Use Of Agarose Gels Research Articles

A novel real-time PCR coupled with high resolution melting analysis as a simple and fast tool for the entomological authentication of honey by targeting Apis mellifera mitochondrial DNA

Honey is one of the foods easily adulterated worldwide. Recently, the analysis of honeybee DNA has been proposed as a useful tool to authenticate the entomological origin of honey. However, the methods proposed so far require more than one polymerase chain reaction (PCR) and the use of agarose gels, making the authentication process laborious and lengthy. In this work, a novel real-time PCR coupled with high-resolution melting (HRM) analysis of a 150 bp fragment of the cytochrome c oxidase I (COI) gene is proposed as a fast and simple tool to assess honey’s entomological origin by discriminating the mitochondrial DNA lineages of European honey bees (A, M and C lineages). In addition, the new tool allowed the differentiation of honeys produced by different mitotypes of C-lineage ancestry. The method showed high analytical performance and was able to successfully identify the entomological origin of honeys of known origin obtained from research apiaries/beekeepers. Therefore, it was applied to 44 commercial honeys from different countries. It confirmed the entomological authenticity of French PDO honeys that should be produced by the Corse ecotype A. m. mellifera. For the remaining honeys, the results were also in good agreement with the declared geographical origin. However, three honeys from Slovenia did not cluster with C2 mitotype A. m. carnica as expected, suggesting the mixture of honeys produced by honeybees of different mitotypes.

Synthesis of Au sponges based on agarose template

Metal-containing porous structures have been widely used in the fields of electrochemical and acid-base catalysis, bio-filtration, and heat-dissipation. Here, we propose a method for the fabrication of robust macroporous metallic sponges using agarose gel and HAuCl4 as soft-sacrificial templates and Au precursor, respectively. We demonstrate that the preparation of self-supporting macroporous Au sponges is practicable by heating the soft-agarose framework in the range of temperatures from 200 to 500°C. Before the calcination of agarose framework, Au precursor was reduced using NaBH4. A series of data obtained from X-ray photoelectron spectroscopy and X-ray diffraction indicates that the Au sponges were well synthesized with high crystallinity. The structural properties of porous Au were methodically investigated using Rietveld refinement and density-functional-theory calculations. The use of agarose gel as a soft template for the fabrication of metallic sponges has various advantages over conventional methods, as this approach is environmentally benign, shape-controllable, inexpensive, and scalable for mass production.

Diffusion characteristics of agarose hydrogel used in diffusive gradients in thin films for measurements of cations and anions

The agarose hydrogel has been increasingly used as a diffusive layer in diffusive gradients in thin films (DGT) measurements. However its diffusive characteristics have not been examined in detail. In this study, the performance of agarose gel was tested in DGT measurements of eight cations (Fe(II), Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Pb(II), and Cd(II)) and eight anions (P(V), As(V), Cr(VI), Mo(VI), Sb(V), Se(VI), V(V), and W(VI)). It was found that the thickness of agarose, a key parameter in the calculation of DGT measured concentration, remained unchanged after hydration followed by storage under the following conditions: pH 2–11, ionic strength 0–1.0 M, temperature 4–40 °C, and with the storage time extending to 300 d. Enrichment of cations and repelling of anions were observed in the gel under the ionic strengths of < 2–3 mM and <1 mM (NaNO3), respectively, which was attributed to the electrostatic interactions of these ions with the fixed negatively charged groups (mainly pyruvate) in the gel. The diffusion coefficients of cations and anions through the agarose gel (plus a PVDF filter membrane) were on average 1.10 ± 0.04 times of the reported diffusion coefficients through the agarose cross-linked polyacrylamide (APA) hydrogel, typically used in DGT technique. The working pH ranges for the agarose gel-assembled DGTs were 4–10 and 5–9 for anions and cations, respectively. The use of agarose gel, either individually or along with different filter membranes, affected the overall diffusion rates of cations and anions. The measured DGT concentrations of cations and anions in filtered natural freshwater and seawater were mostly in line with those measured directly. The results showed that the agarose gel can be used as one of the standard diffusive layers in DGT measurements for a wide range of inorganic and organic analytes.

