基于磁性纳米粒子PCR的高通量单核苷酸多态性分型方法

发布时间：2018-07-01 12:16

  本文选题：磁性纳米粒子 + PCR　； 参考：《湖南工业大学》2007年硕士论文

【摘要】： 单核苷酸多态性(Single Nucleotide Polymorphisms,SNPs)是人类基因序列中最常见的变异,对其研究有助于解释个体的表型差异、不同群体和个体对复杂疾病的易感性、以及对各种药物的耐受性和对环境因子的反应。因此,建立一种快速、准确、高通量且适用于临床的SNP分型方法非常重要。目前已报道的众多SNP分型方法中,绝大多数需要对包含有SNP位点的PCR产物进行纯化、浓缩,这是一个即费时又费力的工作,而且很难满足未来对分型自动化的要求。 随着纳米技术的迅速发展,纳米材料逐渐被应用到生命科学领域,为其研究和发展提供了新的技术和手段。磁性纳米粒子(Magnetic Nanoparticles,MNPs)作为纳米材料的一个重要组成部分,由于其特殊的物理化学性质和生物相容性,目前已被广泛应用于细胞的分离、免疫测定、蛋白质和酶的固定以及DNA的检测等。 本文将磁性纳米粒子的富集功能和生物芯片“并行”读出的高通量特性结合起来,首先发展了一种基于磁性纳米粒子PCR扩增和等位基因特异性双色荧光杂交的分型方法,用以检测亚甲基四氢叶酸还原酶(MTHFR)基因的677位点(C→T)的单核苷酸多态性,应用该方法对126个样本MTHFR基因的C677T多态进行检测。实验表明,当下游引物连接在磁性纳米粒子表面时,能与反应体系中的模板DNA以及上游引物通过PCR扩增出所需的目的片段。当杂交温度在38℃时,所使用的荧光探针能够精确的区分样品的SNP基因型,分型结果经测序得到验证。 为了进一步降低分型成本,在基于本文提出的MNPs-PCR分型方法的基础上,通过使用两条带有野生、突变通用标签(tag)的探针和双色荧光(Cy3、Cy5)标记的通用检测子,进一步降低了样本的分型成本。本文中对96个样本的MTHFR基因C677T位点以及AGT基因的M235T位点用这种新型方法进行了高通量分型检测。上述基于磁性纳米粒子PCR的两种SNP分型方法不仅省略了传统方法中纯化、浓缩PCR产物的繁琐步骤,而且获得了很好的分型结果。接着验证了该方法在自动化工作站中自动检测的准确性,我们将96个样本的AGT基因M235T位点进行自动化检测,整个实验操作过程在96孔PCR板中自动完成,利用通用标签技术,把最终变性下来的荧光标记的通用检测子点在玻片上构成检测芯片,经扫描,能够在几分钟内完成对大量样品的分型,且分型结果直观、准确,正错配信号比值高,是一种操作简单、快速、高通量、高灵敏度的分型方法,有较高的应用价值。
[Abstract]:Single nucleotide polymorphic SNPs (SNPs) are the most common mutations in human gene sequences. The study of SNPs helps to explain the phenotypic differences of individuals and the susceptibility of different populations and individuals to complex diseases. Tolerance to various drugs and response to environmental factors. Therefore, it is very important to establish a rapid, accurate, high throughput and suitable for clinical SNP typing. At present, most of the reported SNP typing methods need to purify and concentrate the PCR products containing SNP sites, which is a time-consuming and laborious task, and it is difficult to meet the requirements of typing automation in the future. With the rapid development of nanotechnology, nanomaterials have been gradually applied to the field of life science, providing new technologies and means for their research and development. As an important part of nanomaterials, magnetic nanoparticles (MNPs) have been widely used in cell separation and immunoassay because of their special physical and chemical properties and biocompatibility. Immobilization of proteins and enzymes and detection of DNA. In this paper, the enrichment function of magnetic nanoparticles is combined with the high throughput characteristics of "parallel" readout by biochip. Firstly, a typing method based on PCR amplification of magnetic nanoparticles and allele-specific two-color fluorescence hybridization is developed. The single nucleotide polymorphism at site 677 (C / T) of methylenetetrahydrofolate reductase (MTHFR) gene was detected by this method. The C677T polymorphism of MTHFR gene in 126 samples was detected by this method. The results showed that when the downstream primers were attached to the surface of the magnetic nanoparticles, the desired target fragments could be amplified by PCR with the template DNA in the reaction system and the upstream primers. When the hybridization temperature was 38 鈩，

本文编号：2087752