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Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface

Gustavsson, Per-Erik LU ; Lemmens, R; Nyhammar, T; Busson, P and Larsson, Per-Olof LU (2004) In Journal of Chromatography A 1038(1-2). p.131-140
Abstract
We have prepared a new type of anion exchanger, which effectively discriminates between RNA and plasmid DNA. The material is based on a Sephacryl S-500 HR matrix provided with quartenary amine anion-exchange groups. A distinguishing feature of the beads is that a thin (2-3 mum) outer layer of the beads lacks ion-exchange groups. In the synthesis of these beads the vinyl groups in the outer layer of vinylalkyl substituted Sephacryl S-500 HR beads are reacted with bromine. The resulting layer of bromoalkyl groups are hydrolysed, creating an inert outer layer of hydroxyalkyl groups. Finally, bromination and trimethylamine reactions of the inner vinyl groups provide the beads with a core of cationic groups. Large plasmid molecules will not... (More)
We have prepared a new type of anion exchanger, which effectively discriminates between RNA and plasmid DNA. The material is based on a Sephacryl S-500 HR matrix provided with quartenary amine anion-exchange groups. A distinguishing feature of the beads is that a thin (2-3 mum) outer layer of the beads lacks ion-exchange groups. In the synthesis of these beads the vinyl groups in the outer layer of vinylalkyl substituted Sephacryl S-500 HR beads are reacted with bromine. The resulting layer of bromoalkyl groups are hydrolysed, creating an inert outer layer of hydroxyalkyl groups. Finally, bromination and trimethylamine reactions of the inner vinyl groups provide the beads with a core of cationic groups. Large plasmid molecules will not bind to such beads since they are too large to enter the pores and therefore cannot come into contact with the charged matrix in the inner parts of the beads. RNA and protein molecules present in a cleared lysate, on the other hand, readily enter the pores and become adsorbed. A two-column strategy was developed for plasmid purification (recombinant pBluescript, 5.9 kilo base pairs, kbp). The first column was packed with the restricted access anion-exchanger beads (lid beads) and the second column with normal ion-exchange material (same ligand density as the lid beads). Diluted (3 x), cleared lysate was pumped through the tandem columns. The first column was subsequently disconnected from the system and the purified plasmid adsorbed on the second column was eluted in a concentrated form (6x) and with 89% recovery. The two-column procedure removed 99.5% of the RNA and 96% of the proteins. (C) 2004 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Restricted access material, Anion exchangers, Stationary phases, LC, Coupled columns, DNA, RNA
in
Journal of Chromatography A
volume
1038
issue
1-2
pages
131 - 140
publisher
Elsevier
external identifiers
  • wos:000221708200016
  • pmid:15233529
  • scopus:2442530783
ISSN
0021-9673
DOI
10.1016/j.chroma.2004.03.050
language
English
LU publication?
yes
id
d7be1e4e-faa0-43ba-a3ae-96ddb109db4d (old id 141027)
date added to LUP
2007-07-16 09:39:42
date last changed
2017-01-01 07:21:00
@article{d7be1e4e-faa0-43ba-a3ae-96ddb109db4d,
  abstract     = {We have prepared a new type of anion exchanger, which effectively discriminates between RNA and plasmid DNA. The material is based on a Sephacryl S-500 HR matrix provided with quartenary amine anion-exchange groups. A distinguishing feature of the beads is that a thin (2-3 mum) outer layer of the beads lacks ion-exchange groups. In the synthesis of these beads the vinyl groups in the outer layer of vinylalkyl substituted Sephacryl S-500 HR beads are reacted with bromine. The resulting layer of bromoalkyl groups are hydrolysed, creating an inert outer layer of hydroxyalkyl groups. Finally, bromination and trimethylamine reactions of the inner vinyl groups provide the beads with a core of cationic groups. Large plasmid molecules will not bind to such beads since they are too large to enter the pores and therefore cannot come into contact with the charged matrix in the inner parts of the beads. RNA and protein molecules present in a cleared lysate, on the other hand, readily enter the pores and become adsorbed. A two-column strategy was developed for plasmid purification (recombinant pBluescript, 5.9 kilo base pairs, kbp). The first column was packed with the restricted access anion-exchanger beads (lid beads) and the second column with normal ion-exchange material (same ligand density as the lid beads). Diluted (3 x), cleared lysate was pumped through the tandem columns. The first column was subsequently disconnected from the system and the purified plasmid adsorbed on the second column was eluted in a concentrated form (6x) and with 89% recovery. The two-column procedure removed 99.5% of the RNA and 96% of the proteins. (C) 2004 Elsevier B.V. All rights reserved.},
  author       = {Gustavsson, Per-Erik and Lemmens, R and Nyhammar, T and Busson, P and Larsson, Per-Olof},
  issn         = {0021-9673},
  keyword      = {Restricted access material,Anion exchangers,Stationary phases,LC,Coupled columns,DNA,RNA},
  language     = {eng},
  number       = {1-2},
  pages        = {131--140},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface},
  url          = {http://dx.doi.org/10.1016/j.chroma.2004.03.050},
  volume       = {1038},
  year         = {2004},
}