Agarose Hydrogels Embedded with pH-Responsive Diblock Copolymer Micelles for Triggered Release of Substances

Hybrid agarose hydrogels embedded with pH-responsive diblock copolymers micelles were developed to achieve functional hydrogels capable of stimulus-triggered drug release. Specifically, a well-defined poly(ethylene oxide) (PEO)-based diblock copolymer, PEO-b-poly(2-(N,N-diisopropylamino)ethyl methacrylate) (PEO(113)-b-PDPAEMA(31), where the subscripts represent the degrees of polymerization of two blocks), was synthesized by atom transfer radical polymerization. PDPAEMA is a pH-responsive polymer with a pKa value of 6.3. The PEO(113)-b-PDPAEMA(31) micelles were formed by a solvent-switching method, and their pH-dependent dissociation behavior was investigated by dynamic light scattering and fluorescence spectroscopy. Both studies indicated that the micelles were completely disassembled at pH = 6.40. The biocompatibility of PEO(113)-b-PDPAEMA(31) micelles was demonstrated by in vitro primary cortical neural culture. Hybrid agarose hydrogels were made by cooling 1.0 wt % agarose solutions that contained various amounts of PEO(113)-b-PDPAEMA(31) micelles at either 2 or 4 °C. Rheological measurements showed that the mechanical properties of gels were not significantly adversely affected by the incorporation of diblock copolymer micelles with a concentration as high as 5.0 mg/g. Using Nile Red as a model hydrophobic drug, its incorporation into the core of diblock copolymer micelles was demonstrated. Characterized by fluorescent spectroscopy, the release of Nile Red from the hybrid hydrogel was shown to be controllable by pH due to the responsiveness of the block copolymer micelles. Based on the prominent use of agarose gels as scaffolds for cell transplantation for neural repair, the hybrid hydrogels embedded with stimuli-responsive block copolymer micelles could allow the controlled delivery of hydrophobic neuroprotective agents to improve survival of transplanted cells in tune with signals from the surrounding pathological environment.

The substitute brain and the potential of the gel model.

This purpose of this paper is to review the recent history of the use of agarose gels. Although originally confined to electrophoresis work, agarose gels have proven themselves useful to a number of disciplines in the modern world, which includes brain infusion studies for research involving the treatment of various neurological conditions, such as Parkinson's Disease. In reviewing the relevant research leading up to the modern day, this paper attempts to track agarose gels through their stages of accuracy verification, highlighting why they are useful to the neurosurgery discipline and characterizing the nature of their use. Agarose gels do have significant limitations, which are also discussed, but they have substantial potential as a modifiable medium or as a basis of comparison for even more accurate models in the future.

Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein‐Free Medium

Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p < 0.05). In contrast, α-MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock-out serum replacement supplementation and a protein-free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange.

Femtosecond laser-induced nucleation of protein in agarose gel

We have performed femtosecond laser-induced nucleation of protein in agarose gel to study the crystallization dynamics. The use of agarose gel facilitates the monitoring of laser-induced crystallization from the focal point, because the gel forms a highly viscous solution at low supersaturation and thereby suppresses the diffusion of laser-induced bubbles and microcrystals. We succeeded in monitoring the crystallization process from the focal point in the time range from microseconds to days. In addition, by combining an agarose gel system with fluorescence imaging, we revealed that the shrinking of laser-induced cavitation bubbles causes a local increase of protein concentration, which could be the trigger for protein nucleation.

Pulsatile dynamic stiffness of cartilage‐like materials and use of agarose gels to validate mechanical methods and models

Stiffness is a fundamental indicator of the functional state of articular cartilage. Reported test modes include compressive incremental strain to determine the equilibrium modulus, and sinusoidal strain to determine the dynamic modulus and stress/strain loss angle. Here, initial development is described for a method recognizing that gait is pulsatile. Agarose gels have been used by others for validation or comparison of mechanical test methods and models for cartilage and proteoglycan aggregate. Accordingly, gels ranging from 0.5 to 20% agarose were prepared. Pulsatile stiffness in both indentation and unconfined compression were closely reproducible. Stiffness as a function of agarose concentration rose exponentially, as found using other methods. Indentation stiffness was higher than for unconfined compression and ranged from approximately 2.0 kPa for 0.5% gel to approximately 3,800 kPa for 20% gel. Pulsatile dynamic stiffness appears to be a useful method, although further development is needed. Agarose gel stiffness values obtained by other methods were reviewed for comparison. Unfortunately, reported values for a given agarose concentration ranged widely (e.g. fourfold) even when test methods were similar. Causes appear to include differences in molecular weight and gel preparation time-temperature regimens. Also, agarose is hygroscopic, leading to unintended variations in gel composition. Agarose gels are problematic materials for validation or comparison of cartilage mechanical test methods and models.

