小鼠SNP遗传检测方法的建立及生化标记位点的分子生物学研究

发布时间：2018-04-29 21:37

本文选题：单核苷酸多态性 + 遗传检测　；参考：《中国药品生物制品检定所》2006年硕士论文

【摘要】：遗传检测技术是遗传质量控制的重要手段之一，现有的检测方法不能满足科学发展的需要。近年来，随着人类基因组研究的不断深入，有关SNP的研究越来越受到重视，但小鼠SNP研究，特别是将SNP应用于近交系小鼠遗传检测的研究比较少。为此，我们建立小鼠SNP遗传检测方法，并对SNP与生化标记位点多态性的关联性进行研究，进一步完善我国实验动物遗传检测技术，提高检测水平。本研究分为两个部分：(1)小鼠SNP遗传检测方法的建立采用单管双向等位基因专一性扩增(single-tube bi-directional allele specific amplification，SB-ASA)方法，，首先对5个国外品系的近交系小鼠的16个SNP位点进行了检测，并进行测序验证，同时设计双盲实验验证该方法的可靠性。结果5个小鼠品系的16个SNP位点都成功地进行了分型，与测序的结果完全一致。双盲实验通过3个SNP位点综合分析，可以准确地鉴别这5个小鼠品系。此外，我们对国内培育的4个品系的近交系小鼠也进行了检测，最终确定了4个品系的16个SNP位点等位基因，而且还绘制了9个品系小鼠的16个SNP位点的等位基因图谱。这些研究不仅为各小鼠品系的遗传鉴定和品系间的鉴别提供了实验依据，而且还为近交系小鼠遗传检测提供了新方法。 (2)生化标记位点的分子生物学研究本实验尝试从分子生物学水平去探索近交系小鼠生化标记位点多态性的机理，以及与SNP的关联性。我们选择了Car2、Gpi1和Hbb3个生化遗传检测位点，分别从生化电泳、DNA、RNA和翻译蛋白4个方面进行了分析研究。结果发现Car2基因第38位Gin／His之间的转换是引起所有小鼠品系形成Car2~a和Car2~b两种电泳表型的原因；Gpi1基因的第247位氨基酸残基Phe／Leu之间的转换可能是形成Gpi1~a和Gpi1~b不同等位基因的原因；Hbb基因的第13、20和139位氨基酸残基Cys／Gly、Ser／Ala和Thr／Ala之间的转换是形成Hbb~d和Hbb~s两种电泳表型的原因。通过以上研究，建立了近交系小鼠的SNP遗传质量检测方法，确定了国内4个品系的16个SNP位点等位基因，绘制了9个品系小鼠的SNP位点等位基因图谱。同时，通过分子生物学手段对生化标记位点多态性的形成机制进行研究，初步阐明三个生化位点多态的形成机理。本研究对提高我国实验动物遗传检测技术水平，促进生命科学发展具有重要意义。
[Abstract]:Genetic detection technology is one of the important means of genetic quality control, the existing detection methods can not meet the needs of scientific development. In recent years, with the development of human genome research, more and more attention has been paid to the study of SNP, but the study of mouse SNP, especially the application of SNP to the genetic detection of inbred mice, is less. Therefore, we established the method of SNP genetic detection in mice, and studied the association between SNP and polymorphism of biochemical marker loci in order to further improve the genetic detection technology of laboratory animals in China and improve the detection level. This study was divided into two parts: 1) Establishment of SNP genetic detection method in mice using single-tube bi-directional allele specific amplification SB-ASAA method. First, 16 SNP loci of 5 inbred strains of foreign strains were detected. The reliability of the method was verified by sequencing and double-blind experiments were designed to verify the reliability of the method. Results 16 SNP loci of 5 mouse strains were successfully typed, and the results were in good agreement with the sequencing results. Three SNP loci were used to identify the five mouse strains. In addition, 16 SNP loci alleles of 4 strains were determined and alleles of 16 SNP loci of 9 strains were plotted. These studies not only provide experimental basis for genetic identification and identification among strains of mice, but also provide a new method for genetic detection of inbred mice. (2) Molecular biology of biochemical marker loci in this experiment, we try to explore the mechanism of the polymorphism of biochemical marker loci in inbred mice and its association with SNP from the molecular biological level. We selected Car2Gpi1 and Hbb3 sites for biochemical genetic analysis, and analyzed them from four aspects of biochemical electrophoretic DNA RNA and translation protein, respectively. It was found that the conversion between the 38th Gin/His of the Car2 gene was the cause of the formation of two electrophoretic phenotypes of Car2~a and Car2~b in all mouse strains. The conversion between the 247th amino acid residues Phe/Leu of the Gpi1 gene may be the formation of different alleles of Gpi1~a and Gpi1~b. The conversion between Cys / Glycine Serr / Ala and Thr/Ala in the 1320 and 139 amino acid residues of HB gene was responsible for the formation of two electrophoretic phenotypes of Hbb~d and Hbb~s. Based on the above studies, the SNP genetic quality detection method of inbred strain mice was established, 16 alleles of SNP loci in 4 strains in China were determined, and the allelic map of SNP loci in 9 strains of mice were plotted. At the same time, the formation mechanism of the polymorphism of biochemical marker loci was studied by molecular biology, and the formation mechanism of the polymorphism of three biochemical loci was preliminarily elucidated. This study is of great significance to improve the technology of genetic detection of laboratory animals and promote the development of life science in China.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332

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