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首页> 外文期刊>Separation and Purification Technology >Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate
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Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate

机译：用PEG 600和柠檬酸钠/硫酸铵的水相两相系统纯化质粒DNA

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Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citrate and ammonium sulfate were used to partially purify plasmid DNA (pDNA) from Escherichia coli alkaline lysates. The effect of pH and lysate load on the binodal curve was analyzed and tie-lines were determined in order to establish the optimal conditions for ATPS formation. A series of extraction experiments were performed at pH 6.9 using a 20% (w/w) lysate load and systems with 16.5% (w/w) salt, 19.0% (w/w) PEG 600, and a tie-line length of 37.1% (w/w). Under these conditions, plasmid DNA was recovered in the salt-rich bottom phase. However, whereas with sodium citrate-based systems recovery yields closer to 100% were obtained, the use of ammonium sulfate enabled higher purification although with lower recoveries. Thus, the performance of ATPS prepared by combining sodium citrate and ammonium sulfate was also evaluated. A mixture of 25% (w/w) ammonium sulfate and 75% (w/w) sodium citrate offered a good compromise between plasmid recovery (91.1%) and purity (17.2%). Multi-step extraction procedures were evaluated in order to improve the process performance. Although the majority of the impurities were removed in the first step, incremental increases in the purity were obtained with the inclusion of extra steps. The top to bottom phase volume ratio was increased in order to increase plasmid concentration in the bottom phase. Although this was achieved using a phase ratio of 4, it was not possible to concentrate plasmid relatively to the starting lysate. Overall, the results show that ammonium sulfate, a salt which has a high environmental impact, can be partially replaced in ATPS by sodium citrate, without significant decrease in the performance of plasmid purification.
机译：基于聚乙二醇（PEG）600，柠檬酸钠和硫酸铵的聚合物/盐水溶液两相系统（ATPS）用于从大肠杆菌碱性裂解物中部分纯化质粒DNA（pDNA）。分析了pH和裂解物负荷对双曲线曲线的影响，并确定了联系线，以建立ATPS形成的最佳条件。使用20％（w / w）的裂解物负载量和含有16.5％（w / w）盐，19.0％（w / w）PEG 600的连接线长度为pH 6.9的系统进行一系列提取实验37.1％（w / w）。在这些条件下，在富盐的底部相中回收质粒DNA。但是，尽管在基于柠檬酸钠的系统中回收率接近100％，但使用硫酸铵可以实现更高的纯化率，但回收率却较低。因此，还评估了通过组合柠檬酸钠和硫酸铵制备的ATPS的性能。 25％（w / w）的硫酸铵和75％（w / w）柠檬酸钠的混合物在质粒回收率（91.1％）和纯度（17.2％）之间提供了很好的折衷方案。对多步萃取程序进行了评估，以提高工艺性能。尽管在第一步中除去了大多数杂质，但是通过增加额外的步骤，纯度得到了逐步提高。增加顶相体积与底相体积的比例，以增加底相中的质粒浓度。尽管这是使用4的相比实现的，但相对于起始裂解液而言，无法浓缩质粒。总体而言，结果表明，具有较高环境影响的盐硫酸铵可被柠檬酸钠部分替代ATPS，而不会显着降低质粒纯化的性能。

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