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Selective partition of plasmid DNA and RNA from crude Escherichia coli cell lysate by aqueous two-phase systems
Biochemical Engineering Journal 55 (2011) 230–232
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Selective partition of plasmid DNA and RNA from crude Escherichia coli cell lysate by aqueous two-phase systems Frank Luechau a,∗ , Tau Chuan Ling b , Andrew Lyddiatt a,c a b c
Biochemical Recovery Group, Department of Chemical Engineering, School of Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Institute of Biological Sciences, Faculty of Science, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia Business School Newcastle University, Citywall, Citygate, St. James Boulevard, Newcastle Upon Tyne NE1 4JH, UK
a r t i c l e
i n f o
Article history: Received 17 March 2011 Received in revised form 19 April 2011 Accepted 27 April 2011 Available online 5 May 2011 Keywords: Bioseparations Aqueous two-phase system Purification Integrated processing RNA DNA
a b s t r a c t In this paper, the partition of plasmid DNA (pDNA) and RNA in polyethylene glycol (PEG) and dipotassium phosphate aqueous two-phase systems (ATPS) by adding up of NaCl salt was studied with crude Escherichia coli (E. coli) cell lysate. The partition of nucleic acid was studied in an ATPS composed of 17% (w/w) PEG300, 14% (w/w) di-potassium phosphate (designated as 17/14) and 40% (w/w) lysate. It is demonstrated that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342 mM (kg ATPS)−1 NaCl were able to selective partition the pDNA to the bottom phase. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The ATPS comprised of two polymers (e.g. PEG and dextran) have been applied to recover nucleic acids [1,2]. It has been shown that the partition is size-dependent [2] and the large difference between the partition coefficient of single and double-stranded nucleic acids has been exploited to isolate adenovirus messenger RNA [3]. For the separation of native and denatured DNA, Alberts [4,5] has developed standard ATPS procedures for preparative and analytical purposes. The buffering action of certain salts has been exploited to isolate DNA, RNA and DNA–protein complexes from bacterial feedstocks [6,7]. The properties of ATPS can be altered by adding one or more types of neutral salt to the systems [1]. In polymer–polymer systems, neutral salts are known to have preferences for a certain phase [8]. Such preferences can effectively render the phases positive or negative which can influence the partition behaviour of biomolecules [9]. Neutral salts are also known to partition to a cer-
∗ Corresponding author. Current address: B. Braun Melsungen AG, Aesculap Division, Vascular Systems, Sieversufer 8, D-12359 Berlin, Germany. Tel.: +49 0 3068989778. E-mail addresses: [email protected], f [email protected] (F. Luechau). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.04.014
tain phase in polymer–salt systems [10]. The use of PEG–salt ATPS for the separation and purification of nucleic acids is not common probably because PEG–Dx systems have worked sufficiently well for preparative and analytical purposes. The PEG–salt ATPS have several benefits over the polymer–polymer systems such as lower viscosities, higher interfacial tensions and less expensive. However, due to the high salt concentration in polymer–salt ATPS, it can be expected that the underlying principle of neutral salt partition in polymer–salt systems is different from that in polymer–polymer systems. The partition of pDNA of E. coli cell paste in polymer–salt (i.e. PEG 300 and di-potassium hydrogen phosphate) ATPS by adding up of NaCl salt has been reported recently [11]. The study verified a change in the selective partition of pDNA from top to bottom phase taking place between 2.1 and 2.6% (w/w) NaCl. However, the application of ATPS in the processing of crude lysate has not yet been explored. ATPS operated challenged with crude lysate would be expected to differ from the cell paste system reported in the previous publication by virtue of its complex content. In this paper, studies with crude lysate were carried out to validate the applicability of the findings obtained for E. coli cell paste. The partition of nucleic acid was studied in an ATPS composed of 17% (w/w) PEG300, 14% (w/w) di-potassium phosphate and 40% (w/w) lysate. Such a system was relatively close to the crude binodial curve, and the initial system without NaCl displayed no interfacial partition [12].
