Size‐selective DNA separation: Recovery spectra help determine the sodium chloride (NaCl) and polyethylene glycol (PEG) concentrations required

In the presence of sodium chloride (NaCl), DNA fragments can be size‐selectively separated by varying the final concentration of polyethylene glycol (PEG). This separation strategy in combination with the use of paramagnetic particles provides a valuable platform for achieving the desired DNA size interval, which is important in automated library preparation for high‐throughput DNA sequencing. Here, we report the establishment of recovery spectra of DNA fragments that enable the determination of suitable NaCl and PEG concentrations for size‐selective separation. Firstly, at a given NaCl concentration, the recovery equation was obtained by fitting the DNA recovery ratios versus the PEG concentrations using the logistic function to determine the required parameters. Secondly, the slope function of the recovery equation was achieved by deducing its first derivative. Therefore, the recovery spectrum can be generated using the slope function based on those parameters. According to the recovery spectra of different length DNA fragments, suitable NaCl and PEG concentrations can be determined, respectively, by calculating their resolution values and recovery ratios. The strategy was effectively applied to the size‐selective separation of 532‐, 400‐, and 307‐bp fragments at the selected reagent concentrations with recoveries of 96.9, 64.7, and 85.9%, respectively. Our method enables good predictions of NaCl and PEG concentrations for size‐selective DNA separation.     

Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge

Correlations to describe the effect of surface hydrophobicity and charge of proteins with their partition coefficient in aqueous two-phase systems were investigated. Polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate, and dextran systems in the presence of low (0.6% w/w) and high (8.8% w/w) levels of NaCl were selected for a systematic study of 12 proteins. The surface hydrophobicity of the proteins was measured by ammonium sulfate precipitation as the inverse of their solubility. The hydrophobicity values measured correlated well with the partition coefficients, K, obtained in the PEG/salt systems at high concentration of NaCl (r = 0.92-0.93). In PEG/citrate systems the partition coefficient correlated well with protein hydrophobicity at low and high concentrations of NaCl (r = 0.81 and 0.93, respectively). The PEG/citrate system also had a higher hydrophobic resolution than other systems to exploit differences in the protein's hydrophobicity. The surface charge and charge density of the proteins was determined over a range of pH (3-9) by electrophoretic titration curves; PEG/salt systems did not discriminate well between proteins of different charge or charge density. In the absence of NaCl, K decreased slightly with increased positive charge. At high NaCl concentration, K increased as a function of positive charge. This suggested that the PEG-rich top phase became more negative as the concentration of NaCl in the systems increased and, therefore, attracted the positively charged proteins. The effect of charge was more important in PEG/dextran systems at low concentrations of NaCl. In the PEG/dextran systems at lower concentration of NaCl, molecular weight appeared to be the prime determinant of partition, whereas no clear effect of molecular weight could be found in PEG/salt systems.     

Size fractionation of double-stranded DNA by precipitation with polyethylene glycol

We show that DNA molecules of differing molecular mass are separable by selective precipitation with polyethylene glycol (PEG+.. Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA. Double-stranded DNA can be fractionated at least in the range of 3 times 10-7 to 1 times 10-5 daltons. The effects on PEG concentration, sodium chloride concentration, DNA concentration, pH, divalent ions, precipitation time, and centrifugal force have been determined. These studies show PEG precipitation offers a size fractionation method for DNA which is convenient, of high capacity, and applicable over a wide range of conditions. However, resolution is not high and separation of two species approaches 100% only if they differ in molecular mass by at least a factor of two.     

Correlations for the partition behavior of proteins in aqueous two-phase systems: effect of overall protein concentration

The effect of protein concentration in partitioning in PEG/salt aqueous two-phase systems has been investigated. PEG 4000/phosphate systems in the presence of 0% w/w and 8.8% w/w NaCl have been evaluated using amyloglucosidase, subtilisin, and trypsin inhibitor. Also, a PEG 4000/phosphate system with 3% w/w NaCl was used for alpha-amylase. The concentration of the protein in each of the phases affected its partition behavior. The pattern for the individual proteins was dependent on their physicochemical properties. In the top phase, maximum protein concentration was determined mainly by a steric exclusion effect of PEG, and hydrophobic interaction between PEG and proteins. In the bottom phase, maximum concentration was determined mainly by a salting-out effect of the salts present. As the ionic strength was increased in the systems the concentration in the top phase increased for all proteins. In the bottom phase an increase in ionic strength increased the salting-out effect. Amyloglucosidase had a very low maximum concentration in the PEG-rich top phase which was probably due to its large size (steric exclusion) and low hydrophobicity, and a high concentration in the salt-rich bottom phase due to its high hydrophilicity. In the case of subtilisin and trypsin inhibitor, their high concentrations in the top phase were due to their hydrophobic nature (hydrophobic interaction with PEG) and small size (negligible steric exclusion). The maximum concentration in the bottom phase for trypsin inhibitor was lower than that of subtilisin which was probably due to its higher hydrophobicity and, hence, a stronger salting-out effect. The protein concentration in each of the two phases was correlated with a "saturation"-type equation. The partition coefficient could be satisfactorily predicted, as a function of the overall protein concentration, by the ratio between the "saturation" equations of the two individual phases. Better correlations were obtained when an empirical sigmoidal Boltzmann equation was fitted to the data, since in virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. (c) 1996 John Wiley & Sons, Inc.     