Rapid and sensitive detection of internal tandem duplication and activating loop mutations of FLT3

Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA-500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.

Fast and reliable sexing of prosimian and human DNA

Molecular sexing of mammals is normally done by PCR amplification of Y chromosomal fragments, or coamplification of homologous fragments from both sex chromosomes. Existing primers are often unreliable for distantly related species due to mutations in primer regions. Currently there are no published primers for the sexing of prosimian DNA. We show that an existing method (using the zinc finger protein) based on a size difference between the X and Y homologs does not work in prosimians. Multiple alignments of distantly related mammalian species from Genbank and genome databases enabled us to identify conserved regions in the amelogenin gene. Using these conserved regions, we can target species that have no sequence information. We designed a single, conserved primer pair that is useful for fast and reliable molecular sexing of prosimian primates. A single PCR yields two fragments in males and only one in females, which are easily separated with the use of agarose gels. Amplification of separable fragments was successful in seven species of lemurs, as well as humans.

The Fibrin Intermediate, Its Place in the Fibrinogen‐Fibrin Transformation

Our preceding study indicated that, in course of coagulation of human fibrinogen by thrombin, substantial production of the fibrin intermediate (alpha-profibrin) lacking only one fibrinopeptide A (FPA) precedes the formation of alpha-fibrin monomer lacking both FPAs. The plateau concentration of alpha-profibrin (20% of initial fibrinogen) appearing in reactions indicated, however, that the second FPA is released four times faster than the first. The study reported here confirms those findings, and provides new insight into the significance of differing rate constants for the production of alpha-profibrin and its conversion to alpha-fibrin. The intermediate could be isolated in a distinct electrophoretic band by electrophoresing partial thrombin digests at high concentrations. Its identity was verified by digesting it with CNBr and by demonstrating that its N-terminal domain, the NDSK fragment, both lacks an FPA and contains an FPA, unlike the NDSKs of the bands from fibrin which contained no FPA or the fibrinogen band that lacked no FPA. The single step isolation also enabled us to confirm the 15-20% plateau level of alpha-profibrin in course of thrombin reactions, well below the 37% maximum that would be expected if release of the first and second FPA proceeded independently with no difference in rate. The 37% maximum is observed in reactions with atroxin, and it is suggested that the abundant production of alpha-profibrin underlies the therapeutic utility of atroxin as a defibrinating agent. Gel chromatography procedures were optimized for isolation of alpha-profibrin/fibrin mixtures free of fibrinogen, the final step of which involves literal use of agarose gel as a filter to remove fibrin aggregates from the fibrinogen free fractions (aggregates are left behind in gel filtration, rather than their moving ahead in gel chromatography). Unlike human fibrinogen, rabbit fibrinogen does not yield much alpha-profibrin in course of its conversion to fibrin, less than 10% as determined by electrophoresis and comparison with abundant production with atroxin. This low production of alpha-profibrin conformed with conclusions from our early studies on the generalized Shwartzman reaction in rabbits, and we now infer that the low production of alpha-profibrin and rapid conversion to fibrin by rabbit fibrinogen underlies the unparalleled susceptibility of these animals toward fibrinoid formation in the generalized Shwartzman reaction.

Persistence of Viruses in Upper Respiratory Tract of Children with Asthma

Objectives: Nasopharyngeal swabs of 50 asthmatic children in the symptom-free period were examined for the presence of adenoviruses, rhinoviruses and coronaviruses. A control group of 20 healthy individuals was included in this study.Methods: A polymerase chain reaction was used to detect adenovirus DNA and rhinovirus and coronavirus complementary DNA. The fragments of amplified genetic material were visualized with the use of agarose gel electrophoresis.Results: Adenovirus DNA was found in 78.4% of asthmatic children, rhinovirus RNA in 32.4% and coronavirus RNA in 2.7%. Adenovirus DNA was detected in one of the 20 nasopharyngeal swabs of healthy controls; the rest of the control samples were negative.Conclusions: The persistent presence of viruses in the upper respiratory tract of asthmatic children shows a possible connection between viral infections and asthma.

