Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membra...Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.展开更多
对结核病核酸疫苗的质粒DNA的制备性纯化过程进行了初步研究。此工艺主要包括以简化的碱裂解法处理大肠杆菌、超滤与离心法除大部分杂质并浓缩样品、Q-Sepharose阴离子交换层析、DNA B ioSepharTMFF亲和层析等步骤,以去除细胞RNA和蛋白...对结核病核酸疫苗的质粒DNA的制备性纯化过程进行了初步研究。此工艺主要包括以简化的碱裂解法处理大肠杆菌、超滤与离心法除大部分杂质并浓缩样品、Q-Sepharose阴离子交换层析、DNA B ioSepharTMFF亲和层析等步骤,以去除细胞RNA和蛋白等杂质,并对不同分子结构的质粒DNA作分离。结果表明:以碱裂液中质粒DNA为计算基准的得率为56.61%、超螺旋质粒DNA的电泳纯度100%。展开更多
基金supported in part by research grants from the China Postdoctoral Science Foundation(2019M663446 to ZZ)the Postdoctoral Program of the Natural Science Foundation of Chongqing,China(cstc2019jcyj-bsh0006 to ZZ)+6 种基金WW was supported by the Medical Scientist Training Program of the National Institutes of Health(T32 GM007281)This project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430TCH was supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund.Funding sources were not involved in the study designin the collection,analysis and interpretation of datain the writing of the reportand in the decision to submit the paper for publication.
文摘Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.
文摘对结核病核酸疫苗的质粒DNA的制备性纯化过程进行了初步研究。此工艺主要包括以简化的碱裂解法处理大肠杆菌、超滤与离心法除大部分杂质并浓缩样品、Q-Sepharose阴离子交换层析、DNA B ioSepharTMFF亲和层析等步骤,以去除细胞RNA和蛋白等杂质,并对不同分子结构的质粒DNA作分离。结果表明:以碱裂液中质粒DNA为计算基准的得率为56.61%、超螺旋质粒DNA的电泳纯度100%。
