Adsorptive purification of pDNA on superporous rigid cross-linked cellulose matrix.
Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS-PAGE.
Deshmukh
N R
NR
Chemical Engineering Division, Mumbai University Institute of Chemical Technology, Matunga, Mumbai 400019, India.
Lali
A M
AM
D000327:Adsorption; D002482:Cellulose; D002845:Chromatography / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000302:isolation & purification*; D057927:Hydrophobic and Hydrophilic Interactions; D010957:Plasmids / Q000302:isolation & purification*
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D002482:Cellulose