Improved purification and PCR amplification of DNA from environmental samples

Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.     

Comparison of different methods for the isolation and purification of total community DNA from soil

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.     

硫酸鱼精蛋白去除Vero细胞乙脑疫苗残余DNA方法的条件优化

Vero细胞规模化生产疫苗时利用硫酸鱼精蛋白可去除乙型脑炎纯化疫苗中Vero细胞的DNA,实验中对不同条件进行了比较。首先在Vero细胞冻融浓缩液中分别按终浓度2mg/ml、1mg/ml、0.5mg/ml、0.2mg/ml、0.1mg/ml加入鱼精蛋白去除DNA,确定0.2~2mg/ml均有良好去除效果;其次将Vero细胞培养的乙脑病毒浓缩液分3组:第1组按2mg/ml、1mg/ml和0.5mg/ml分别沉淀一次、两次;第2组加入终浓度1mg/ml PS后调pH值为pH6.0、6.4、6.8、7.2、7.6;第3组将NaCl浓度调整至0.029mol/L、0.145mol/L、0.725mol/L,加入终浓度1mg/mlPS。确定低浓度多次沉淀、pH值6.8~7.2、盐浓度0.145mol/L条件下沉淀效果最好。     

Application of magnetic hydroxyapatite nanoparticles for solid phase extraction of plasmid DNA

We developed a facile method for plasmid DNA (pDNA) extraction from crude Escherichia coli lysate using magnetic hydroxyapatite nanoparticles (MHapNPs) in the presence of polyethylene glycol (PEG)/NaCl. DNA condensation induced by PEG/NaCl is a prerequisite for achieving pronounced DNA recovery. The quality and quantity of MHapNP-purified pDNA under optimal binding buffer conditions (0.5 volume of 20% PEG 8000/2M NaCl) were comparable to those obtained using organic solvents or commercial kits. This MHapNP technique is rapid, simple, cost-effective, and environmentally friendly and has the potential to extract DNA from other cell lysates.     

盐分和水分胁迫对菊芋幼苗离子吸收及叶片酶活性的影响

采用砂培试验,用不同浓度的NaCl和等渗PEG6000(聚乙二醇6000,渗透势约为-0．44MPa)处理生长20d的菊芋幼苗,3d后分别测定其根、茎、叶中的Na^ 、K^ 、Cl^-含量以及叶片SOD、POD活性。结果表明,在NaCl和PEG胁迫下,根、茎、叶的Na^ 、Cl^-含量不断升高,而K^ 含量保持稳定。其中,茎中Na^ 含量高于根和叶。NaCl胁迫下,根、茎、叶的SX、Na值随胁迫强度的增加而递增,茎中SK、Na值小于根和叶。随着NaCl胁迫强度的增加,菊芋幼苗叶片的SOD和POD活性先上升后下降;PEG处理下,SOD活性分别高于对照和等渗NaCl处理31．1％和27．1％;而POD活性却分别低于对照和等渗NaCl处理26．0％和36．1％。     

Effect of zinc chloride and PEG concentrations on the critical flux during tangential flow microfiltration of BSA precipitates

There is renewed interest in the possibility of using precipitation for initial capture of high value therapeutic proteins as part of an integrated continuous downstream process. These precipitates can be continuously washed using tangential flow filtration, with long term operation achieved by operating the membrane modules below the critical filtrate flux for fouling. Our hypothesis was that the critical flux for the precipitated protein would be a function of the properties of the precipitate as determined by the precipitation conditions. We evaluated the critical flux using a flux‐stepping procedure for model protein precipitates (bovine serum albumin) generated using a combination of a crosslinking agent (zinc chloride) and an excluded volume precipitant (polyethylene glycol [PEG]). The critical flux varied with shear rate to approximately the 1/3 power, consistent with predictions of the classical polarization model. The critical flux increased significantly with increasing zinc chloride concentration, going from 60 L/m²/h for a 2 mM ZnCl₂ solution to 200 L/m²/h for an 8 mM ZnCl₂ solution. In contrast, the critical flux achieved a maximum value at an intermediate PEG concentration. Independent measurements of the effective size and viscosity of the protein precipitates were used to obtain additional understanding of the effects of ZnCl₂ and PEG on the precipitation and the critical flux. These results provide important insights into the development of effective tangential flow filtration systems for processing large quantities of precipitated protein as would be required for large scale continuous protein purification by precipitation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1561–1567, 2017     

