Concept: Agarose gel electrophoresis
The protective efficacy of a DNA construct containing extra small virus antisense (XSVAS) gene of nodavirus encapsulated with chitosan nanoparticles (NPs) was investigated in giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879). The delivery was carried out using oral and immersion methods. A plasmid concentration of 100 ng μL(-1) when conjugated with chitosan NPs was found to be more effective in increasing the survivability of the infected prawn. The particle mean size, zeta potential and loading efficiency percentage were 297 nm, 27 mV and 85%, respectively. The ability of the chitosan to form a complex with the plasmid was studied by agarose gel electrophoresis. The NPs were characterized by atomic force microscopy (AFM). Persistence study showed the presence of the DNA construct up to 30th day post-treatment. The oral treatment was found to be better than the immersion treatment for delivery of the chitosan-conjugated DNA construct. This is probably the first report on the delivery of nanoconjugated DNA construct in M. rosenbergii, against nodavirus.
- Journal of molecular microbiology and biotechnology
- Published over 5 years ago
In this work we present a new option to identify 11 rickettsial species that cause human rickettsioses, with some advantages over the previous methods described. Using rickettsial isolates from 11 Rickettsia species as a sample, we used the polymerase chain reaction to amplify a 990- to 1,000-bp DNA fragment from the ompB gene, common for the 11 Rickettsia species analyzed in this study, which were digested with AluI restriction enzyme to obtain different digestion patterns. This restriction pattern can be visualized using a polyacrylamide gel electrophoresis technique. Using this method we could differentiate between the 11 Rickettsia species analyzed regardless of the group to which the Rickettsia belonged. We developed a simple method to identify 11 Rickettsia species which cause human rickettsioses using polymerase chain reaction and restriction fragment length polymorphism techniques with the advantage that it only needs one amplicon and only one restriction enzyme to obtain the restriction pattern. The identification of the species infecting vectors, reservoirs, and humans is essential to establish the ecological and behavioral ecosystem involved in its maintenance and transmission in nature in the specific region where the pathogen is circulating. This method is very helpful to identify Rickettsia species in a short time.
One dimensional (1D) serum protein electrophoresis (SPE) on agarose gels is a frequently used diagnostic tool for canine diseases; however, little is known regarding the precise composition of the different protein fractions in normal or diseased animals. In this study, to analyse the canine serum proteome in more detail, conventional 1D SPE was combined with second dimension (2D) polyacrylamide gel electrophoresis (PAGE), followed by tandem mass spectrometry (MS). One dimensional SPE was performed on the sera of 17 healthy dogs to establish normal reference ranges for the albumin and globulin sub-fractions. Two representative serum samples from healthy dogs were further separated using a novel method of 2D PAGE, leading to the generation of 26 distinct bands across the six main sub-fractions, which were subjected to MS analysis. Thirty-two proteins were identified, most of which were found in both dogs. Twenty proteins belonged specifically to the species Canis lupus familiaris, with the remaining 12 proteins belonging to other mammalian species, likely reflecting incomplete sequencing knowledge of canine proteins. Two dimensional electrophoresis and MS allowed identification of canine serum albumin precursor, serpin peptidase inhibitor, kininogen-1, vitamin D binding protein, haemopexin, complement C4 and a variety of immunoglobulin class molecules, along with localisation of these proteins within serum protein subfractions.
Gold nanoparticles (Au NPs) coupled with Tollens reagent were used for measuring formaldehyde. Au@Ag core-shell NPs were formed along with distinct color changes from pink to deep yellow. This colorimetric system was further immobilized into an agarose gel, which was used for monitoring of gaseous formaldehyde.
Formulation of nanoparticulate DNA vaccines requires the assessment of stability and integrity of the components implicated. Stability of cationic nanoparticles made of N-trimethyl chitosan and chondroitin sulfate (TMC nanoparticles) was investigated in aqueous solution and after freeze-drying by characterization of their size, polydispersity index (PDI), and zeta potential. Furthermore, the structural integrity of plasmid DNA (pDNA) on adsorption to the nanoparticle surface was investigated. Agarose gel electrophoresis showed DNA retention when applied with the nanocarrier, suggesting that pDNA adsorption on nanoparticles took place. In circular dichroism (CD) spectra, ellipticity of pDNA decreased at 280 nm and increased at 245 nm, and the maximum wavelength shifted from 275 nm to 285 nm when nanoparticles were present. Once released from the particles, the secondary structure of the plasmid was retained in its native form. pDNA release from pDNA-TMC nanoparticles was indicated by a rise in zeta potential from initially -32 mV (pDNA adsorbed to particles) to 14 mV during one hour, and to 36 mV after 24 hours. Unloaded TMC nanoparticles remained stable in suspension for 24 hours, maintaining diameters of around 200 nm, and zeta potential values of approximately 38 mV. Freeze-drying with sucrose could ensure storage for 30 days, with minimal increase in size (291 nm) and charge (62 mV). In conclusion, TMC nanoparticles may potentially be freeze-dried in the presence of sucrose to be stored for prolonged periods of time. Furthermore, pDNA was successfully adsorbed to the cationic nanoparticles and remains intact after being released.
Competitive dye displacement titration has previously been used to characterize chitosan-DNA interactions using ethidium bromide. In this work, we aim to develop a fast and reliable method using SYBR Gold as a fluorescent probe to evaluate the binding affinity between ssRNA and chitosan. The interaction of chitosan with ssRNA was investigated as a function of temperature, molecular weight and degree of acetylation of chitosan, using competitive dye displacement titrations with fluorescence quenching. Affinity constants are reported, showing the high sensitivity of the interaction to the degree of acetylation of chitosan and barely dependent on the molecular weight. We propose that the mechanism of SYBR Gold fluorescence quenching is governed by both static and dynamic quenching.
Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within ~1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n = 54; heterozygous for the mutation, n = 37; homozygous for the mutation, n = 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ ABCB1 genotyping with less equipment than existing methods.
Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs.
Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female-specific 250-bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.
DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen. In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established. Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits.