本文選題：突變敏感性分子開關(guān) + 氨基糖苷類耳聾�。� 參考：《臨床檢驗(yàn)雜志》2016年03期

【摘要】：目的對(duì)高保真酶介導(dǎo)的突變敏感性分子開關(guān)技術(shù)檢測(cè)線粒體DNA(mt DNA)A1555G位點(diǎn)條件進(jìn)行優(yōu)化。方法利用3'硫代磷酸化修飾的突變型引物和野生型引物作為下游引物,在其上游設(shè)計(jì)一條公共引物分別構(gòu)成突變引物對(duì)和野生引物對(duì),以構(gòu)建好的包含mt DNA A1555G位點(diǎn)的突變型質(zhì)粒和野生型質(zhì)粒為模板,進(jìn)行高保真聚合酶介導(dǎo)的雙向引物延伸反應(yīng),對(duì)PCR體系中的退火溫度、引物濃度、模板濃度等條件優(yōu)化,通過凝膠成像系統(tǒng)對(duì)其PCR結(jié)果進(jìn)行分析確定最佳反應(yīng)條件。結(jié)果分子開關(guān)技術(shù)檢測(cè)mt DNA A1555G位點(diǎn)的最佳PCR條件:退火溫度為61.0℃,引物濃度為0.6μmol/L,檢測(cè)模板濃度為103~106copies/μL。結(jié)論確定了分子開關(guān)技術(shù)檢測(cè)mt DNA A1555G位點(diǎn)的最佳反應(yīng)條件,為該技術(shù)在線粒體DNA的突變篩查中的應(yīng)用提供依據(jù)。
[Abstract]:Objective to optimize the conditions for the detection of mtDNA A1555G site in mitochondrial DNA by high fidelity enzyme mediated mutagenic molecular switch technique. Methods 3'thiophosphorylation modified mutant primers and wild primers were used as downstream primers, and a common primer was designed to form mutant primer pair and wild primer pair, respectively. The mutant plasmids and wild type plasmids containing mt DNA A1555G site were used as templates to carry out bidirectional primer extension mediated by high fidelity polymerase. The conditions of annealing temperature, primer concentration and template concentration in PCR system were optimized. The PCR results were analyzed by gel imaging system to determine the optimal reaction conditions. Results the optimal conditions for detecting mt DNA A1555G locus by molecular switch technique were as follows: annealing temperature was 61.0 鈩，

本文編號(hào)：2007168