔黏膜上皮细胞DNA的提取方法。作为人类基因组中最常见的遗传变异,单核苷酸多态性与疾病的发生、发展、预后以及疾病治疗过程中药物的疗效和毒副作用之间的关系日益受到重视。对SNPs的准确分型有助于疾病的预防、早期诊断和治疗。虽然目前有很多SNPs的分型方法,但有的需要特殊的设备、有的需要昂贵的试剂、有的成本较高。如果能用普通PAGE直接对SNPs进行分型,可大大降低实验成本,提高效率。目的：1、建立一种安全、快速、简便、经济、可靠的口腔拭子DNA提取方法。2、建立一种能在普通实验室开展的准确、简便、快速、低成本、高通量的SNPs分型方法。3、应用建立的SNPs分型方法对临床样本进行分析。方法：1、以在本研究室做实验的志愿者提供的10支口腔拭子为例,用自己配制的试剂进行DNA提取。用细胞裂解液和蛋白酶K消化口腔上皮细胞,然后用5mol/L NaCl沉淀蛋白质,用异丙醇沉淀DNA,最后用70%乙醇洗涤得到的DNA并将其溶于TE中。用紫外分光光度计对得到的DNA进行浓度和纯度检测。以得到的DNA为模板,用聚合酶链反应-限制性片段长度多态性技术对两个SNPs进行分型。对不同基因型的样本测序验证。2、用自己建立的盐析法快速提取口腔拭子DNA的方法,对130例志愿者提供的口腔拭子进行DNA提取。以2个方面来源的DNA为模板,对13个SNPs(MTHFR基因A1298C、CYP19A1基因rs10046和rs4646.CYP3A5基因A6986G、TP53基因rs1042522. NQO1基因C609T、GCKR基因rs780094、RGS4基因rs951436、CHRNA3基因rs12910984、MYOC基因-1081A/G和-998C/G、MDR1基因3489+59T>G和3489+80C>T)进行分析。对在SNPs附近有内切酶酶切位点的5个SNPs(TP53基因rs1042522、GCKR基因rs780094、NQO1基因rs1800566、MTHFR基因A1298C和CHRNA3基因的rs12910984)进行酶切分析。对可能影响SNPs分型的DNA片段自身因素、PCR扩增条件和电泳条件进行优化,建立普通聚丙烯酰胺凝胶电泳分析SNPs的方法。在检测条件优化的基础上,对13个SNPs进行检测。为了进行高通量检测,还进行了复合扩增和1块胶上分10层上样。对所有的SNPs进行测序验证。对聚丙烯酰胺凝胶电泳结果、酶切结果和测序结果进行比较。3、用建立的SNPs分型方法对104例使用5-FU为基础的化疗的晚期胃癌患者样品的3个SNPs位点(MTHFR基因A1298C、MDR1基因3489+59T>G和3489+80C>T)进行分析,并研究这3个位点多态性与化疗有效率之间的关系。结果：1、建立的盐析法提取口腔拭子DNA所需时间在1小时左右,提取单支口腔拭子DNA的全部费用不超过1.5元。单支口腔拭子得到的DNA量在0.68μg-2.56μg之间,OD260/OD280比值在1.77-1.94之间。经PCR扩增和酶切消化,10个样本的两个单核苷酸多态性都得到了清楚分型。酶切结果与测序结果吻合。2、5个SNPs的酶切都得到了清楚分型。聚丙烯酰胺凝胶电泳分析SNPs的优化结果显示：GC含量和碱基组成对电泳的影响不大,而SNPs在片段中的位置和扩增片段长度可能是影响聚丙烯酰胺凝胶电泳对SNPs分型的重要因素。就PCR扩增来说,模板量、DNA聚合酶、退火温度、循环数都可能会影响聚丙烯酰胺凝胶电泳对SNPs的分型。把这种以构象不同为基础的对SNPs分型的方法称为构象差异凝胶电泳。对13个SNPs的分析显示有8个SNPs可以被直接分型,2个SNPs不能直接被分型,3个SNPs因样本量小不能确定。此外,在CHRNA3基因上还发现1个新的SNPs。10例样品NQO1基因C609T多态性PCR产物10层上样电泳图显示至少有7层可以清楚地区分3种基因型。4个SNPs的复合扩增也都得到了清楚的结果。DNA测序结果较好,证实了各扩增片段为目的片段。构象差异凝胶电泳结果和酶切、DNA测序结果完全一致,证明了构象差异凝胶电泳对SNPs分型的可靠性。与酶切相比,构象差异凝胶电泳对SNPs的分型不需要内切酶,因此成本较低。与单链构象多态性相比,由于构象差异凝胶电泳在PCR扩增后不需要对PCR产物进行变性,可以直接上样,因此较单链构象多态性成本更低、操作更简便。3、构象差异凝胶电泳上3个位点(MTHFR基因A1298C、MDR1基因3489+59T>G和3489+80C>T)的不同基因型都得到了清楚区分。3个位点的基因型分布均符合Hardy-Weinberg平衡。104例患者的总有效率为41.3%。不同基因型与化疗有效率之间的关系：①MTHFR基因A1298C：AA基因型有效率为34.8%,AC基因型有效率为50.0%,CC基因型有效率为66.7%。②MDR1基因3489+59T>G：GG基因型有效率为56.0%,GT基因型有效率为39.1%,TT基因型有效率为31.3%。③MDR1基因3489+80C>T：CC基因型有效率为27.3%,CT基因型有效率为36.2%,TT基因型有效率为48.9%。统计学分析表明化疗有效率与3个位点的多态性均无关。结论：1、盐析法可以快速提取口腔拭子基因组DNA,得到DNA的质量较高,可以用于分子流行病学、遗传学和法医学等领域的研究。2、构象差异凝胶电泳对SNPs的分型是一种能在普通实验室开展的准确、简便、快速、低成本、高通量的SNPs分型方法。3、晚期胃癌患者MTHFR基因A1298C、MDR1基因3489+59T>G和3489+80C>T多态性均与以5-FU为基础的化疗方案的有效率无关。更多还原

【Abstract】 With the accomplishment of Human Genome Project, more and more researches are being aware of the importance for the application of gene variation on medicine. Baccal mucosa epithelial cells are most common sources for DNA extraction in molecular epidemiology, forensic medicine, and genetic studies. Although there are different methods for DNA extraction from buccal mucosa epithelial cells, none of which can meet the standards of safety, rapidity, convenience, economy and reliability. Salting out method is a commonly used method for genomic DNA extraction from blood, which is characteristic for its lots of advantages. Whether salting out method can be used for genomic DNA extraction from buccal mucosa epithelial cells remains uncertain.Single nucleotide polymorphisms (SNPs), as