A simple and sensitive assay of nucleic acids based on the enhanced resonance light scattering of zwitterionics

A new assay of nucleic acids at nanogram level was established based on the enhanced resonance light scattering (RLS) signals of two zwitterionics cocamidopropyl hydroxysultaine (HSB) and lauryl betaine (BS-12). Under optimum conditions, the weak RLS signal of HSB is enhanced by nucleic acids, and the enhanced RLS intensity is proportional to the concentration of nucleic acids in the range of 0.02–7.3 mg l⁻¹ for calf thymus DNA and 0.01–8.6 mg l⁻¹ for fish sperm DNA. The detection limits were 1.5 ng ml⁻¹ for calf thymus DNA and 1.9 ng ml⁻¹ for fish sperm DNA. Plasmid DNA extracted from K-12-HB101 colt was determined with satisfactory results.     

Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization

Chromatography is one of the key operations in the downstream processing of plasmid DNA (pDNA). However, the increased demand for highly purified pDNA experienced in recent years has made clear the need for alternative processes capable of retaining the advantages of conventional chromatography, such as selectivity, while providing increased throughput at a lower cost. The work presented in this article outlines the development and optimization of an alternative hydrophobic interaction membrane chromatography process for the purification of pDNA. The studies included the modification of functionalized membrane supports with a linear alkyl chain ligand and the testing of chromatographic performance of these membranes. Three modification procedures were tested and the membranes were screened for their capacity and selectivity. The modified membranes could separate the model plasmid pVAX1‐LacZ (6050 bp) from impurities in clarified Escherichia coli cell lysates (specifically RNA), with good resolution. Subsequent optimization of elution profiles with the best‐performing modified membrane, resulted in a high purification factor of 4.7, competitive with its bead process counterpart, and a plasmid yield of 73%.     

Co2+、Zn2+、Cd2+的阴离子交换色谱法分离研究

本文对钴与锌、镉等金属离子的阴离子交换色谱法分离进行了研究．采用201×7型阴离子交换树脂经氧化铵溶液饱和后,在pH＝4．0时,使钴与其它金属离子分离,然后分别用0．02mol／L的盐酸溶液及蒸馏水将锌、镉等金属离子从阴离子交换树脂上洗脱．此法分离效果好,操作简单方便．     

The chromatographic behavior of arsenic compounds on anion exchange columns with binary organic acids as mobile phases

Summary Identification and quantification of arsenic compunds was performed with high- performance liquid chromatography (HPLC) and flame atomic absorption spectrometry (FAAS) as element-specific detector. Arsenous acid, methylarsonic acid, dimethylarsinic acid, arsenic acid, arsenobetaine, and arsenocholine were separated on two anion-exchange columns (Synchropak Q 300 and PRP-X 100) with different binary organic acids as mobile phases. The infleunce of chromatographic parameters, such as pH and the concentration of the mobile phase were investigated. An unusual chromatographic behavior of arsenous acid was observed when tartaric acid was used as mobile phase.     

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial‐scale systems aiming at plasmid DNA purification.     

A convenient tool for studying the stability of proteins and nucleic acids

The aim of this work is to discuss the thermodynamic properties, obtained by differential scanning calorimetry (DSC), of the thermal transition of proteins and nucleic acids and to analyze these data using statistical thermodynamic relations. The denaturation of the ordered, specific structures of biological macromolecules is a cooperative process and in many cases the macromolecules undergo a two-state transition. Differential scanning calorimetry, giving direct thermodynamic information, has proved to be very useful in clarifying the energetics of macromolecule transitions and in characterizing their thermal stability. Here, various examples are discussed: i) the equilibrium thermal denaturation of ribonuclease A, a model for the use of DSC by following the temperature-unfolding of the proteins, a monomolecular transition; ii) the equilibrium thermal dissociation of a DNA double helix in two strands, an example of how DSC is used to follow a bimolecular process; iii) an example of the use of DSC for studying the melting of unimolecular and tetramolecular DNA quadruple-helices.     

Application of monoliths for plasmid DNA purification development and transfer to production

The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.     

Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids

Hybridization probes are often inefficient in the analysis of single‐stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real‐time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T_m>80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure‐folded analytes, whereas a MB‐based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.     

