本文選題：磁性納米粒子 + PCR�。� 參考：《湖南工業(yè)大學(xué)》2007年碩士論文

【摘要】： 單核苷酸多態(tài)性(Single Nucleotide Polymorphisms,SNPs)是人類基因序列中最常見的變異,對(duì)其研究有助于解釋個(gè)體的表型差異、不同群體和個(gè)體對(duì)復(fù)雜疾病的易感性、以及對(duì)各種藥物的耐受性和對(duì)環(huán)境因子的反應(yīng)。因此,建立一種快速、準(zhǔn)確、高通量且適用于臨床的SNP分型方法非常重要。目前已報(bào)道的眾多SNP分型方法中,絕大多數(shù)需要對(duì)包含有SNP位點(diǎn)的PCR產(chǎn)物進(jìn)行純化、濃縮,這是一個(gè)即費(fèi)時(shí)又費(fèi)力的工作,而且很難滿足未來(lái)對(duì)分型自動(dòng)化的要求。 隨著納米技術(shù)的迅速發(fā)展,納米材料逐漸被應(yīng)用到生命科學(xué)領(lǐng)域,為其研究和發(fā)展提供了新的技術(shù)和手段。磁性納米粒子(Magnetic Nanoparticles,MNPs)作為納米材料的一個(gè)重要組成部分,由于其特殊的物理化學(xué)性質(zhì)和生物相容性,目前已被廣泛應(yīng)用于細(xì)胞的分離、免疫測(cè)定、蛋白質(zhì)和酶的固定以及DNA的檢測(cè)等。 本文將磁性納米粒子的富集功能和生物芯片“并行”讀出的高通量特性結(jié)合起來(lái),首先發(fā)展了一種基于磁性納米粒子PCR擴(kuò)增和等位基因特異性雙色熒光雜交的分型方法,用以檢測(cè)亞甲基四氫葉酸還原酶(MTHFR)基因的677位點(diǎn)(C→T)的單核苷酸多態(tài)性,應(yīng)用該方法對(duì)126個(gè)樣本MTHFR基因的C677T多態(tài)進(jìn)行檢測(cè)。實(shí)驗(yàn)表明,當(dāng)下游引物連接在磁性納米粒子表面時(shí),能與反應(yīng)體系中的模板DNA以及上游引物通過PCR擴(kuò)增出所需的目的片段。當(dāng)雜交溫度在38℃時(shí),所使用的熒光探針能夠精確的區(qū)分樣品的SNP基因型,分型結(jié)果經(jīng)測(cè)序得到驗(yàn)證。 為了進(jìn)一步降低分型成本,在基于本文提出的MNPs-PCR分型方法的基礎(chǔ)上,通過使用兩條帶有野生、突變通用標(biāo)簽(tag)的探針和雙色熒光(Cy3、Cy5)標(biāo)記的通用檢測(cè)子,進(jìn)一步降低了樣本的分型成本。本文中對(duì)96個(gè)樣本的MTHFR基因C677T位點(diǎn)以及AGT基因的M235T位點(diǎn)用這種新型方法進(jìn)行了高通量分型檢測(cè)。上述基于磁性納米粒子PCR的兩種SNP分型方法不僅省略了傳統(tǒng)方法中純化、濃縮PCR產(chǎn)物的繁瑣步驟,而且獲得了很好的分型結(jié)果。接著驗(yàn)證了該方法在自動(dòng)化工作站中自動(dòng)檢測(cè)的準(zhǔn)確性,我們將96個(gè)樣本的AGT基因M235T位點(diǎn)進(jìn)行自動(dòng)化檢測(cè),整個(gè)實(shí)驗(yàn)操作過程在96孔PCR板中自動(dòng)完成,利用通用標(biāo)簽技術(shù),把最終變性下來(lái)的熒光標(biāo)記的通用檢測(cè)子點(diǎn)在玻片上構(gòu)成檢測(cè)芯片,經(jīng)掃描,能夠在幾分鐘內(nèi)完成對(duì)大量樣品的分型,且分型結(jié)果直觀、準(zhǔn)確,正錯(cuò)配信號(hào)比值高,是一種操作簡(jiǎn)單、快速、高通量、高靈敏度的分型方法,有較高的應(yīng)用價(jià)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，

本文編號(hào)：2087752