酵母合成基因电路的计算设计

Mario Andrea Marchisio
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引用次数: 1

摘要

在本研究中,我们首次提出了结合引物延伸(pett)和金纳米颗粒的横向流动法对柴鸭tmigd1基因进行SNP分型,该方法具有操作简单、成本低、省时等优点。采用25℃盐老化法,用巯基-(dT)30对金纳米颗粒进行尾化处理,作为横向流动实验的标记物。横向流动装置主要由含链霉亲和素和d(A)30的硝化纤维素膜上的试验区和控制区组成。当特定SNP存在时,相应的引物可以被延长,然后在PEXT过程中,由于生物素- dutp的引入,反应产物可以在测试区被链霉亲和素捕获。金纳米粒子将与反应产物杂交,使其变得可见。本文报道了PEXT反应中Mg2+和膜上链霉亲和素的优化参数,以检测信号特异性。此外,我们发现增加PCR产物的数量和pex反应周期数会导致信号强度的增加,但信号特异性没有明显变化。
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Computational design of yeast synthetic gene circuits
I this work we firstly proposed lateral-flow assay combined with primer extension (PEXT) and gold nanoparticles to SNP genotyping of tmigd1 gene in Tsaiya duck which has advantages of easy operation, cost effective and time saving. The gold nanoparticles were tailed with thiol-(dT)30 using salt-aging method at 25 oC and used as label of lateral-flow assay. The lateral-flow device is mainly composed of test and control zone on the nitrocellulose membrane containing streptavidin and d(A)30, respectively. When the specific SNP exists, the corresponding primers can be extended, and then the reaction product will be able to be captured by streptavidin in the test zone due to the introduction of biotin-dUTP into the reaction product during PEXT. Gold nanoparticles will hybridize with reaction product to make it become visible. Here we reported the optimized parameters of Mg2+ in PEXT reaction and streptavidin on membranes to detect the signal specificity. In addition, it is found that increasing amount of PCR product and PEXT reaction cycle number result in the increase of the signal intensity without observable change of signal specificity.
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