基于高密度单核苷酸多态性微阵列的胚胎植入前全面遗传检测

发布时间：2018-02-28 23:26

本文关键词： 胚胎植入前遗传检测单核苷酸多态性微阵列染色体异常多重置换单细胞全基因组扩增　出处：《天津大学》2014年硕士论文　论文类型：学位论文

【摘要】：目前鉴于不孕不育人群的增加，辅助生殖技术，包括体外受精和胞浆内精子注射技术的临床操作逐渐增加。但与自然生产相比，这些技术由于鉴别异常胚胎缺陷的能力有限，以及植入多胚胎以确保受孕率等操作的使用，会增加胎儿出生缺陷的风险，影响婴儿的出生率。胚胎植入前遗传诊断是在常规体外受精将胚胎植入母体之前，通过一系列的遗传检测获得胚胎的遗传信息，发现遗传缺陷，确保植入更少但质量更优的胚胎。现在胚胎植入前遗传检测普遍采用的遗传检测方法包括PCR和荧光原位杂交，用以检测单基因突变，以及有限分辨率的染色体异常。本研究旨在建立一种能全面分析胚胎遗传信息的遗传检测方法，采用的是含有约300,000检测位点的单核苷酸多态性微阵列的方法。高密度芯片的位点覆盖全基因的23对染色体，对于染色体片段的平均分辨长度约10.6kb，因此可在一次检测中探测到从整条染色体到染色体微小片段的缺失、增加、插入、非平衡易位和染色体数目异常等。本研究的第一步是要建立多重置换单细胞全基因组扩增方法，，将胚胎植入前活体取样的单个细胞基因组扩增到单核苷酸多态性微阵列可以检测的量。经过产物浓度检测、琼脂糖凝胶电泳和384位点单核苷酸多态性微阵列验证，扩增平均产量为2.70μg，产物片段长度大于2kb，全基因组覆盖率近99%，在数量和质量上均完全符合下游遗传检测的要求第二步采用12个DNA样品，建立并验证了用以检测细胞染色体异常的单核苷酸多态性微阵列高密度芯片方法。12个DNA样品包括人类染色体异常细胞系的标准gDNA样品，以及经医院初步诊断的病理样品。人类染色体异常细胞系的标准gDNA样品的通过本研究的遗传检测方法的染色体异常结果与其已知的测序结果相符，确证了高密度芯片检测的准确性；病理样品的检测结果与医院的初步诊断已知，但更加具体和准确。
[Abstract]:In view of the increasing number of infertile people, the clinical practice of assisted reproductive techniques, including in vitro fertilization and intracytoplasmic sperm injection, is gradually increasing. However, compared with natural production, these techniques are limited in their ability to identify abnormal embryo defects, And the use of procedures such as implantation of multiple embryos to ensure pregnancy rates increases the risk of birth defects and affects the birth rate of babies. Preimplantation genetic diagnosis is before conventional in vitro fertilization implants the embryo into the mother. Genetic information of embryos is obtained through a series of genetic tests, genetic defects are found, and fewer but better-quality embryos are implanted. The current genetic methods commonly used in pre-implantation genetic testing include PCR and fluorescence in situ hybridization to detect single gene mutations. The purpose of this study is to establish a genetic detection method that can comprehensively analyze the genetic information of embryos. A single nucleotide polymorphic microarray containing about 300,000 loci was used. The loci of the high density microarray covered 23 pairs of chromosomes of the entire gene. The average resolution length of chromosomal fragments is about 10.6kb, so the deletion, increase, insertion, non-equilibrium translocation and abnormal number of chromosomes can be detected in a single test. The first step of this study was to establish a multiplex replacement single cell genome amplification method, in which the genome of a single cell sampled in vivo before embryo implantation was amplified to the amount that could be detected by single nucleotide polymorphism microarray. Agarose gel electrophoresis and microarray verification of 384 locus single nucleotide polymorphism showed that the average amplification yield was 2.70 渭 g, the length of the product was more than 2 kb, the whole genome coverage was nearly 99%, and the quantity and quality of the product met the requirements of downstream genetic detection. In the second step, 12 DNA samples were used to establish and verify the single-nucleotide polymorphism microarray microarray microarray method for the detection of chromosomal abnormalities in cells. 12 DNA samples included standard gDNA samples of human chromosomal abnormal cell lines. Standard gDNA samples of human chromosomal abnormal cell lines. The results of chromosomal abnormalities obtained by the genetic detection method in this study are consistent with the results of known sequencing. The accuracy of high density microarray detection was confirmed, and the detection results of pathological samples and the preliminary diagnosis of hospital were known, but more specific and accurate.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

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