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Isolation of PCR DNA fragments using Aqueous Two-Phase Systems

Matos, Tiago LU ; Johansson, Hans-Olof LU ; Queiroz, João A. and Bülow, Leif LU (2014) In Separation and Purification Technology 122. p.144-148
Abstract
Aqueous two-phase systems have frequently been utilized as an efficient bioseparation tool in the downstream processing of different biomolecules. The poly(ethyleneglycol)/poly(acrylate)/salt system, already explored successfully for purification of proteins and plasmid DNA, is here presented as an alternative approach for the isolation of small DNA fragments generated during in vitro DNA polymerase chain reactions. The polymerase chain reaction (PCR) is one of the most versatile laboratory techniques, but the purification of the amplified fragments often represents a major bottleneck. In this work we describe a simple and cost-effective method for isolation of DNA fragments obtained from PCR mixtures. The composition of the aqueous... (More)
Aqueous two-phase systems have frequently been utilized as an efficient bioseparation tool in the downstream processing of different biomolecules. The poly(ethyleneglycol)/poly(acrylate)/salt system, already explored successfully for purification of proteins and plasmid DNA, is here presented as an alternative approach for the isolation of small DNA fragments generated during in vitro DNA polymerase chain reactions. The polymerase chain reaction (PCR) is one of the most versatile laboratory techniques, but the purification of the amplified fragments often represents a major bottleneck. In this work we describe a simple and cost-effective method for isolation of DNA fragments obtained from PCR mixtures. The composition of the aqueous two-phase system in this work has been chosen to precipitate DNA molecules larger than 5000–7000 bp in the interphase, while the PCR products are partitioned very strongly between the phases in a two-step extraction process. In the first step, the DNA is partitioned quantitatively to the poly(ethyleneglycol)-phase. In the second, i.e. the back-extraction step, the DNA is strongly partitioned to a salt-rich phase that contains only low amounts of polymer. This system promotes rapid and high yields of purified small DNA products, less than 4000 bp, without contamination of proteins or large DNA templates present in the reaction mixture. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Separation and Purification Technology
volume
122
pages
144 - 148
publisher
Elsevier
external identifiers
  • wos:000331422800021
  • scopus:84888615757
ISSN
1873-3794
DOI
10.1016/j.seppur.2013.11.014
language
English
LU publication?
yes
id
d392f31c-59e0-4c42-8572-0cfee0609ad0 (old id 4228180)
date added to LUP
2014-01-30 12:01:35
date last changed
2017-07-23 03:27:44
@article{d392f31c-59e0-4c42-8572-0cfee0609ad0,
  abstract     = {Aqueous two-phase systems have frequently been utilized as an efficient bioseparation tool in the downstream processing of different biomolecules. The poly(ethyleneglycol)/poly(acrylate)/salt system, already explored successfully for purification of proteins and plasmid DNA, is here presented as an alternative approach for the isolation of small DNA fragments generated during in vitro DNA polymerase chain reactions. The polymerase chain reaction (PCR) is one of the most versatile laboratory techniques, but the purification of the amplified fragments often represents a major bottleneck. In this work we describe a simple and cost-effective method for isolation of DNA fragments obtained from PCR mixtures. The composition of the aqueous two-phase system in this work has been chosen to precipitate DNA molecules larger than 5000–7000 bp in the interphase, while the PCR products are partitioned very strongly between the phases in a two-step extraction process. In the first step, the DNA is partitioned quantitatively to the poly(ethyleneglycol)-phase. In the second, i.e. the back-extraction step, the DNA is strongly partitioned to a salt-rich phase that contains only low amounts of polymer. This system promotes rapid and high yields of purified small DNA products, less than 4000 bp, without contamination of proteins or large DNA templates present in the reaction mixture.},
  author       = {Matos, Tiago and Johansson, Hans-Olof and Queiroz, João A. and Bülow, Leif},
  issn         = {1873-3794},
  language     = {eng},
  pages        = {144--148},
  publisher    = {Elsevier},
  series       = {Separation and Purification Technology},
  title        = {Isolation of PCR DNA fragments using Aqueous Two-Phase Systems},
  url          = {http://dx.doi.org/10.1016/j.seppur.2013.11.014},
  volume       = {122},
  year         = {2014},
}