F. Luechau et al. / Biochemical Engineering Journal 55 (2011) 230–232
160
2
3
1 Volume ratio
80
0
2
Volume ratio
120
ln Knucleic acids
Nucleic acid concentration in (µg pDNA equivalent) ml-1
Bottom phase concentration
1
Top phase concentration
40
-1 ln K
0
-2 0
0
1 2 NaCl concentration in % w/w 200
0
3
400
NaCl concentration in mMoles (kg ATPS)-1 Fig. 1. Selective partition of plasmid DNA and RNA from crude E. coli cell lysate in 17/14 ATPS. To an ATPS composed of 17% (w/w) PEG 300, 14% (w/w) potassium phosphate and 40% (w/w) freshly prepared lysate (without ribonuclease A), NaCl was added at 0, 0.5, 1, 2 and 2.5% (w/w). The pH was not controlled and it decreased from 8.1 (no NaCl) to 7.8 (2.5%, w/w NaCl) for the suspended phases. The study was carried out at 20 ◦ C and the phases were separated by centrifugation at 1000 × g for 3 min. The data reported in this study are the mean of two independent experiments with an estimated error of ±5%. (a) Volume ratios () were recorded and nucleic acid contents were determined by PicoGreenTM analysis. The measured concentrations in top phases () and bottom phases () were expressed in (g pDNA equivalent) ml−1 and the logarithmic partition coefficient for nucleic acids (䊉) was calculated.
2. Materials and methods 2.1. Materials The plasmid purification kit (QIAprep® Spin Miniprep) was supplied by QIAGEN Ltd. (Crawley, UK). PicoGreenTM double-stranded (ds) DNA quantification reagent was sourced from Molecular Probes Inc. (Eugene, OR). Ribonucleic acid type II, lambda DNA BSTE II Digest, ribonuclease A (Type I-AS, from bovine pancreas) were purchased from Torula yeast. All of the chemicals were of analytical grade and purchased from Sigma–Aldrich Ltd. (Dorset, UK). 2.2. ATPS with crude lysate To an ATPS composed of 17% (w/w) PEG 300, 14% (w/w) potassium phosphate and 40% (w/w) freshly prepared lysate (without ribonuclease A), NaCl was added at 0, 0.5, 1, 2 and 2.5% (w/w). The
231
pH was not controlled and it decreased from 8.1 (no NaCl) to 7.8 (2.5%, w/w NaCl) for the both top and bottom phases. The ATPS study was carried out at 20 ◦ C and the phases were separated by centrifugation at 1000 × g for 3 min. Volume ratios were recorded and nucleic acid contents were estimated by PicoGreenTM analysis [13]. Volume ratio is the ratio between the volume of the salute in the top phase (VT ) and in the bottom phase (VB ) [1]. The measured concentrations in top phases and bottom phases were expressed in (g pDNA equivalent) ml−1 and the logarithmic partition coefficient for nucleic acids was determined. The partition coefficient (K) is the ratio between the concentration of the solute in the top phase (CT ) and in the bottom phase (CB ) [1]. The nucleic acid composition of top and bottom phases was visualised by 1% agarose gel analysis according to Sambrook et al. and the procedure has been described previously [14,15]. The data reported in this paper are the mean of two independent experiments with an estimated error of ± 5%. 3. Results and discussion In this study, the pH of the suspended phases was not manipulated and it decreased from pH 8.1 (no NaCl) to 7.8 (2.5%, w/w NaCl) for the both top and bottom phases. On addition of NaCl, a small degree of nucleic acids partitioned to the interface. However, the degree was not quantifiable in this study and in order to make the analysis of pDNA partition results easier, they were harvested in conjunction with the bottom phase for bulk analysis [14]. Figs. 1 and 2 show the partition results and nucleic acid analysis of top and bottom phase samples. The volume ratio decreased from 2.76 to 2.06 for NaCl concentrations between 0 and 1% (w/w). As a result, the effect of NaCl on the volume ratio appeared to be similar to that observed earlier in clean 19/16 ATPS [14]. The initial nucleic acid concentration (directly measured) was 61.5 (g pDNA equivalent ml−1 ). The measured nucleic acid concentration in the top phase decreased from 58 to 43 (g pDNA equivalent ml−1 ), while the concentration in the bottom phase increased from 12 to 131 (g pDNA equivalent ml−1 ). As a result, the resulting logarithmic partition coefficient ln Knucleic acids decreased from 1.6 to −1.1. Apparently, the bulk of nucleic acids had changed their top phase preference to a bottom preference at 1% (w/w) NaCl. However, agarose gel analysis of top and bottom phases shown in Fig. 2 revealed that NaCl concentrations between 1 and 2% (w/w) were required to ensure that the top phase did not contain any pDNA. It was concluded that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342 mM (kg ATPS)−1 NaCl were required to partition pDNA to the bottom phase.