Preparation of methacrylate-based anion-exchange monolithic microbore column for chromatographic separation of DNA fragments and oligonucleotides

In this paper, we report on the preparation of a microbore-scale (1 mm i.d.) anion-exchange monolithic column suitable not only for analytical purposes but also for potentially preparative applications. In order to meet the conflicting requirements of high permeability and good mechanical strength, the following two-step procedure was applied. First, an epoxy-containing monolith was synthesized by in situ copolymerization of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) within the confines of a silicosteel tubing of 1.02 mm i.d. and 1/16″ o.d. in the presence of a ternary porogenic mixture of 1-propanol, 1,4-butanediol, and water. The monolithic matrix was subsequently converted into weak anion-exchanger via the ring-opening reaction of epoxy group with diethyl amine. The dynamic binding capacity was 21.4 mg mL⁻¹ for bovine serum albumin (BSA) at 10% breakthrough. The morphology and porous structure of this monolith were assessed by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). To optimize the separation efficiency, the effects of various chromatographic parameters upon the separation of DNA fragments were investigated. The resulting monolithic anion exchanger demonstrated good potential for the separation of both single- and double-stranded DNA molecules using a gradient elution with NaCl in Tris–HCl buffer (20 mM). Oligodeoxythymidylic acids (dT₁₂–dT₁₈) were successfully resolved at pH 8, while the fragments of 20 bp DNA ladder, 100 bp DNA ladder, and pBR322-HaeIII digest were efficiently separated at pH 9.     

Sepabeads FP series: New packing materials for industrial-scale separation of biopolymers

Summary Sepabeads FP series with a variety of anion-exhanger type functional groups have been examined as largescale chromatographic separation media for proteins. The adsorption-desorption behavior of proteins has been found to be dependent mainly on the type and partly on the content of the attached amino ligands. The effect of the attached groups are discussed using batch measurement data. It has been found that the existence of ethanol residue on the nitrogen atom incrases the hydrophilic nature of quaternary ammonium type packings and thus gives a higher protein adsorption capacity. Among weakly basic type anionexchangers, polyethyleneimine-modified packings showed very large protein capacities. Presented at HPLC '87, Amsterdam, 1987.     

Group separation of non-sulphated and 3-sulphated bile acids by disposable anion-exchange cartridges

Summary A rapid and simple method has been developed for the group fractionation of the major unsulphated and mono-sulphated bile acids in human body fluids. After extraction with Bond Elut C₁₈ cartridges, the bile acids are separated into the unconjugated, glycine-, taurine- and sulphate-conjugated forms on pre-packed Bond Elut SAX columns by increasing the ionic strength of the methanol-acetate buffer eluent. The procedure was found to be accurate and reproducible and to give complete resolution between the different groups. The levels of 3-sulphate bile acids in human serum and urine from patients with liver disease were determined by high-performance liquid chromatography, after group separation and solvolysis of the sulphate fraction.     

Implementing a two-layer feed-forward catalytic DNA circuit for enzyme-free and colorimetric detection of nucleic acids

In the present study, a highly sensitive and specific bio-sensing platform for enzyme-free and colorimetric detection of nucleic acids has been developed. The biosensor is composed of two DNA nanostructures and two fuel strands that construct the foundation of a feed-forward catalytic DNA circuit. Upon binding the target strand to a specific DNA nanostructure, the circuit is run in order that at the end a hemin-binding aptamer, with the ability to convert a colorless substrate into a colored substance is released. Based on this strategy, 4 pM of the target DNA can be easily detected in serum samples by naked eyes after only a two-hour incubation with the circuit; meanwhile, if the incubation time is extended to 3 h, the biosensor can detect 1 pM of the target DNA. Besides the elevated sensitivity, the circuit can truly discriminate a spurious target containing one nucleotide mismatch with high specificity. Overall, the enzyme-free catalytic DNA circuit can be used as a sensitive alternative method to enzyme-based biosensors for the specific and cost-effective detection of nucleic acids.     

Spectroscopic study of the interaction of pazelliptine with nucleic acids

The antitumor drug pazelliptine (PZE) binds to natural and synthetic DNA sequences at 100 mM NaCl, pH 7.0, as deduced from the absorption and fluorescence data. Scatchard plots constructed from the results obtained with poly(dG-dC)-poly(dG-dC) give binding constants of base pairs in the range (2–6) × 10⁵ M⁻¹. The modifications in the absorption and fluorescence spectra observed when PZE binds to various polynucleotides, namely poly(dA-dT)-poly(dA-dT), poly(dA)-poly(dT), poly(dG-dC)-poly(dG-dC) and calf thymus DNA. reveal a change in the protonation state of the drug upon binding, increasing the apparent pK_a of its 9-N nitrogen atom. The PZE excited state properties serve as a sensitive probe to distinguish between homo and hetero A-T sites as well as between AT and GC sites. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to the bound PZE chromophore, a result consistent with an intercalative mode of binding of the drug to DNA. The emission is enhanced when PZE is bound to A-T base pairs ( 30% increase of φ_F) whereas it is quenched in the vicinity of G-C base pairs ( 90% decrease of φ_F). Furthermore, the fluorescence spectrum obtained with calf thymus DNA is hardly distinguishable from that obtained with poly(dG-dC)-polu(dG-dC), suggesting a binding of PZE to G-C rich regions.     