one of the most common genetic variation in human genome, gains more and more attention on its relationship with the occurrence, development and prognosis of diseases, drugs efficacy and toxic side effects during disease therapy. Therefore, accurate detection of SNPs is helpful for early diagnosis and appropriate treatment of disease. There are many SNPs genotyping methods in which either complicated process, specific equipment, expensive reagents or experienced skills are needed. Therefore, if normal polyacrylamide gel electrophoresis (PAGE) can be applied for directly SNPs genotyping, it will significantly reduce cost and increase detection efficiency.Objects:To establish a rapid, simple, economic and reliable method for genomic DNA extraction from buccal mucosa swabs.To set up an accurate, simple, fast, low-cost and high-throughput SNPs genotyping method which can be applied in normal laboratories.Clinical samples would be analysed by the new method.Methods:10buccal mucosa swabs from volunteers in our lab were collected and DNA extracted with reagents made ourselves. Buccal mucosa epithelial cells were digested with cell lysate solution and proteinase K solution. Then the proteins were removed by salting out and centrifugation and DNA was precipitated with isopropyl alcohol, The precipitations of DNA were washed with70%ethanol, resuspended in TE. DNA was detected by UV spectrophotometer. The rs1042522loci of TP53gene and rs12910984loci of CHRNA3gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The samples with different genotypes were confirmed by direct sequencing analysis.Genomic DNA was extracted from130volunteers’ baccal swabs with previously well constructed salting out method.13SNPs (MTHFR gene A1298C, CYP19A1gene rs10046and rs4646, CYP3A5gene A6986G, TP53gene rs1042522, NQO1gene C609T, GCKR gene rs780094, RGS4gene rs951436, CHRNA3gene rs12910984, MYOC-1081A/G and-998C/G, MDR1gene3489+59T>G and3489+80C>T) were analyzed by PAGE.5of these13SNPs (TP53gene rs1042522, GCKR gene rs780094, NQO1gene rs1800566, MTHFR gene A1298C, and CHRNA3gene rs12910984), where had endonuclease restriction sites, were digested by restriction endonucleases. The own factors of DNA fragments and the conditions of PCR amplification and electrophoresis which might affect SNPs genotyping were optimized, to set up a method to analyze SNPs using normal PAGE. Based on the optimization of detecting conditions,13SNPs were detected. For high-throughput detection, samples were multiplex amplificated and loaded into10layers on1single gel. SNPs were confirmed by direct sequencing analysis. Then the results of PAGE, enzyme digestion and direct sequencing analysis were compared with each other.3SNPs(MTHFR gene A1298C、MDR1gene3489+59T>G and3489+80CT) were genotyped in104advanced gastric cancer samples in the treatment of fluorouracil based chemotherapy with the new method. The relationship between genotypes and response rate of chemotherapy were analysed.Results:Time and cost for the established salting out method for DNA extraction from baccal swabs were about1h and1.5Yuan per single buccal swab, respectively. The DNA yield of single buccal swab