Fig. 2. Agarose gel analysis of top and bottom phase samples for selective partition of nucleic acids from lysate in 17/14 ATPS. The nucleic acid composition of top and bottom phases was visualised by 1% agarose gel analysis according to Sambrook et al. [15]. The experiments are described in the legend for Fig. 1. oc – open circular plasmid DNA; sc – supercoiled plasmid DNA.
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F. Luechau et al. / Biochemical Engineering Journal 55 (2011) 230–232
4. Conclusions The study indicated that the use of polymer–salt ATPS could be developed to the selective removal of RNA from pDNA in crude E. coli cell lysate upon the adding up of NaCl to the operation. It is demonstrated that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342 mM (kg ATPS)−1 NaCl were required to partition pDNA to the bottom phase. The result indicated that the NaCl concentrations required to achieve an identical selective partition effect were of similar magnitude with cell paste [11]. The finding obtained in this study demonstrated that the excluded volume effect in the top phase was probably responsible for the selective partition of pDNA and RNA in the crude E. coli cell lysate [14]. Acknowledgement The authors thank Cobra Therapeutics Ltd. (Keele, UK) for their sponsorship. References [1] P.A. Albertsson, Partition of Cell Particles and Macromolecules, Wiley, New York, 1986. [2] W. Müller, A. Eigel, DNA fractionation by two-phase partition with aid of a base-specific macroligand, Anal. Biochem. 118 (1981) 269–277. [3] S. Mak, B. Öberg, K. Johansson, L. Philipson, Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system, Biochemistry 15 (1976) 5754–5761.
[4] B.M. Alberts, in: K.M. Lawrence Grossman (Ed.), Fractionation of Nucleic Acids by Dextran–polyethylene Glycol Two-phase Systems, Method Enzymol Academic Press, 1967, pp. 566–581. [5] B.M. Alberts, Efficient separation of single-stranded and double-stranded deoxyribonucleic acid in a dextran–polyethylene glycol two-phase system, Biochemistry 6 (1967) 2527–2532. [6] J. Favre, D.E. Pettijohn, A method for extracting purified DNA or protein–DNA complex from Escherichia coli, Eur. J. Biochem. 3 (1967) 33–41. [7] L. Rudin, P.Å. Albertsson, A new method for the isolation of deoxyribonucleic acid from microorganisms, BBA-Nucleic Acids Protein Synth. 134 (1967) 37–44. [8] L.M. Miheeva, E.D. Maximova, Y.P. Aleschko-Ozhevskii, B.Y. Zaslavsky, Partitioning of alkali halides in aqueous dextran–ficoll two-phase system, J. Solut. Chem. 20 (1991) 607–611. [9] D.E.B.H. Walter, D. Fisher, Partitioning in Aqueous Two-phase Systems. Theory, Methods, Uses, and Applications to Biotechnology, Academic Press Inc., London, 1985. [10] T.I. Zvarova, V.M. Shkinev, G.A. Vorob’eva, B.Y. Spivakov, Y.A. Zolotov, Liquid–liquid extraction in the absence of usual organic solvents: application of two-phase aqueous systems based on a water-soluble polymer, Mikrochim. Acta 84 (1984) 449–458. [11] F. Luechau, T.C. Ling, A. Lyddiatt, Primary capture of high molecular weight nucleic acids using aqueous two-phase systems, Sep. Purif. Technol. 66 (2009) 202–207. [12] F. Luechau, T.C. Ling, A. Lyddiatt, Partition of plasmid DNA and RNA in polymer–salt aqueous two-phase, Sep. Purif. Technol. 66 (2009) 397–404. [13] E. Thwaites, S.C. Burton, A. Lyddiatt, Impact of the physical and topographical characteristics of adsorbent solid-phases upon the fluidised bed recovery of plasmid DNA from Escherichia coli lysates, J. Chromatogr. A 943 (2002) 77–90. [14] F. Luechau, T.C. Ling, A. Lyddiatt, Selective partition of plasmid DNA and RNA in aqueous two-phase systems by the addition of neutral salt, Sep. Purif. Technol. 68 (2009) 114–118. [15] T. Maniatis, E.F. Fritsch, J. Sambrook, in: J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989.