Electrochemical analysis of nucleic acids as potential cancer biomarkers

In recent years, a huge progress has been made regarding the development of electrochemical (EC) assays for detection of nucleic acids — DNA or RNA — as potential cancer biomarkers. Various ingenious strategies for determination of DNA methylation of gene promoters, circulating tumor DNAs, viral nucleic acids, or short noncoding microRNAs were presented, many of them showing remarkable sensitivities. However, a majority of these assays were not applied into clinical samples from patients, which is crucial should the electrochemistry compete with conventional, routinely used techniques. In this review, we critically evaluate strengths and weaknesses of EC assays that recognized this necessity and successfully determined endogenous DNA or RNA in patient samples with various forms of tumors.     

Synthesis of ferrocenylcarbodiimide as a convenient electrochemically active labeling reagent for nucleic acids

Ferrocenylcarbodiimides carrying different redox potentials, 1 and 2, were designed and synthesized as convenient electrochemically active labeling reagents for nucleic acids, which may be used as dually labeling reagents of nucleic acids like Cy3 and Cy5 dyes. These reagents could react with the imino unit of thymine or guanine base on DNA or of uracil base on RNA under a basic buffer condition to yield a labeled product quantitatively in a short period of time. The current responses of the labeled DNAs in square wave voltammetric (SWV) measurement showed a good linear correlation with the amount of the hybridized ones. DNAs labeled with the two different reagents, 1 and 2, could be detected electrochemically at different potentials after hybridization with a DNA probe-immobilized gold electrode.     

Functionalized cryogel monoliths for fast and selective separation of nucleic acids directly from crude lysate

The fast and selective separation of nucleic acids has been attractive recently because of their wide number of applications in the biomedical field such as the development of vaccines for infectious diseases, gene therapy, and diagnosis. Traditional approaches of nucleic acids separation are costlier, lengthy, and associated with possible denaturation because of the use of organic solvents in the elution step. Under this perspective, cryogels represent an attractive choice as a monolith stationary phase in column chromatography, which have proven efficient in recent chromatographic studies. Cryogels are the macroporous hydrogels with interconnecting properties between the pores. They allow the easy flow of large biomolecules with minimum mass transfer resistance. They are spongy in nature and possess good mechanical strength. Current article represents different developed functionalized cryogel monoliths for nucleic acids separation, their separation strategies, and challenges associated with further advancement in separation science.     

High-Speed Large Scale Chromatographic Purification of Plasmid DNA with a Novel Giant-Pore Stationary Phase

A rigid spherical giant-pore poly (glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension–polymerization on the basis of a novel porogenic mode using superfine particles of calcium carbonate as a solid porogen. Scanning electron microscopy reveals that the bead has pores as large as 10 μm. The hydrodynamic properties show that this polymeric material has good strength and a low back pressure of 1.0 MPa at a flow velocity of 3,000 cm h⁻¹. After being modified to be an anion-exchange material, high dynamic binding capacity of plasmid DNA of above 1,000 μg plasmid per mL of bed by a column of this material, could be obtained comparing to the 150 μg plasmid per mL of bed with a Q-Sepharose FF column at the same flow rate. Large-scale preparative plasmid separations (2–20 mL) from cell lysate were investigated. A 75% yield and 94.9% purity of SC plasmid DNA were obtained by a 20 mL column of giant-pore beads at a flow rate of 600 cm h⁻¹.     

Hydrophobic solid phase extraction using n-alkanoic acid-immobilized anion-exchange membranes as adsorbents

In this study, n-alkanoic acids (C7-COOH, C11-COOH, and C17-COOH) were immobilized onto strong basic anion-exchange membranes to form a stationary phase for hydrophobic solid phase extraction (SPE) application. The effects of feed surfactant amount, membrane counter ions, and surfactant chain length were investigated. Immobilized surfactant capacity increased with increasing feed surfactant amount, decreasing chain length, and the existence of OH⁻ counter ions. Moreover, according to TGA analysis, a surfactant bi-layer was formed on the membrane surface. Following successful surfactant immobilization, batch adsorption experiments for doxepin (feed concentration of 0.2 mg/mL) were conducted. The adsorbed doxepin amount increased with the use of longer-chain surfactants, indicating that doxepin adsorption was dominated by hydrophobic interaction with the immobilized surfactant. An optimal desorption performance was achieved using 1 M NaCl in 50% ethanol for both C7-COOH and C11-COOH-immobilized membranes. In the SPE process with one C11-COOH-immobilized membrane, a concentration factor of 2 and complete doxepin recovery was achieved from 10 mL of a 0.1 ppm load.     

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and pharmaceutical applications. Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.