微卫星DNA在近交系小鼠遗传监测中的应用

发布时间：2018-04-19 22:02

  本文选题：聚合酶链反应 + 遗传监测　； 参考：《郑州大学》2007年硕士论文

【摘要】： 近交系小鼠(Inbred strain mice)是指经连续20代以上的全同胞兄妹或亲子交配培育而成，品系内所有个体都可追溯到起源于第20代或以后代数的一对共同祖先的遗传群。采用近交系的动物可以提高实验结果的准确性、可比性和可重复性，减少实验动物数量和实验重复次数。近交系小鼠的显著特征是基因纯合性及遗传稳定性，但在培育、保种及繁殖生产过程中，存在着发生遗传污染或遗传变异的可能，所以有必要对近交系小鼠进行严格、长期、定时的遗传监测。一些传统的方法如：皮肤移植、生化标记等通过检测其表型变化来推测其基因变化的方法有明显的局限性。 微卫星DNA是核苷酸短串联重复序列(short tandem repeats)，具有分布广、数目多、稳定、易于扩增等特点，采用PCR扩增微卫星DNA可直接检测DNA水平的变化，能够全面地反映出基因组的遗传概貌及变异情况，是近交系动物理想的遗传监测标记。本研究采用10条染色体上的20个微卫星DNA位点对近交系小鼠的DNA多态性进行研究：提取小鼠基因组DNA并利用微卫星引物PCR扩增，然后通过变性聚丙烯酰胺凝胶电泳及银染显色等技术显示微卫星DNA扩增产物。最后采用遗传学数据分析方法进行分析，以期筛选出具有显著多态性的微卫星位点，用于近交系小鼠的遗传监测。 方法：选用七品系近交系小鼠(DBA/2、CBA、C57、C3H/HeJ、BALB/C、FVB/NJ、YYHL小鼠)及一个封闭群小鼠(昆明小鼠)各五只。用酚—氯仿抽提法提取小鼠的基因组DNA，用PCR扩增以下位点：D4 mit9、D5mit31、D6mit15、D6mit16、D3mit36、D7mit12、D7mit29、D8mit11、D8mit22、D8mit24、D8mit30、D9mit21、 D10mit29、D11mit35、D11mit36、D12mit8、D12mit29、D12mit34、D12mit35、D13mit18，将PCR产物与等位基因分型标准物同步高压4-6％变性聚丙烯酰胺凝胶电泳，电泳后经过固定、染色、显色观察带型。根据DNA在变性聚丙烯酰胺凝胶上泳动的距离，通过微卫星基因座扩增条带与对照等位基因分型标准物的比较，进行结果判读并统计八品系小鼠在这些位点上表现出多态性条带的数目。扩增结果按Lynth氏法计算相似系数并分析其相似性。 结果： 1．近交系小鼠纯合性的分析：不同近交系品系及同品系不同个体小鼠之间的扩增产物中，同一微卫星位点呈现一清晰条带，表明河南省及全国目前有关这些近交系小鼠及其群体没有发生遗传污染或遗传变异，符合近交系的要求。 2．不同品系近交系小鼠之间微卫星多态性比较的结果： (1)：不同品系近交系小鼠之间微卫星多态性比较：在20个微卫星位点中有16个位点：D4Mit9、D5Mit31、D6Mit15、D6Mit16、D6Mit36、D7Mit12、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit8、D12Mit29、D12Mit34、D12Mit35、D13Mit18表现出多态性，，其中13个位点：D4Mit9、D5Mit31、D6Mit15、D6Mit36、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit29、D12Mit34、D12Mit35、D13Mit18表现出显著多态性，其余4个位点：D7mit29、D8mit11、D8mit22、D10mit29扩增产物电泳距离一致，表现为单态性，即不具有多态性。 (2)：不同品系小鼠之间的相似系数：近交系小鼠中CBA与DBA/2，FVB/NJ与C3H/HeJ的相似系数分别为0.650，反映出二者亲缘关系较近；其次是FVB/NJ与DBA/2和CBA的相似系数为0.600；亲缘关系最远的是CBA与BALB/C，其相似系数分别为0.350。而封闭群昆明小鼠与BALB/C的相似系数为0.700，可见二者亲缘关系最近；昆明小鼠与C3H/HeJ的相似系数为0.450，二者亲缘关系最远。昆明小鼠与YYHL小鼠、DBA、C57的相似系数中等，为0.550。 3．同一品系内不同个体之间微卫星多态性分析：在20个位点上同一品系内的小鼠在同一位点扩增的产物条带动距离相同，表现为单态性：各位点呈现一条清晰图带，且电泳距离一致。 结论： 1．经过20个位点检测，7个近交系小鼠符合近交系要求，说明河南省及周边 地区有关这些近交系小鼠及其群体没有发生遗传污染或遗传变异，符合近交系的要求。 2．可以运用PCR稳定、特异、有效地扩增8个品系小鼠的微卫星DNA，微卫星DNA技术的建立可以对近交系小鼠的品系鉴别、个体识别、遗传背景监测等提供一个更好的方法和手段。 3．在不同近交系小鼠之间，筛选出的16个微卫星位点：D4Mit9、D5Mit31、D6Mit15、D6Mit16、D6Mit36、D7Mit12、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit8、D12Mit29、D12Mit34、D12Mit35、D13Mit18具有多态性，其中13个位点：D4Mit9、D5Mit31、D6Mit15、D6Mit36、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit29、D12Mit34、D12Mit35、D13Mit18表现出显著多态性，可利用这些位点快速、经济地对有关近交系小鼠进行遗传监测。 4．各品系小鼠之间的相似系数，反映了其祖代之间的亲缘关系。
[Abstract]:The inbred mouse (Inbred strain mice) refers to the mating and breeding of all sibling siblings or parents of more than 20 generations. All of the individuals in the strain can be traced to a pair of common ancestor that originated in the twentieth generation or later algebra. The significant characteristics of mice in the inbred line are gene homozygosity and genetic stability, but there is a possibility of genetic and genetic variation in breeding, breeding and reproduction. Therefore, it is necessary to carry out strict, long-term and regular genetic monitoring of inbred mice. Some traditional methods are necessary. Method: skin transplantation, biochemical markers and other methods of detecting their gene changes by detecting their phenotypic changes have obvious limitations.
Microsatellite DNA is the nucleotide short tandem repeat (short tandem repeats), which has the characteristics of wide distribution, large number, stable and easy amplification. The PCR amplification of microsatellite DNA can directly detect the changes of DNA level, and can fully reflect the genetic profile and variation of the genome. This is an ideal genetic monitoring marker for the inbred animal. The study used 20 microsatellite DNA loci on 10 chromosomes to study the DNA polymorphism of inbred mice. The genomic DNA of mice was extracted and the microsatellite primer PCR was used to amplify the microsatellite primers. Then the microsatellite DNA expansion was displayed by denatured polyacrylamide gel electrophoresis and silver staining and other techniques. Finally, the genetic data analysis was used. Analysis was performed to identify microsatellite loci with significant polymorphisms for genetic monitoring in inbred mice.
Methods: five mice (DBA/2, CBA, C57, C3H/HeJ, BALB/C, FVB/NJ, YYHL mice) and a closed group of mice (Kunming mice) were selected. The genomic DNA of mice was extracted with phenol chloroform extraction method, and the following loci were amplified by PCR. 30, D9mit21,
D10mit29, D11mit35, D11mit36, D12mit8, D12mit29, D12mit34, D12mit35, D13mit18, synchronize high pressure 4-6% denatured polyacrylamide gel electrophoresis with PCR products and allele typing standards. After electrophoresis, the electrophoresis is fixed, stained and coloured to observe the band pattern. According to the distance of DNA on denatured polyacrylamide gel, the microsatellite loci are expanded. The number of polymorphic bands in the eight strain mice showed the number of polymorphic bands. The results of the amplification were calculated by Lynth's method and the similarity was analyzed.
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