發(fā)布時間：2018-04-21 14:54

  本文選題：二代測序 + 多重競爭性PCR��； 參考：《東華大學(xué)》2017年碩士論文

【摘要】：復(fù)雜性狀的系統(tǒng)遺傳學(xué)研究中,分析差異表達(dá)基因,可從中獲取基因轉(zhuǎn)錄,基因調(diào)控,信號轉(zhuǎn)導(dǎo)通路及其相互聯(lián)系等相關(guān)信息;進(jìn)而揭示基因在時間、空間上的表達(dá)模式。常用的技術(shù)有傳統(tǒng)的競爭性PCR、實時熒光定量PCR、基因芯片表達(dá)譜以及近年來蓬勃發(fā)展的基于二代測序平臺的各種基因表達(dá)分析技術(shù)等,研究者常常根據(jù)具體實驗需求而選取適當(dāng)?shù)募夹g(shù)。本研究結(jié)合多重競爭性PCR和Ion proton二代測序平臺,開發(fā)了一種針對大樣本量的目的基因差異表達(dá)分析方案,并應(yīng)用于野生來源1號染色體小鼠血脂調(diào)控相關(guān)系統(tǒng)遺傳學(xué)研究中,為小鼠血脂異常這一復(fù)雜性狀研究提供了豐富的基因表達(dá)數(shù)據(jù)�；�于二代測序的多重競爭性PCR技術(shù)包含如下關(guān)鍵點:1)競爭性模板的構(gòu)建,采用單堿基突變的特異性逆轉(zhuǎn)錄引物,通過逆轉(zhuǎn)錄反應(yīng)快捷制備競爭性模板;2)PCR擴(kuò)增效率標(biāo)準(zhǔn)曲線,競爭性內(nèi)參模板和目標(biāo)模板以不同比例梯度混合,通過標(biāo)準(zhǔn)曲線校正引物實際擴(kuò)增效率帶來的定量偏差;3)多重PCR均一性的調(diào)整,針對同管反應(yīng)不同的擴(kuò)增子,調(diào)整特異性引物濃度比例,采用實時熒光定量PCR方式進(jìn)行定量,每個產(chǎn)物對應(yīng)一個通用性上游引物和一個特異性下游引物,從而保證多個擴(kuò)增子間的均一性;4)Ion proton平臺測序,基于該平臺的數(shù)據(jù)特點,本研究開發(fā)了一套匹配檢測基因、分離不同的競爭性模板和reads數(shù)目統(tǒng)計分析的完整流程,可快速準(zhǔn)確統(tǒng)計結(jié)果。首先用8個基因作為小試,對該技術(shù)方案的可行性進(jìn)行評估,結(jié)果顯示:1)標(biāo)準(zhǔn)曲線擬合度系數(shù)R2均大于0.98,優(yōu)于real-time PCR的擬合度(0.97-0.99),可更精確的校正引物擴(kuò)增效率偏差;2)計算3個實驗重復(fù)的標(biāo)準(zhǔn)方差SD表明,該方案和real-time PCR具有相同的穩(wěn)定性(SD0.05);3)檢測2倍基因表達(dá)差異能力,該方案和real-time PCR結(jié)果有良好的一致性(R20.98),證明該方案優(yōu)秀的靈敏度和準(zhǔn)確度。基于上述結(jié)果,將該方案應(yīng)用于172個小鼠樣本113個基因差異表達(dá)分析中,發(fā)現(xiàn)在不同品系中的小鼠檢測基因有顯著的表達(dá)差異,提示轉(zhuǎn)錄水平的差異可能與血脂水平差異相關(guān),為本課題組今后的系統(tǒng)遺傳學(xué)研究奠定了數(shù)據(jù)基礎(chǔ);同時,檢測113個擴(kuò)增子的均一性,90%以上reads差異在30倍以內(nèi),解決了擴(kuò)增子測序中過量測序(over-sequencing)和低表達(dá)轉(zhuǎn)錄本測序深度不足的難題,將目標(biāo)片段測序成本大大降低。本研究建立的基于二代測序的多重競爭性PCR技術(shù),具有優(yōu)秀的穩(wěn)定性,高靈敏度和高準(zhǔn)確度,在小鼠血脂調(diào)控相關(guān)系統(tǒng)遺傳學(xué)研究中的應(yīng)用證明了其低廉的成本,以及快捷的后續(xù)數(shù)據(jù)分析。在今后復(fù)雜性狀疾病的系統(tǒng)遺傳學(xué)研究中,基于二代測序的多重競爭性PCR一定可以成為眾多研究者的熱門選擇技術(shù)。
[Abstract]:In the systematic genetics of complex traits, the differential expression genes can be analyzed to obtain related information such as gene transcription, gene regulation, signal transduction pathways and their interrelationships, and thus reveal the expression patterns of genes in time and space. The commonly used techniques include traditional competitive PCRs, real-time fluorescent quantitative PCRs, microarray expression profiles and various gene expression analysis techniques based on the second-generation sequencing platform. Researchers often select appropriate techniques according to specific experimental needs. In this study, combined with multiple competitive PCR and Ion proton second-generation sequencing platforms, a novel gene differential expression analysis scheme for large sample size was developed and applied to the genetic studies on lipid regulation in mice with chromosome 1 from wild origin. It provides abundant gene expression data for the complex study of dyslipidemia in mice. The multiplex competitive PCR technique based on second generation sequencing consisted of the following key points: 1) Construction of competitive template. Using specific reverse transcription primers of single base mutation, the competitive template was quickly prepared by reverse transcription reaction to prepare the standard curve of amplification efficiency of competitive template. The competitive internal reference template and the target template were mixed with different proportion gradients, and the quantitative deviation caused by the actual amplification efficiency of the primers was corrected by standard curve to adjust the homogeneity of multiple PCR. The ratio of specific primer concentration was adjusted, and real-time fluorescent quantitative PCR was used to quantify each product. Each product was matched with a universal upstream primer and a specific downstream primer, so as to ensure the homogeneity of multiple amplifiers on the proton platform. Based on the data characteristics of the platform, a set of matching detection genes was developed to separate different competitive templates and a complete process of reads number statistical analysis, which can quickly and accurately calculate the results. The feasibility of the technique was evaluated using eight genes as a pilot test. The results showed that the fitting coefficient R2 of the standard curve was greater than 0.98, which was better than that of real-time PCR (0.97-0.99g), which could more accurately calibrate the amplification efficiency deviation of primer. This scheme has the same ability as real-time PCR to detect the difference of 2 times gene expression. It has good agreement with the result of real-time PCR, which proves the excellent sensitivity and accuracy of the scheme. Based on the above results, 113 differentially expressed genes were analyzed in 172 mouse samples. The results showed that there were significant differences in the expression of the detected genes in different strains of mice, suggesting that the difference of transcription level may be related to the difference of blood lipid level. The results provided a data basis for the future systematic genetics research of our group, and the difference of reads among 113 amplifiers was less than 30 times, and 90% of the homogeneity of the three amplifiers was less than 30 times. The problem of excessive sequencing over-sequencing in Amplifier sequencing and insufficient sequencing depth of low expression transcripts were solved, and the cost of target fragment sequencing was greatly reduced. The multiplex competitive PCR technique based on second generation sequencing has excellent stability, high sensitivity and high accuracy. It has been proved to be low cost by its application in the genetics of mouse blood lipid regulation system. And quick follow-up data analysis. In the future, multiplex competitive PCR based on second-generation sequencing will be a hot choice for many researchers in the field of systemic genetics of complex diseases.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78

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本文編號：1782910