High‐throughput screening techniques for rapid PEG‐based precipitation of IgG4 mAb from clarified cell culture supernatant

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high‐throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG₄ monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96‐microwell filter plates combined with high‐throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG₄ precipitation. To aid the DoE, a set of preliminary range‐finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG₄ as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG₄ concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high‐throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Structural transitions of chromatin at low salt concentrations: a flow linear dichroism study

We have studied the structure of nuclease-solubilized chromatin from Ehrlich ascites cells by flow linear dichroism (LD) using the anisotropic absorption of the DNA bases and of two intercalated dyes, ethidium bromide and methylene blue. It is confirmed that intercalation occurs preferentially in the linker part of the chromatin fiber, at binding ratios (dye/base) below 0.020. Using this information, we determined the orientation of the linker in relation to the average DNA organization in chromatin. The LD measurements indicate that the conformation of chromatin is considerably changed in the ionic strength interval 0.1-10 mM NaCl: with increasing salt concentration, the LD of the intrinsic DNA base absorption changes signs, from negative to positive, at approximately 2.5 mM NaCl. The LD of the intercalated dyes also changes signs, however, at a somewhat higher salt concentration. The results are analyzed in terms of possible allowed combinations of tilt angles of nucleosomes and pitch or tilt angles of linker DNA sections relative to the fiber axis, at different salt concentrations in the interval 0.1-10 mM NaCl. Two models for the salt-induced structural change of chromatin are discussed.     

Precipitation of S,M, X and Y potato viruses by polyethyleneglycols with different molecular weights

The minimum concentration of polyethyleneglycol (PEG) with molecular weights 4000, 6000, and 15000 necessary for precipitation of S, M, X and Y potato viruses was determined. An excessive amount of PEG causes the precipitation of other protein compounds from potato leaf cell sap. In order to obtain highly purified samples, it is necessary to use just the minimum sufficient amount of PEG. Using the minimum quantity of PEG is, also, advisable from an economical point of view. The minimum concentration of PEG of given molecular weight differs for different potato disease viruses. The concentration of PEG necessary for precipitation of a given potato virus depends on the molecular weight of PEG used—4000, 6000 and 15000. As the molecular weight increases, the concentration of PEG necessary for precipitation decreases.     

Salt-induced changes in plasmid DNA transmission through ultrafiltration membranes

Recent studies have demonstrated the feasibility of using ultrafiltration for the purification of plasmid DNA, but there is still little understanding of the factors governing DNA transmission. Experimental data were obtained for the transmission of a 3.0 kbp supercoiled plasmid DNA through composite regenerated cellulose ultrafiltration membranes as a function of solution ionic environment in a stirred ultrafiltration cell. The dependence on salt concentration was quite dramatic, with the sieving coefficient increasing by more than 80-fold as the NaCl concentration increased from 1 to 150 mM at a fixed filtrate flux. At the same total ionic strength, the sieving coefficient in an MgCl2 solution was significantly larger than that evaluated in NaCl. The sieving results are consistent with independent studies showing a reduction in the effective plasmid size due to salt specific shielding of intramolecular electrostatic interactions. DNA transmission was also a strong function of the filtrate flux, with negligible transmission below a critical value of the flux. The predicted values of the critical filtrate flux determined using a modified elongational flow model were in excellent agreement with the experimental data. These results clearly demonstrate that salt-induced changes in plasmid DNA structure have a significant effect on plasmid DNA transmission through ultrafiltration membranes.     

A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions

Using the single cell eukaryote Tetrahymena thermophila, a simple method was developed for studying protein-DNA associations by cross-linking proteins to DNA with formaldehyde and immunoprecipitating the solubilized chromatin fragments with a specific antiserum. The protocol uses crude antiserum and involves only three steps: cross-linking, shearing to solubilize the chromatin, and immunoprecipitation. Methods for optimizing certain critical parameters, such as fixation time and NaCl concentration, are described. The method is likely to be generally useful for a variety of nuclear antigens.     

Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments

Twenty-one DNA restriction fragments ranging in size from 12 to 880 base pairs (bp) were purified to homogeneity in milligram amounts. The developments which facilitated this work were (a) procedures for the rapid preparation of gram quantities of pure recombinant plasmid DNAs, (b) selective poly(ethylene glycol) (PEG) precipitation of DNAs according to broad classes of lengths, and (c) large-scale high-pressure liquid chromatography on RPC-5 for the purification of fragments to homogeneity. The 95- and 301-bp sequences from the lactose control region of Escherichia coli were cloned into the single EcoRI site of pVH51 in up to four copies per plasmid. These tandem inserts are separated by EcoRI sites and have a head to tail orientation in all cases. A total of 50 and 90 mg of th 95- and 301-bp fragments, respectively, were prepared from 300-L fermentations of E. coli cells transformed with these plasmids. A rapid and improved method, which can easily be scaled up, for the purification of plasmids and DNA restriction fragments was developed. Also, the linear pVH51 vector DNA was digested with HaeIII to yield fragments ranging in size from 12 to 880 bp. The five smaller fragments (from 12 to 180 bp) were purified quantitatively by a selective PEG precipitation enrichment step followed by RPC-5 column fractionation. The larger fragments (245-880 bp) were prepared in milligram amounts. Ten subfragments from the 301-bp lac fragment were prepared by HpaII, HinfI, or HaeIII/AluI digestions followed by separation of the reaction products on RPC-5.     

Branched polyethylene glycol for protein precipitation

The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion.     

在cDNA文库构建中探究癌症病人血浆外泌体miRNA的特异性扩增

血浆外泌体微小核糖核酸(microRNAs,miRNAs)与癌症的发生、诊断和治疗密切相关,但其分子机制尚不明晰。本研究探讨了癌症病人血浆外泌体miRNAs在cDNA文库构建中非特异性扩增的解决方案。在酶切法中,采用核酸外切酶T (exonuclease T, EXOT)和phi29 DNA聚合酶降解引物;在磁珠法中,利用DNA结合磁珠分离模板和引物。随后,采取琼脂糖凝胶电泳和变性聚丙烯酰胺凝胶电泳检测磁珠分离情况,运用RT-qPCR检测癌症病人血浆外泌体miRNA和不同连接物的含量变化。结果显示,非特异性扩增来源于miRNA的连接物USR5SR;核酸外切酶T (EXOT)和phi29 DNA聚合酶虽可降解USR5SR,但模板链也会发生降解;磁珠分离法中以9%PEG沉淀引物片段、15%PEG沉淀模板链效果最佳。综上所述,磁珠分离法能够高效解决cDNA文库构建中的非特异性扩增,从而实现293T细胞和癌症病人血浆外泌体miRNA cDNA文库的成功构建。     

DNA length and concentration dependencies of anisotropic phase transitions of DNA solutions

Critical concentrations for the isotropic to cholesteric phase transitions of double-stranded DNA fragments in simple buffered saline (0.1 M NaCl) solutions were determined as a function of DNA contour length ranging from approximately 50 nm to 2700 nm, by solid-state 31P NMR spectroscopy and polarized light microscopy. As expected for semirigid chains, the critical concentrations decrease sharply with increasing DNA length near the persistence length in the range from 50 to 110 nm, and approach a plateau when the contour length exceeds 190 nm. The biphasic region is substantially wider than observed for xanthan, another semirigid polyelectrolyte approximately twice as stiff as DNA, primarily because of low critical concentrations for first appearance of the anisotropic phase, C(i)*, in DNA samples > or =110 nm (320 base pairs) long. The limiting C(i)* for DNA > or =490 nm long is exceptionally low (only 13 mg/ml) and is substantially lower than the C(i)* of approximately 40 mg/ml reported for the stiffer xanthan polyelectrolyte. The much higher values of the critical concentrations, C(a)*, for the disappearance of the isotropic DNA phase (> or =67 mg/ml) are modestly higher than those observed for xanthan and are predicted reasonably well by a theory that has been applied to other semirigid polymers, if a DNA persistence length in the consensus range of 50-100 nm is assumed. By contrast, the broad biphasic region and low C(i)* values of DNA fragments > or =190 nm long could only be reconciled with theory by assuming persistence lengths of 200-400 nm. The latter discrepancies are presumed to reflect some combination of deficiencies in current theory as applied to chiral, strong polyelectrolytes such as DNA, and sequence-dependent variations in DNA properties such as flexibility, curvature, or interaction potential. The propensity of DNA to spontaneously self-order at low concentrations well in the physiological range may have biological significance.