發(fā)布時(shí)間：2018-03-08 07:47

  本文選題：SNP　切入點(diǎn)：等位基因特異性PCR　出處：《南京醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 建立高保真DNA聚合酶介導(dǎo)的硫化修飾等位基因特異性PCR(allele-specific PCR, AS-PCR),并將其應(yīng)用于線粒體DNA(mitochondrial DNA, mtDNA)編碼區(qū)單核苷酸多態(tài)性(single nucleotide polymorphism, SNP)的研究工作,探討其實(shí)際應(yīng)用價(jià)值;同時(shí)調(diào)查60例中國(guó)江蘇無(wú)關(guān)漢族個(gè)體mtDNA編碼區(qū)4個(gè)SNP基因座等位基因頻率和單倍型分布情況,為群體遺傳學(xué)提供新的數(shù)據(jù)。 方法 1根據(jù)線粒體DNA編碼區(qū)10400(T/C)位點(diǎn)設(shè)計(jì)未修飾及3′末端硫化修飾的等位基因特異性引物,采用低保真Taq和高保真Pfu DNA聚合酶進(jìn)行引物延伸反應(yīng),2%瓊脂糖凝膠電泳比較擴(kuò)增結(jié)果。 2從線粒體DNA編碼區(qū)篩選多態(tài)性較好的4個(gè)SNP(C12705T、G8701A、G8584A、C10400T)基因座。針對(duì)每個(gè)SNP基因座設(shè)計(jì)兩條片段相差4個(gè)堿基的3′末端硫化修飾等位基因特異性引物和一條公共引物。血液標(biāo)本采自中國(guó)江蘇地區(qū)60名漢族無(wú)關(guān)、健康個(gè)體。采用Chelex-100法提取模板mtDNA,利用Pfu高保真DNA聚合酶進(jìn)行擴(kuò)增反應(yīng)。擴(kuò)增產(chǎn)物經(jīng)聚丙烯酰胺凝膠電泳、銀染顯帶后分析樣本的基因型并應(yīng)用直接測(cè)序技術(shù)驗(yàn)證分型結(jié)果的準(zhǔn)確性。 結(jié)果 1與傳統(tǒng)AS-PCR相比,高保真Pfu DNA聚合酶介導(dǎo)的硫化修飾等位基因特異性PCR獨(dú)具的開(kāi)關(guān)效應(yīng)使引物延伸的特異性大大提高,降低了SNP分析的假陽(yáng)性率,提高了對(duì)SNP檢測(cè)的準(zhǔn)確性和可靠性。 2應(yīng)用上述方法對(duì)江蘇地區(qū)漢族群體60名無(wú)關(guān)個(gè)體4個(gè)mtDNA-SNP基因座進(jìn)行了準(zhǔn)確分型,不同等位基因型各為一條長(zhǎng)度不同的單一譜帶,分型結(jié)果與直接測(cè)序完全一致。4個(gè)mtDNA-SNP基因座C12705T、G8701A、G8584A、C10400T等位基因頻率分別為0.37/0.63、0.47/0.53、0.83/0.17、0.53/0.47,共檢出6種單倍型,單倍型的基因多樣性為0.7438,偶合概率值為0.2686。 結(jié)論 本研究成功建立了由高保真DNA聚合酶和3′末端硫化修飾等位基因特異性引物共同構(gòu)成的SNP敏感性分子開(kāi)關(guān)技術(shù),并應(yīng)用于mtDNA編碼區(qū)單核苷酸多態(tài)性的群體遺傳學(xué)研究,證實(shí)該技術(shù)檢測(cè)SNP的準(zhǔn)確性和優(yōu)越性,具有極高的臨床實(shí)用價(jià)值,值得在基因SNP研究中進(jìn)一步推廣;同時(shí)為線粒體SNP基因座在法醫(yī)學(xué)等方面的應(yīng)用提供了方法學(xué)和群體遺傳學(xué)基礎(chǔ)。
[Abstract]:Purpose. A high fidelity DNA polymerase mediated sulphide modified allele-specific PCR(allele-specific PCR, AS-PCRN, was established and applied to the study of single nucleotide polymorphisms (SNPs) in the coding region of mitochondrial DNA(mitochondrial DNA (mtDNA), and its practical application value was discussed. The allelic frequency and haplotype distribution of 4 SNP loci in the mtDNA coding region of 60 unrelated Han individuals from Jiangsu Province of China were also investigated in order to provide new data for population genetics. Method. 1 the allele specific primers of unmodified and 3 '-terminal vulcanization modification were designed according to the 10400 T / C site of mitochondrial DNA coding region. The amplification results were compared by using low fidelity Taq and high fidelity Pfu DNA polymerase for primer extension reaction of 2% agarose gel electrophoresis. 2 four SNPs C12705TnG8701AG8584AG10400T loci were screened from the mitochondrial DNA coding region. A 3'terminal sulphide modified allele specific primer and a common blood primer were designed for each SNP locus. The liquid samples were collected from 60 Han nationality in Jiangsu, China. The template mtDNA was extracted by Chelex-100 method and amplified by Pfu high fidelity DNA polymerase. The amplified products were analyzed by polyacrylamide gel electrophoresis. The genotypes of the samples were analyzed after silver staining and the accuracy of the typing results was verified by direct sequencing. Results. 1 compared with traditional AS-PCR, the unique switch effect of high fidelity Pfu DNA polymerase mediated sulphide modified allele-specific PCR greatly improved the specificity of primer extension and reduced the false positive rate of SNP analysis. The accuracy and reliability of SNP detection are improved. (2) the four mtDNA-SNP loci of 60 unrelated individuals in the Han population of Jiangsu province were accurately typed by the above method. The genotypes of each allele were a single band with different lengths. The allele frequencies of four mtDNA-SNP loci C12705TnG8701AnG8584AH10400T were 0.37 / 0.630.47 / 0.530.83 / 0.170.53 / 0.47, respectively. Six haplotypes were detected, the genetic diversity of haplotypes was 0.7438, and the probability of coupling was 0.2686. Conclusion. In this study, a novel SNP sensitive molecular switch technique, composed of high fidelity DNA polymerase and 3 'terminal vulcanized modified allele specific primers, was successfully established and applied to the population genetics of single nucleotide polymorphisms in the mtDNA coding region. The accuracy and superiority of this technique in detecting SNP are proved to be of high clinical and practical value. It is worthy to be further popularized in the study of gene SNP. It also provides methodology and population genetic basis for the application of mitochondrial SNP locus in forensic science.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341

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