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(kāi)關(guān)”技術(shù)建立先天性耳聾4個(gè)基因10個(gè)熱點(diǎn)突變的檢測(cè)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(jì)測(cè)定其濃度;對(duì)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(cè)序,明確其基因序列,這些樣本作為建立“分子開(kāi)關(guān)”技術(shù)檢測(cè)10個(gè)熱點(diǎn)突變方法的標(biāo)準(zhǔn)樣本;構(gòu)建10個(gè)位點(diǎn)的野生型和突變型質(zhì)粒,作為檢測(cè)的野生型和突變型模板;根據(jù)該10個(gè)位點(diǎn)的突變特點(diǎn),設(shè)計(jì)多對(duì)突變檢測(cè)引物,通過(guò)實(shí)驗(yàn)選擇產(chǎn)物特異性高的引物作為突變檢測(cè)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(cè)引物對(duì)相應(yīng)的野生型和突變模板進(jìn)行擴(kuò)增并測(cè)序驗(yàn)證擴(kuò)增產(chǎn)物;優(yōu)化PCR擴(kuò)增條件,提高“分子開(kāi)關(guān)”對(duì)突變位點(diǎn)的識(shí)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(cè)步驟,嘗試建立一種簡(jiǎn)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(cè)。 結(jié)果:對(duì)不同的熱點(diǎn)突變位點(diǎn)設(shè)計(jì)的多對(duì)突變檢測(cè)引物,通過(guò)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(cè)引物對(duì)突變模板擴(kuò)增得到產(chǎn)物,對(duì)正常模板無(wú)擴(kuò)增產(chǎn)物,顯示“分子開(kāi)關(guān)”對(duì)耳聾基因熱點(diǎn)突變位點(diǎn)的特異性識(shí)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(kāi)關(guān)”技術(shù)建立了耳聾基因10個(gè)熱點(diǎn)突變的檢測(cè)方法;“分子開(kāi)關(guān)”技術(shù)是一種很有應(yīng)用價(jià)值的點(diǎn)突變檢測(cè)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(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R764.43

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