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Selective partition of plasmid DNA and RNA from crude Escherichia coli cell lysate by aqueous two-phase systems Frank Luechau a,∗ , Tau Chuan Ling b , Andrew Lyddiatt a,c a b c
Biochemical Recovery Group, Department of Chemical Engineering, School of Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Institute of Biological Sciences, Faculty of Science, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia Business School Newcastle University, Citywall, Citygate, St. James Boulevard, Newcastle Upon Tyne NE1 4JH, UK
a r t i c l e
i n f o
Article history: Received 17 March 2011 Received in revised form 19 April 2011 Accepted 27 April 2011 Available online 5 May 2011 Keywords: Bioseparations Aqueous two-phase system Purification Integrated processing RNA DNA
a b s t r a c t In this paper, the partition of plasmid DNA (pDNA) and RNA in polyethylene glycol (PEG) and dipotassium phosphate aqueous two-phase systems (ATPS) by adding up of NaCl salt was studied with crude Escherichia coli (E. coli) cell lysate. The partition of nucleic acid was studied in an ATPS composed of 17% (w/w) PEG300, 14% (w/w) di-potassium phosphate (designated as 17/14) and 40% (w/w) lysate. It is demonstrated that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342 mM (kg ATPS)−1 NaCl were able to selective partition the pDNA to the bottom phase. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The ATPS comprised of two polymers (e.g. PEG and dextran) have been applied to recover nucleic acids [1,2]. It has been shown that the partition is size-dependent [2] and the large difference between the partition coefficient of single and double-stranded nucleic acids has been exploited to isolate adenovirus messenger RNA [3]. For the separation of native and denatured DNA, Alberts [4,5] has developed standard ATPS procedures for preparative and analytical purposes. The buffering action of certain salts has been exploited to isolate DNA, RNA and DNA–protein complexes from bacterial feedstocks [6,7]. The properties of ATPS can be altered by adding one or more types of neutral salt to the systems [1]. In polymer–polymer systems, neutral salts are known to have preferences for a certain phase [8]. Such preferences can effectively render the phases positive or negative which can influence the partition behaviour of biomolecules [9]. Neutral salts are also known to partition to a cer-
∗ Corresponding author. Current address: B. Braun Melsungen AG, Aesculap Division, Vascular Systems, Sieversufer 8, D-12359 Berlin, Germany. Tel.: +49 0 3068989778. E-mail addresses: [email protected], f [email protected] (F. Luechau). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.04.014
tain phase in polymer–salt systems [10]. The use of PEG–salt ATPS for the separation and purification of nucleic acids is not common probably because PEG–Dx systems have worked sufficiently well for preparative and analytical purposes. The PEG–salt ATPS have several benefits over the polymer–polymer systems such as lower viscosities, higher interfacial tensions and less expensive. However, due to the high salt concentration in polymer–salt ATPS, it can be expected that the underlying principle of neutral salt partition in polymer–salt systems is different from that in polymer–polymer systems. The partition of pDNA of E. coli cell paste in polymer–salt (i.e. PEG 300 and di-potassium hydrogen phosphate) ATPS by adding up of NaCl salt has been reported recently [11]. The study verified a change in the selective partition of pDNA from top to bottom phase taking place between 2.1 and 2.6% (w/w) NaCl. However, the application of ATPS in the processing of crude lysate has not yet been explored. ATPS operated challenged with crude lysate would be expected to differ from the cell paste system reported in the previous publication by virtue of its complex content. In this paper, studies with crude lysate were carried out to validate the applicability of the findings obtained for E. coli cell paste. The partition of nucleic acid was studied in an ATPS composed of 17% (w/w) PEG300, 14% (w/w) di-potassium phosphate and 40% (w/w) lysate. Such a system was relatively close to the crude binodial curve, and the initial system without NaCl displayed no interfacial partition [12].
