Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction.
In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.
Frerix
Andreas
A
Institut für Biotechnologie 2, Forschungszentrum Jülich, 52426 Jülich, Germany.
Geilenkirchen
Petra
P
Müller
Markus
M
Kula
Maria-Regina
MR
Hubbuch
Jürgen
J
D005591:Chemical Fractionation / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004270:DNA, Circular / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D044367:Phase Transition; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D014462:Ultrafiltration / Q000379:methods*; D014867:Water / Q000737:chemistry
D004270:DNA, Circular; D014867:Water; D012313:RNA; D004247:DNA