本文選題：耳聾 + 高保真酶　； 參考：《蘇州大學(xué)》2011年碩士論文

【摘要】：目的:利用全血PCR結(jié)合Pfu高保真DNA聚合酶介導(dǎo)的突變敏感性“分子開關(guān)”技術(shù)建立先天性耳聾4個基因10個熱點(diǎn)突變的檢測方法。 方法:采集臨床健康體檢病人及診斷為神經(jīng)性耳聾病人的血液標(biāo)本,提取血液白細(xì)胞中的基因組DNA,分光光度計測定其濃度;對健康體檢病人的DNA樣本的耳聾基因GJB2、GJB3,SLC26 A4、線粒體DNA熱點(diǎn)突變所在的外顯子進(jìn)行PCR擴(kuò)增并測序,明確其基因序列,這些樣本作為建立“分子開關(guān)”技術(shù)檢測10個熱點(diǎn)突變方法的標(biāo)準(zhǔn)樣本;構(gòu)建10個位點(diǎn)的野生型和突變型質(zhì)粒,作為檢測的野生型和突變型模板;根據(jù)該10個位點(diǎn)的突變特點(diǎn),設(shè)計多對突變檢測引物,通過實(shí)驗(yàn)選擇產(chǎn)物特異性高的引物作為突變檢測引物;利用Pfu高保真DNA聚合酶和3’末端硫化修飾的突變檢測引物對相應(yīng)的野生型和突變模板進(jìn)行擴(kuò)增并測序驗(yàn)證擴(kuò)增產(chǎn)物;優(yōu)化PCR擴(kuò)增條件,提高“分子開關(guān)”對突變位點(diǎn)的識別能力。分別以基因組DNA和全血作為DNA模板,重復(fù)上述檢測步驟,嘗試建立一種簡化的PCR方法。將該方法用于20例健康人及耳聾患者的熱點(diǎn)突變檢測。 結(jié)果:對不同的熱點(diǎn)突變位點(diǎn)設(shè)計的多對突變檢測引物,通過實(shí)驗(yàn)篩選到特異性引物;在高保真DNA聚合酶介導(dǎo)的PCR反應(yīng)體系中,3’末端硫化修飾突變檢測引物對突變模板擴(kuò)增得到產(chǎn)物,對正常模板無擴(kuò)增產(chǎn)物,顯示“分子開關(guān)”對耳聾基因熱點(diǎn)突變位點(diǎn)的特異性識別。 結(jié)論:成功應(yīng)用全血PCR結(jié)合高保真DNA聚合酶介導(dǎo)的“分子開關(guān)”技術(shù)建立了耳聾基因10個熱點(diǎn)突變的檢測方法;“分子開關(guān)”技術(shù)是一種很有應(yīng)用價值的點(diǎn)突變檢測技術(shù),全血PCR方法也是一種值得推廣的方法。
[Abstract]:Aim: to establish a method for detecting 10 hot spot mutations in 4 genes of congenital deafness by means of whole blood PCR and Pfu high fidelity DNA polymerase mediated mutagenic "molecular switch" technique. Methods: the blood samples of patients with clinical health examination and those diagnosed as neurodeafness were collected, the genomic DNA in the blood leukocytes was extracted, and the concentration of the DNA was measured by spectrophotometer. The deafness gene GJB2GJB3OSLC26A4 was amplified by PCR and sequenced in the exon of mitochondrial DNA hot spot mutation. These samples serve as standard samples for the establishment of a "molecular switch" technique for the detection of 10 hot spot mutations; construct wild and mutant plasmids of 10 loci as templates for detection of wild and mutant types; and according to the mutation characteristics of the 10 loci, Several pairs of mutation detection primers were designed and the primers with high product specificity were selected as mutation detection primers. The corresponding wild type and mutation template were amplified by Pfu high-fidelity DNA polymerase and 3'terminal vulcanization modified mutation detection primer, and the amplified products were verified by sequencing, and the conditions of PCR amplification were optimized. To improve the ability of molecular switch to recognize mutation sites. Genomic DNA and whole blood were used as DNA templates to repeat the above steps and to establish a simplified PCR method. The method was applied to the detection of hot spot mutations in 20 healthy persons and deafness patients. Results: multiple pairs of primers were designed for different hot spot mutation sites, and specific primers were screened by experiments. In the PCR reaction system mediated by high fidelity DNA polymerase, the product was obtained by the amplification of the mutated template by using the primers for the detection of vulcanized mutagenesis at the 3'end, but no amplification product was obtained for the normal template. The results showed that the molecular switch was specific to the hot spot mutation of deafness gene. Conclusion: the whole blood PCR combined with high fidelity DNA polymerase mediated "molecular switch" technique has been successfully used to detect 10 hot spot mutations of deafness gene, and "molecular switch" technique is a valuable point mutation detection technique. The whole blood PCR method is also worth popularizing.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R764.43

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