ranged from0.68μg to2.56μg; the OD26o/OD28o value ranged from1.77to1.94. After PCR amplification and enzyme digestion, two SNPs of10samples were clearly genotyped. The results of PCR-RFLP agreed well with the results of direct sequencing.5SNPs of enzyme digesting samples were clearly genotyped. The optimization results of analyzing SNPs with PAGE showed that, GC content and base composition had little effect on the electrophoresis, while the positions of SNPs in fragments and the length of amplified fragments might be the key influencing factors of SNPs genotyping with PAGE. For PCR amplification, template amount, DNA polymerase, annealing temperature and number of cycles might all affect the results of SNPs genotyping. Becaused this SNPs genotyping method is based on conformational differences, we call it conformation-difference gel electrophoresis (CDGE).13SNPs with CDGE showed that8of them were genotyped directly, while2SNPs could not directly be genotyped, and3SNPs were not clear due to the small sample size. In addition, one new SNPs was found in CHRNA3gene. Based on the results of electrophoresis with sample-loading10layers of NQO1C609T PCR pruducts of10samples, it showed that at least sample-loading with7layers could clearly separate three kinds of genotypes. The results of multiplex amplification of4SNPs were also clear. Due to the good results of direct sequencing, it was confirmed that the amplified fragments were the target ones. Based on the results of CDGE agreeing well with the results of enzyme digestion and direct sequencing, it confirmed the reliability of CDGE for SNPs genotyping. In comparision with enzyme digestion, CDGE did not need restriction endonucleases and was low-cost. And in contrast with single-strand conformation polymorphism (SSCP), CDGE was low-cost and convenient owing to no need for denature of PCR products and direct sample loading after PCR amplification.Different genotypes of3SNPs were distinguished clearly on CDGE.The genotypes distribution of3SNPs were accorded with Hardy-Weinberg equilibrium. Of all the104cases, the overall response rate was41.3%. The relationship between different genotypes and the response rate:①MTHFR gene A1298C, the response rates to chemotherapy of AA, AC, CC genotypes were34.8%,50.0%and66.7%, respectively.②MDR1gene3489+59T>G, the response rates to chemotherapy of GG, GT, TT genotypes were34.8%,50.0%and66.7%, respectively.③MDR1gene3489+80C>T, the response rates to chemotherapy of CC, CT, TT genotypes were27.3%,36.2%and48.9%, respectively. No significant difference in response rate to chemotherapy was observed according to the3SNPs genotypes.Conclusions:High quality DNA can be rapidly obtained with salting out method from baccal swabs and applied in molecular epidemiology, forensic medicine, and genetic studies.CDGE is an accurate, simple, fast, low-cost and high-throughput SNPs genotyping method which can be used in normal laboratories.No significant difference in response rate to advanced gastric cancer samples in the treatment of fluorouracil based chemotherapy was observed according to the MTHFR gene A1298C, MDR1gene3489+59T>G and3489+80C>T genotypes.更多还原