F. Luechau et al. / Biochemical Engineering Journal 55 (2011) 230–232
160
2
3
1 Volume ratio
80
0
2
Volume ratio
120
ln Knucleic acids
Nucleic acid concentration in (µg pDNA equivalent) ml-1
Bottom phase concentration
1
Top phase concentration
40
-1 ln K
0
-2 0
0
1 2 NaCl concentration in % w/w 200
0
3
400
NaCl concentration in mMoles (kg ATPS)-1 Fig. 1. Selective partition of plasmid DNA and RNA from crude E. coli cell lysate in 17/14 ATPS. To an ATPS composed of 17% (w/w) PEG 300, 14% (w/w) potassium phosphate and 40% (w/w) freshly prepared lysate (without ribonuclease A), NaCl was added at 0, 0.5, 1, 2 and 2.5% (w/w). The pH was not controlled and it decreased from 8.1 (no NaCl) to 7.8 (2.5%, w/w NaCl) for the suspended phases. The study was carried out at 20 ◦ C and the phases were separated by centrifugation at 1000 × g for 3 min. The data reported in this study are the mean of two independent experiments with an estimated error of ±5%. (a) Volume ratios () were recorded and nucleic acid contents were determined by PicoGreenTM analysis. The measured concentrations in top phases () and bottom phases () were expressed in (g pDNA equivalent) ml−1 and the logarithmic partition coefficient for nucleic acids (䊉) was calculated.
2. Materials and methods 2.1. Materials The plasmid purification kit (QIAprep® Spin Miniprep) was supplied by QIAGEN Ltd. (Crawley, UK). PicoGreenTM double-stranded (ds) DNA quantification reagent was sourced from Molecular Probes Inc. (Eugene, OR). Ribonucleic acid type II, lambda DNA BSTE II Digest, ribonuclease A (Type I-AS, from bovine pancreas) were purchased from Torula yeast. All of the chemicals were of analytical grade and purchased from Sigma–Aldrich Ltd. (Dorset, UK). 2.2. ATPS with crude lysate To an ATPS composed of 17% (w/w) PEG 300, 14% (w/w) potassium phosphate and 40% (w/w) freshly prepared lysate (without ribonuclease A), NaCl was added at 0, 0.5, 1, 2 and 2.5% (w/w). The
231
pH was not controlled and it decreased from 8.1 (no NaCl) to 7.8 (2.5%, w/w NaCl) for the both top and bottom phases. The ATPS study was carried out at 20 ◦ C and the phases were separated by centrifugation at 1000 × g for 3 min. Volume ratios were recorded and nucleic acid contents were estimated by PicoGreenTM analysis [13]. Volume ratio is the ratio between the volume of the salute in the top phase (VT ) and in the bottom phase (VB ) [1]. The measured concentrations in top phases and bottom phases were expressed in (g pDNA equivalent) ml−1 and the logarithmic partition coefficient for nucleic acids was determined. The partition coefficient (K) is the ratio between the concentration of the solute in the top phase (CT ) and in the bottom phase (CB ) [1]. The nucleic acid composition of top and bottom phases was visualised by 1% agarose gel analysis according to Sambrook et al. and the procedure has been described previously [14,15]. The data reported in this paper are the mean of two independent experiments with an estimated error of ± 5%. 3. Results and discussion In this study, the pH of the suspended phases was not manipulated and it decreased from pH 8.1 (no NaCl) to 7.8 (2.5%, w/w NaCl) for the both top and bottom phases. On addition of NaCl, a small degree of nucleic acids partitioned to the interface. However, the degree was not quantifiable in this study and in order to make the analysis of pDNA partition results easier, they were harvested in conjunction with the bottom phase for bulk analysis [14]. Figs. 1 and 2 show the partition results and nucleic acid analysis of top and bottom phase samples. The volume ratio decreased from 2.76 to 2.06 for NaCl concentrations between 0 and 1% (w/w). As a result, the effect of NaCl on the volume ratio appeared to be similar to that observed earlier in clean 19/16 ATPS [14]. The initial nucleic acid concentration (directly measured) was 61.5 (g pDNA equivalent ml−1 ). The measured nucleic acid concentration in the top phase decreased from 58 to 43 (g pDNA equivalent ml−1 ), while the concentration in the bottom phase increased from 12 to 131 (g pDNA equivalent ml−1 ). As a result, the resulting logarithmic partition coefficient ln Knucleic acids decreased from 1.6 to −1.1. Apparently, the bulk of nucleic acids had changed their top phase preference to a bottom preference at 1% (w/w) NaCl. However, agarose gel analysis of top and bottom phases shown in Fig. 2 revealed that NaCl concentrations between 1 and 2% (w/w) were required to ensure that the top phase did not contain any pDNA. It was concluded that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342 mM (kg ATPS)−1 NaCl were required to partition pDNA to the bottom phase.
