基于荧光淬灭的SNP检测方法的建立及应用

发布时间：2018-06-12 23:17

本文选题：SNP检测 + 方法学　；参考：《华东理工大学》2016年博士论文

【摘要】：当前,检测基因SNP已成为分析判断遗传病、个体化药物反应特征的重要手段,也是预测和预防许多疾病的重要手段。现今的单核苷酸多态性(Single nucleotide polymorphism, SNP)检测方法,比如测序法和Taq探针法都存在一定的弊端。因此我们开发了一种高效的基于荧光淬灭的SNP检测方法——Quenching-PCR法。该方法是利用标记有荧光淬灭分子的特异序列探针与有荧光分子标记的双脱氧核苷三磷酸在待测基因的特异位点上进行单碱基延伸,然后通过检测在探针末端延伸的荧光分子标记的碱基的荧光淬灭值,来确定探针末端延伸的碱基类型,亦即待测基因特异位点的碱基类型。与金标准测序法相比,Quenching-PCR法结果的一致率为93.40%,TaqMan探针法结果的一致率为92.45%,由此可见,Quenching-PCR法与TaqMan探针法在准确度方面无明显差异。但在检测成本、检测时间和探针设计等方面都远优于TaqMan探针法,因此Quenching-PCR法更易于在临床诊断中应用,具有重要意义。在建立的基于荧光淬灭的SNP检测方法的基础上,我们将该方法用于临床相关肿瘤个体化用药常规位点的检测。选取与酪氨酸激酶抑制剂作用靶点——表皮生长因子受体基因(epidermal growth factor receptor, EGFR)相关位点的检测, EGFR第19号外显子中的2235_2249位的插入缺失和第21号外显子的L858R与药物效应密切相关；同时还用于与铂类药物使用相关的多药耐药相关蛋白2基因(Multidrug resistance-associated protein 2, MRP2) Val417Ile位点、核苷酸切除修复交叉互补基团1(Excision repair cross-complementation group 1, ERCC1)rs3212986位点和X线修复交叉互补基团1基因(X-rayrepaircrosscomplementing 1, XRCC1) Arg 194T rp位点的检测。结果发现Quenching PCR能很好用于肿瘤个体化用药相关位点SNP的检测,同时还发现该方法不但能用于SNP的检测,还能应用于插入／缺失(Insert/Delete, InDel)的检测。肿瘤异质性与肿瘤转移密切相关,近年来成为肿瘤研究的热点。结肠癌是世界三大恶性肿瘤之一,是一种高度异质性的肿瘤,形态学、免疫表型、分子遗传学上的差异导致了结肠癌对临床治疗的不同反应及预后。肿瘤个体之间存在异质性,在同一个体不同肿瘤区域以及转移灶的相关基因组也存在一定差异。本课题将通过对不同结肠癌病人原位肿瘤组织、癌旁组织以及转移灶进行全基因组SNP检测,并通过生物信息学解析明确和发现个体癌细胞异质性程度是否与病程和转移具有相关性。同时,利用建立的基于荧光淬灭的SNP检测方法对相关提示与结肠癌异质性与肿瘤转移相关SNP进行验证。结果发现结肠癌的转移灶的的拷贝数变异(Copy number variation, CNV)率显著高于癌旁组织与原发灶(p0.01)。同时通过GO分析发现差异CNVs涉及的相关通路约有四分之一与粘附和免疫相关。而将肿瘤的转移灶与原发灶发生基因编码区域SNP改变所涉及的基因进行GO分析,所涉及的基因二分之一与转移和免疫相关,且涉及粘附和免疫调节的相关通路也占到了约五分之一。同样将肿瘤的转移灶与原发灶的差异miRNAs涉及的靶基因进行GO分析,发现涉及的基因约三分之一与转移和免疫相关,涉及粘附和免疫调节的相关通路也占到了约五分之一。上述结果提示粘附与免疫调节可能是调控肿瘤发生与发展的关键因素。通过芯片上转移灶与原发灶的SNP比较分析,并通过Quenching PCR扩大验证,发现位于第14号染色体上的rs12881063位点在原发灶和癌旁组织中几乎没有产生突变,而在转移灶中则出现比例较高的突变,在近端转移中,突变率达到了66.7%,而在远端转移中,突变率达到了83.3%。上述结果提示该位点的突变与可能与肿瘤的转移的能力相关在所有样本的转移灶中都产生了突变,这可能为临床肿瘤分期诊断的潜在标志物,并提供一定的依据。
[Abstract]:At present, the detection of gene SNP has become an important means to analyze and judge the characteristics of genetic diseases and individualized drug reactions. It is also an important means to predict and prevent many diseases. The current single nucleotide polymorphism (Single nucleotide polymorphism, SNP) detection methods, such as sequencing and Taq probe, have some drawbacks. A highly efficient fluorescence quenching based SNP method, Quenching-PCR method, is used to extend the single base of a specific sequence probe labeled with fluorescence quenching molecules with a fluorescent molecule labeled double deoxynucleoside three phosphoric acid on the specific loci of the gene, and then by detecting the fluorescence extended at the tip of the probe. The fluorescence quenching value of the base of the molecular marker is used to determine the base type of the extension of the tip of the probe, that is, the base type of the special site of the gene. Compared with the gold standard sequencing method, the consistency rate of the results of Quenching-PCR method is 93.40% and the agreement rate of the TaqMan probe method is 92.45%. Thus, the Quenching-PCR method and the TaqMan probe method can be seen in the criterion. There is no obvious difference in accuracy, but it is far superior to TaqMan probe in detection cost, detection time and probe design. Therefore, Quenching-PCR method is more easily applied in clinical diagnosis and is of great significance. On the basis of SNP detection method based on fluorescence quenching, we apply this method to the individualization of clinical related tumors. Detection of the routine site of drug use. The detection of epidermal growth factor receptor (EGFR) related loci with tyrosine kinase inhibitors, the insertion deletion of 2235_2249 in exon nineteenth of EGFR and the L858R of exon twenty-first in exon are closely related to the drug effect; meanwhile, it is also used. The multidrug resistance related protein 2 gene (Multidrug resistance-associated protein 2, MRP2) Val417Ile loci associated with the use of platinum drugs, the nucleotide excision and repair of the cross complementing group 1 (Excision repair cross-complementation group 1, ERCC1) rs3212986 site and the X line repair cross complementary group 1 gene The detection of Arg 194T RP site of nting 1, XRCC1). The results showed that Quenching PCR could be used for the detection of SNP in individual oncology loci, and also found that this method can be used not only for detection of SNP but also for the detection of insertion / deletion (Insert/Delete, InDel). The heterogeneity of tumor is closely related to tumor metastasis, and has been developed in recent years. Colon cancer is one of the three most malignant tumors in the world. It is a highly heterogeneous tumor. The differences in morphology, immunophenotype and molecular genetics lead to the different responses and prognosis of colon cancer to clinical treatment. Heterogeneity exists between individuals, in the same individual tumor regions and in metastatic foci. There are certain differences in the genomes. This topic will be detected by genomic SNP in tumor tissues, para cancer tissues and metastatic foci in different colon cancer patients. Through bioinformatics analysis, it is clear whether the degree of individual cancer cell heterogeneity is related to the course and metastasis. The quench SNP detection method verified the correlation with the heterogeneity of colon cancer and tumor metastasis associated with SNP. The results showed that the copy number variation (Copy number variation, CNV) in the metastasis of colon cancer was significantly higher than that of the para cancerous tissues and primary foci (P0.01). At the same time, the related pathways involved in the difference CNVs were found to be about four points by GO analysis. One is associated with adhesion and immunity, and GO analysis of the genes involved in the SNP change of the gene encoding region of the primary foci, the gene 1/2 involved is related to metastasis and immunity, and the related pathways involved in adhesion and immunomodulation are also about 1/5. The difference miRNAs involved in GO analysis of target genes, and found that about 1/3 of the genes involved were related to metastasis and immunity, and the related pathways involved in adhesion and immunomodulation were also about 1/5. The results suggested that adhesion and immunoregulation may be the key factor in regulating the occurrence and development of tumor. SNP comparative analysis of primary foci, and Quenching PCR enlargement, found that the rs12881063 loci on chromosome fourteenth had little mutation in the primary and paracancerous tissues, but a higher mutation in the metastasis, and the mutation rate reached 66.7% in the proximal metastasis, and the rate of mutation reached the distal metastasis. The above results suggest that mutations in the site may be associated with the ability of the metastasis of the tumor to produce mutations in all the metastases of the samples, which may be a potential marker for the diagnosis of clinical oncology and provide some basis for the 83.3%..
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.4;R440

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