A rapid method for the isolation of circular DNA using an aqueous two-phase partition system

Ohlsson, Rolf; Hentschel, Christopher C.; Williams, Jeffrey

doi:10.1093/nar/5.2.583

Cited by 48 publications

(21 citation statements)

References 9 publications

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“…Although nucleic acids have been separated, or partitioned, in two-phase aqueous polymer systems (Alberts, 1967;Ohlsson et al, 1978;Ribeiro et al, 2002), to the best of our knowledge, this work represents the first reported investigation of DNA partitioning in two-phase aqueous micellar systems. These micellar systems are composed of mainly water and surfactants.…”

Section: Introductionmentioning

confidence: 99%

Concentration of mammalian genomic DNA using two‐phase aqueous micellar systems

Mashayekhi

Meyer

Shiigi

et al. 2008

Biotech & Bioengineering

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The concentration of biomarkers, such as DNA, prior to a subsequent detection step may facilitate the early detection of cancer, which could significantly increase chances for survival. In this study, the partitioning behavior of mammalian genomic DNA fragments in a two-phase aqueous micellar system was investigated using both experiment and theory. The micellar system was generated using the nonionic surfactant Triton X-114 and phosphate-buffered saline (PBS). Partition coefficients were measured under a variety of conditions and compared with our theoretical predictions. With this comparison, we demonstrated that the partitioning behavior of DNA fragments in this system is primarily driven by repulsive, steric, excluded-volume interactions that operate between the micelles and the DNA fragments, but is limited by the entrainment of micelle-poor, DNA-rich domains in the macroscopic micelle-rich phase. Furthermore, the volume ratio, that is, the volume of the top, micelle-poor phase divided by that of the bottom, micelle-rich phase, was manipulated to concentrate DNA fragments in the top phase. Specifically, by decreasing the volume ratio from 1 to 1/10, we demonstrated proof-of-principle that the concentration of DNA fragments in the top phase could be increased two- to nine-fold in a predictive manner.

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Section: Introductionmentioning

confidence: 99%

Concentration of mammalian genomic DNA using two‐phase aqueous micellar systems

Mashayekhi

Meyer

Shiigi

et al. 2008

Biotech & Bioengineering

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“…Aqueous two-phase system (ATPS) is widely used in biochemistry and biotechnology for purification of proteins (Diamond and Hsu, 1992;Balasubramaniam et al, 2003), enzymes (Pan et al, 2001;Bim and Franco, 2000), amino acids (Li et al, 2002), antibiotics (Yang et al, 1994;Guan et al, 1994), nucleic acids (Ohlsson et al, 1978;Kimura, 2000), aroma compounds (Marco et al, 2000) and lactic acid (Planas et al, 1998). ATPS is mainly composed of two-incompatible polymers (e.g.…”

Section: Introductionmentioning

confidence: 99%

Effective extraction of elastase from Bacillus sp. fermentation broth using aqueous two-phase system

Li³

2005

J Zheijang Univ Sci B

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This paper presents the evaluation of an aqueous two-phase system (ATPS) for extracting elastase produced by Bacillus sp. EL31410. The elastase and cell partition behavior in polyethylene glycol (PEG)/salt systems was investigated. The suitable system for elastase extraction was PEG/KH 2 PO 4 -K 2 HPO 4 , in which elastase is mainly partitioned into the PEG-rich phase, while the cells remained in the other phase. The influence of defined system parameters (e.g. PEG molecular mass, pH, NaCl addition) on the partitioning behavior of elastase is described. The concentration of phase forming components, PEG and KH 2 PO 4 -K 2 HPO 4 , was optimized for elastase recovery by means of response surface methodology, and it was found that they greatly influenced extraction recovery. The optimal ATPS was 23.1% (w/w) PEG 2 000 and 11.7% (w/w) KH 2 PO 4 -K 2 HPO 4 . The predicted recovery was about 89.5%, so this process is suggested to be a rapid and convenient method for elastase extraction.

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“…The technique later proved to be of immense utility in analytical, biochemical, and environmental research and applications (Hatti-Kaul, 1999). As opposed to proteins, plasmid purification by aqueous twophase extraction has evolved very little and few related references have appeared in the literature in the last years (Cole, 1991;Ohlsson et al, 1978).…”

Section: Introductionmentioning

confidence: 99%

Isolation of plasmid DNA from cell lysates by aqueous two‐phase systems

Ribeiro

Monteiro

Cabral

et al. 2002

Biotech & Bioengineering

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This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.

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A rapid method for the isolation of circular DNA using an aqueous two-phase partition system

Cited by 48 publications

References 9 publications

Concentration of mammalian genomic DNA using two‐phase aqueous micellar systems

Concentration of mammalian genomic DNA using two‐phase aqueous micellar systems

Effective extraction of elastase from Bacillus sp. fermentation broth using aqueous two-phase system

Isolation of plasmid DNA from cell lysates by aqueous two‐phase systems

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