Fig. 2. Agarose gel analysis of top and bottom phase samples for selective partition of nucleic acids from lysate in 17/14 ATPS. The nucleic acid composition of top and bottom phases was visualised by 1% agarose gel analysis according to Sambrook et al. [15]. The experiments are described in the legend for Fig. 1. oc – open circular plasmid DNA; sc – supercoiled plasmid DNA.
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F. Luechau et al. / Biochemical Engineering Journal 55 (2011) 230–232
4. Conclusions The study indicated that the use of polymer–salt ATPS could be developed to the selective removal of RNA from pDNA in crude E. coli cell lysate upon the adding up of NaCl to the operation. It is demonstrated that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342 mM (kg ATPS)−1 NaCl were required to partition pDNA to the bottom phase. The result indicated that the NaCl concentrations required to achieve an identical selective partition effect were of similar magnitude with cell paste [11]. The finding obtained in this study demonstrated that the excluded volume effect in the top phase was probably responsible for the selective partition of pDNA and RNA in the crude E. coli cell lysate [14]. Acknowledgement The authors thank Cobra Therapeutics Ltd. (Keele, UK) for their sponsorship. References [1] P.A. Albertsson, Partition of Cell Particles and Macromolecules, Wiley, New York, 1986. [2] W. Müller, A. Eigel, DNA fractionation by two-phase partition with aid of a base-specific macroligand, Anal. Biochem. 118 (1981) 269–277. [3] S. Mak, B. Öberg, K. Johansson, L. Philipson, Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system, Biochemistry 15 (1976) 5754–5761.
[4] B.M. Alberts, in: K.M. Lawrence Grossman (Ed.), Fractionation of Nucleic Acids by Dextran–polyethylene Glycol Two-phase Systems, Method Enzymol Academic Press, 1967, pp. 566–581. [5] B.M. Alberts, Efficient separation of single-stranded and double-stranded deoxyribonucleic acid in a dextran–polyethylene glycol two-phase system, Biochemistry 6 (1967) 2527–2532. [6] J. Favre, D.E. Pettijohn, A method for extracting purified DNA or protein–DNA complex from Escherichia coli, Eur. J. Biochem. 3 (1967) 33–41. [7] L. Rudin, P.Å. Albertsson, A new method for the isolation of deoxyribonucleic acid from microorganisms, BBA-Nucleic Acids Protein Synth. 134 (1967) 37–44. [8] L.M. Miheeva, E.D. Maximova, Y.P. Aleschko-Ozhevskii, B.Y. Zaslavsky, Partitioning of alkali halides in aqueous dextran–ficoll two-phase system, J. Solut. Chem. 20 (1991) 607–611. [9] D.E.B.H. Walter, D. Fisher, Partitioning in Aqueous Two-phase Systems. Theory, Methods, Uses, and Applications to Biotechnology, Academic Press Inc., London, 1985. [10] T.I. Zvarova, V.M. Shkinev, G.A. Vorob’eva, B.Y. Spivakov, Y.A. Zolotov, Liquid–liquid extraction in the absence of usual organic solvents: application of two-phase aqueous systems based on a water-soluble polymer, Mikrochim. Acta 84 (1984) 449–458. [11] F. Luechau, T.C. Ling, A. Lyddiatt, Primary capture of high molecular weight nucleic acids using aqueous two-phase systems, Sep. Purif. Technol. 66 (2009) 202–207. [12] F. Luechau, T.C. Ling, A. Lyddiatt, Partition of plasmid DNA and RNA in polymer–salt aqueous two-phase, Sep. Purif. Technol. 66 (2009) 397–404. [13] E. Thwaites, S.C. Burton, A. Lyddiatt, Impact of the physical and topographical characteristics of adsorbent solid-phases upon the fluidised bed recovery of plasmid DNA from Escherichia coli lysates, J. Chromatogr. A 943 (2002) 77–90. [14] F. Luechau, T.C. Ling, A. Lyddiatt, Selective partition of plasmid DNA and RNA in aqueous two-phase systems by the addition of neutral salt, Sep. Purif. Technol. 68 (2009) 114–118. [15] T. Maniatis, E.F. Fritsch, J. Sambrook, in: J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989.