Electrophoresis of Neurochordins, a Family of High Molecular Weight Neural Tissue Glycoproteins, on Horizontal Submerged Agarose Gels in the Presence of Dodecyl Sulfate

We have developed a procedure for the use of agarose gels to separate electrophoretically neurochordins, a family of high molecular weight neural tissue glycoproteins. This procedure is a modification of the method of horizontal submerged electrophoresis on agarose gels routinely applied for the separation of DNA restrictions. For protein analysis we prepared gels of higher agarose concentration (3%), and 0.1% of sodium dodecyl sulfate was present in the buffer used both for gel formation and as an electrode buffer. After electrophoresis agarose gels can be directly stained with Coomassie blue, or proteins can be transferred from the gel to nitrocellulose or nylon filters for immunostaining. The main advantage of the proposed method compared to the method of electrophoresis on large-pore, composite agarose/polyacrylamide gels is its simplicity. Other proteins of very high molecular weight can also be successfully separated by this technique.

The use of agarose gels for quantitative determination of fluid saturations in porous media

The use of agarose gel reference standards for quantifying petrophysical properties in porous media is described. The specific interest is to determine the values of fluid saturations and porosity in oil bearing rocks; the MRI methodology for estimating these properties is discussed. It is shown that the relaxation times of the gel reference standard and the relaxation times of the fluid contained in the porous media affect the estimation process. The determination of porosity and fluid saturations can be greatly simplified if the relaxation times of the reference standard and the relaxation times of the fluid are closely matched. Gel concentration of paramagnetic impurities in the form of copper ions is used to control the longitudinal relaxation properties. The relaxation properties of agarose gels, as a function of agarose and paramagnetic impurity concentrations, have been measured at 2.0 T. The data are well fitted by a simple polynomial in agarose concentration and paramagnetic impurity concentration. Finally, a one-dimensional imaging example of use of agarose gels as reference phantoms is discussed.

32] An agarose gel electrophoresis assay for the detection of DNA-binding activities in yeast cell extracts

This chapter discusses the use of an agarose gel electrophoresis assay for the detection of deoxyribonucleic acid (DNA)-binding activities in yeast cell extracts. The gel electrophoresis DNA-binding assay is a simple and versatile method for the quantitative detection and analysis of specific protein–DNA interactions. Recently, the polyacrylamide gel binding assay has been used to detect specific binding proteins in crude lysates of the cells of the African green monkey, Drosophila , and Escherichia coli . The agarose gel electrophoresis DNA-binding assay described in the chapter is used to identify and partially characterize a number of activities, with different DNA-binding specificities, present in yeast cell lysates. The use of agarose gels allows whole plasmids, digested into a number of restriction fragments, to be used as substrates in the assay. Specific DNA-binding activity is observed as a change in the mobility of one specific DNA fragment containing the sequence of interest; nonspecific DNA-binding activities, observed as the altered mobility of all the plasmid fragments, can be minimized by the use of unlabeled carder DNA. Competition studies with plasmid DNA containing the specifically bound DNA fragment can also be used to identify and delimit the binding substrate. Binding to genomic DNA sequences can also be detected using an adaptation of this method—the genomic-blot binding assay. In this assay, unlabeled yeast genomic DNA is used as a substrate in the binding reactions and the specific DNA mobility shifts are detected by the hybridization of a DNA probe to the DNA in a Southern blot of the gel.

Preparative isoelectric focusing in agarose

The use of agarose gels as supporting media for flat-bed preparative isoelectric focusing was applied to the fractionation of serum proteins in the pH range 3.5–6, and red cell hemolysates in the pH range 3–8. The agarose gels are easy to prepare, give linear pH gradients, and do not appear to produce molecular sieving effects. Up to 1 g serum proteins can be loaded on the gels, with recoveries between 68 and 82%. Nucleoside phosphorylase from red cell lysates was recovered with 76% yield, indicating that no appreciable denaturation of this enzyme had occurred. Preparative isoelectric focusing in agarose gels provides a useful alternative to existing techniques of preparative isoelectric focusing in sucrose gradients or granulated gels.

The use of agarose gels for the assay of the deoxyribonuclease activity associated with the soluble penton of adenoviruses.

Evidence is presented that the excess pool of penton complexes purified from adenovirus type 1-infected HEp-2 cells also carry an endonucleolytic activity. The effect was measured with both E. coli and adenovirus type 1 double-stranded DNA substrates. The use of agarose gels for assaying the endonuclease activity allowed the approximative calculation of the number of penton molecules which might produce one DNA double-strand break under the experimental conditions selected.