目的: 建立可定量检测质粒DNA 超螺旋构象相对百分含量的毛细管凝胶电泳法（CGE）,并开展方法学验证研究及初步应用探讨。方法: 选用涂层毛细管,采用荧光检测器CGE 法分离质粒DNA,首先通过限制性内切酶和缺刻酶处理法确认质粒DNA 超螺旋（covalently closed circular,ccc）、线性（linear）及开环（open circular,oc）这3 种构象在电泳图谱中的位置,然后通过对荧光染料、样品上样浓度等条件进行优化,建立质粒DNA 构象的检测方法。对所建方法进行专属性、准确性、线性、精密度、检测下限和定量下限验证,并通过将质粒DNA 置于不同保存温度、反复冻融及紫外照射后再进行质粒DNA 构象的检测,初步分析该方法应用的可行性。结果: 采用总长度为40 cm、有效长度为30.2 cm 涂层毛细管,毛细管和样品上样温度分别为20 ℃和4 ℃,质粒浓度为4 ng·μL^-1 以1 378.95 Pa,4 s 上样分离,CGE 法可准确地定量质粒DNA 3 种构象所占的比例,且分离度良好。通过对3 种常用的质粒保存液基质的检测结果显示,这些基质不会干扰检测结果,专属性较好。质粒DNA 3 种构象的线性范围均在0.25~8 ng·μL^-1,构象峰面积与上样浓度具有良好的线性关系,相关系数分别是0.997 7、0.999 4、0.992 7。将线性化质粒及开环质粒分别以3%~50% 添加至原质粒中,可检出的线性及开环构象的峰面积与添加浓度呈现良好的线性关系,相关系数分别为0.999 9 和0.997 2,线性构象的回收率在104%~120%,准确性良好;开环构象的回收率稍差,为68%~94%。将质粒DNA 分别以高（4 μg·mL^-1）、中（2 μg·mL^-1）、低（0.5 μg·mL^-1）3 个浓度验证实验的重复性,结果显示,试验内重复性良好,质粒DNA 超螺旋构象迁移时间的RSD 在0.22%~0.54%,相对百分含量的RSD 在0.38%~2.1%; 中间精密度也较好,迁移时间的RSD 在0.52%~1.1%;相对百分含量的精密度在质量浓度为2 ng·μL^-1 和 4 ng·μL^-1 时,RSD 值分别为3.6%、0.25%。灵敏度的验证结果显示,定量下限及检测下限分别为0.5 ng·μL^-1 和0.25 ng·μL^-1。用该方法分别检测质粒DNA 在不同保存条件、冻融及紫外照射后的构象变化,结果显示,质粒DNA 在-80 ℃、-20 ℃保存1 周,超螺旋构象无明显变化,但在室温保存1 周后,质粒DNA 开环构象含量从1.39% 增加到4.16%;将质粒DNA 反复冻融5 次,未观察到明显的构象变化,但冻融7 次后,质粒DNA 开环构象相对百分含量从1.3% 左右增加到1.93%;质粒DNA 质量浓度在1 mg·mL^-1 时,紫外照射 15 min 到3 h,质粒DNA 的超螺旋构象相对百分含量从96.97% 下降到54.38%,同时线性及开环构象明显增加。结论: 本研究建立的毛细管凝胶电泳法可以很好地将质粒DNA 的超螺旋、线性及oc 3 种构象分离,各构象分离度良好,可准确定量不同构象相对百分含量,线性相关系数不低于0.99,灵敏度高,可重复性好,且可灵敏地反映出质粒DNA 构象的变化,可用于质粒DNA 构象纯度检测的质量控制。

Objective： To establish and validate a kind of capillary gel electrophoresis（CGE） method which can quantitate the content of plasmid DNA isoforms,and explore its application. Methods： Firstly using the coated capillary,the plasmid DNA（ p16E25） was separated by CGE method. Then the plasmid p16E25 was digested by restriction and nicking endonuclease respectively and separated by CGE to determine which migration peak was supercoiled closed circular（ ccc）,linear or open circular（oc） form. Secondly,CGE method for plasmid DNA isoforms separation was further optimized for fluorescent dyes and loading concentration and then validated for its the specificity,accuracy,linearity,precision,limit of detection（ LOD） and limit of quantitation（ LOQ）. Finally,the method was used to detect the change of plasmid DNA isoforms after treatment for being kept at varied temperature,several freeze-thawed cycles and UV irradiation. Results： After optimization,the content percentage of plasmid DNA isoforms could be well quantitated by CGE method with LIF detector using following parameters, including coated capillary whose total length was 40 cm, the 30.2 cm effective length（ inlet to window）,loading the 4 ng·μL^-1 of plasmid DNA at 4 ℃ with capillary temperature at 20 ℃,and injection at 4 s at 1 378.95 Pa. The established CGE method could accurately quantify the content percentage of three plasmid DNA isoforms（ ccc, linear and oc isoforms） with good resolution. The method had good specificity asthere was no observed interference from substances which were often used as plasmid DNA excipients. Linear range of the method for each of plasmid isoforms was from 0.25 ng·μL^-1 to 8 ng·μL^-1. A good linearity was exhibited between content percentage and loading concentration of each isoform,and the correlation coefficient were 0.997 7,0.999 4 and 0.992 7 for linear,oc and ccc isoforms of plasmid DNA,respectively. Spiking 3%-50% of linear or oc isoform plasmid to the original plasmid,the content percentage of the linear or plasmid exhibited good linearity,and the correlation coefficients were 0.999 9 and 0.997 2,respectively. The linear isoform had good recovery ratio ranged from 104% to 120%,which was better than that of oc isoform,rangingfrom 68% to 94%. The repeatability was determined by detecting three concentrations of high,medium and low concentrations of plasmid DNA （4,2 and 0.5 ng·μL^-1）. The RSD of migration time and content percentage of ccc plasmid were 0.22%-0.54% and 0.38%-2.1%, respectively. The results indicated the method had a good intra-assay repeatability. The intermediate precision was also good. The RSD of migration time was 0.52%-1.1%,and the RSD of content percentage were 3.6% and 0.25% corresponding to the medium and high loading concentration of plasmid DNA,respectively. Precision was better when the concentrations were 2 ng·μL^-1 and 4 ng·μL^-1,respectively. After sensitivity validation,LOQ and LOD of the method were 0.5 and 0.25 ng·μL^-1,respectively. Finally,after full validation,the established CGE method could be used for intended purpose. The method was used to determine the change of plasmid DNA isoforms under different conditions,such as storage temperature,freeze-thawed cycles and UV irradiation. The results showed that the content percentage of ccc plasmid DNA isoform was not affected by stored at -80 ℃ and -20 ℃ for one week. However,after stored at room temperature for one week,the content percentage of oc plasmid DNA increased from 1.39% to 4.16%. Plasmid isoforms alteration was not obviously observed after five freeze-thawed cycles, but the content percentage of oc plasmid DNA was increased from about 1.3% to 1.93% after seven freeze-thawed cycles. When the 1 mg·mL^-1 plasmid was exposed to UV irradiation for 15 min to 3 h,the content percentage of ccc plasmid DNA was markedly decreased from 96.97% to 54.38% along with content percentage of linear and oc plasmid DNA significantly increased. Conclusion： The established capillary gel electrophoresis method is capable of well separating the ccc,linear and oc isoforms of plasmid DNA,with high resolution. The method can be used to accurately quantify the content of different isoforms in a plasmid DNA,and it could detect the change of plasmid DNA isoforms with high sensitivity. Therefore,it will be helpful for control of plasmid DNA purity.