Rapid Genomic DNA Extraction Research Articles

Rapid genomic DNA extraction for Soybean (Glycine max L. Merr) using modified CTAB protocol to obtain high-quality DNA

Rapid genomic DNA extraction for Soybean (Glycine max L. Merr) using modified CTAB protocol to obtain high-quality DNA

Comparative Study of Genomic DNA Extraction Methods for Common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is one of the significant grains used in the human diet, accounting for half of all grain legumes consumed globally. To enhance production, conventional breeding and molecular approaches have been used so far. An efficient and rapid genomic DNA extraction method is required for these molecular approaches. The aim of this study was to compare and optimize an efficient and rapid DNA extraction protocol for common bean. Modified cetyl trimethylammonium bromide (CTAB) and potassium chloride (KCL) extraction methods were used. The mean DNA yield per nanoliter was 209 µg from modified CTAB and 150.3 µg from the KCL method. The concentration of gDNA was significantly (P< 0.05) higher for the KCL method, which was 5.01 µg/µl and 2.09 µg/µl for the CTAB method. The obtained DNA was also pure, with an absorbance ratio at 260 nm to an absorbance of 280 nm (A260/280) of 1.75-2.23 for the KCL method and 1.86-2.09 for the modified CTAB method. Gel electrophoresis separation was used to evaluate the quality of the total DNA extracted by the present protocols. The results showed that intense bands close to the gel wells were obtained from both extraction methods. DNA isolated with the two methods was successfully used for PCR-based downstream analysis, which includes random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. In this study, it took approximately 150 minutes for KCL and 240 minutes for the CTAB for whole process. In contrast to the CTAB method, the KCL method uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. Therefore, it is more convenient and economical than the traditional technique.

Rapid and Sensitive Detection of Toxigenic Fusarium asiaticum Integrating Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Techniques.

Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. Both Fusarium graminearum and F. asiaticum are the causal agents of FHB in China. F. asiaticum is the predominant species in the Middle-Lower Reaches of the Yangtze River (MLRYR) and southwest China. Therefore, detecting F. asiaticum in a timely manner is crucial for controlling the disease and preventing mycotoxins from entering the food chain. Here, we combined rapid genomic DNA extraction, recombinase polymerase amplification, Cas12a cleavage, and lateral flow detection techniques to develop a method for the rapid detection of F. asiaticum. The reaction conditions were optimized to provide a rapid, sensitive, and cost-effective method for F. asiaticum detection. The optimized method demonstrated exceptional specificity in detecting F. asiaticum while not detecting any of the 14 other Fusarium strains and 3 non-Fusarium species. Additionally, it could detect F. asiaticum DNA at concentrations as low as 20 ag/μL, allowing for the diagnosis of F. asiaticum infection in maize and wheat kernels even after 3 days of inoculation. The developed assay will provide an efficient and robust detection platform to accelerate plant pathogen detection.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Automated multi-sample DNA extraction for genotyping live Xenopus embryos.

Xenopus frogs are used extensively for modeling genetic diseases owing to characteristics such as the abundance of eggs combined with their large size, allowing easy manipulation, and rapid external embryo development enabling the examination of cellular and phenotypic alterations throughout embryogenesis. However, genotyping of mutant animals is currently done either as part of a large group, requiring many embryos, or late in development with welfare effects. Therefore, we adapted the Zebrafish Embryonic Genotyper for rapid genomic DNA extraction from Xenopus tropicalis and Xenopus laevis at early stages. Sufficient and good quality DNA was extracted as early as the Nieuwkoop and Faber stage 19 and, importantly, no detrimental effects of the extraction process on the subsequent tadpole development, behavior, or morphology were observed. Amplicons of up to 800 bp were successfully amplified and used for further analyses such as gel electrophoresis, T7 endonuclease I assay and Sanger sequencing. This method allows rapid genotyping during the early stages of Xenopus development, which enables safe identification of mutants. These can be analyzed at early developmental stages or selected for raising without the need for invasive genotyping later, with resource savings and ethical gains in line with the 3Rs principles.

Genomic DNA extraction from minimal amount of dried mushroom samples

Aims: To develop a simple and rapid genomic DNA extraction technique for dried (≈ 1 year old) mushroom fruiting bodies that yields high-quality DNA, suitable for use by post-graduate institutions. Method: Small amounts (0.04 g) of pulverized dried mushroom sample were incubated in a Tris /EDTA/SDS lysis buffer (100mM:10mM: 2%) at 65°C to lyse the chitinous fungal cell walls. Genomic DNA purification was performed using chloroform isoamyl alcohol (24:1), and DNA was precipitated using 100% ethanol. Results: Genomic DNA was successfully extracted under 70 minutes from 16 samples morphologically identified as Panaeolus, Copelandia, Gymnopilus, Pluteus and Favolus species. DNA concentrations were on average of 696.9ng/µL. PCR successfully amplified the ITS-5.8S region. The protocol has been successfully used by numerous post-graduate students in our research programme. Conclusion: The rapid and easy protocol produced high-quality genomic DNA void of any inhibitors that is suitable for downstream molecular implications across multiple mushroom genera. Noticeably, this method requires only minute quantities (0.04g) of starting material and is ideal for student training in higher academic institutions.

A one step method for isolation of genomic DNA using multi-amino modified magnetic nanoparticles.

A simple and efficient approach for the rapid extraction of genomic DNA from blood using various amino-modified magnetic nanoparticles (AMNPs) has been described. The salmon sperm DNA was isolated from aqueous solution based on electrostatic interaction between the positively charged amino-groups of AMNPs and the negatively charged phosphate groups of the DNA. The results of ultraviolet-visible (UV-Vis) spectrometry showed that increasing number of amino groups on the AMNPs surface resulted in an improvement in DNA adsorption efficiency. Several variables including the extraction pH, adsorption time, ionic strength and quantity of AMNPs were optimized to achieve the best extraction efficiency with the proposed method. Acceptable adsorption efficiency of 92% and recovery of 91% were achieved using multi-amino modified MNPs (mAMNPs) with an extraction time of 10 min and an overall processing time of 30 min. The mAMNPs enabled genomic DNA capture from human whole blood, and the resulting mAMNP/DNA complexes could be directly used as templates for PCR amplification without the need for complex and time-consuming DNA elution and purification steps. Our results imply that this method can be used as an effective strategy for genomic DNA extraction and may be extended to other types of biological samples.

A Rapid Colorimetric Assay for On-Site Authentication of Cephalopod Species.

A colorimetric assay, exploiting the combination of loop-mediated isothermal amplification (LAMP) with DNA barcoding, was developed to address the authentication of some cephalopod species, a relevant group in the context of seafood traceability, due to the intensive processing from the fishing sites to the shelf. The discriminating strategy relies on accurate design of species-specific LAMP primers within the conventional 5’ end of the mitochondrial COI DNA barcode region and allows for the identification of Loligo vulgaris among two closely related and less valuable species. The assay, coupled to rapid genomic DNA extraction, is suitable for large-scale screenings and on-site applications due to its easy procedures, with fast (30 min) and visual readout.

A diagnostic test that uses isothermal amplification and lateral flow detection sdaA can detect tuberculosis in 60min.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is now the leading cause of death from infectious disease, thus rapid diagnostic and screening techniques for TB are urgently needed. Here, a detection of MTB using multiple cross displacement amplification coupling with nanoparticles-based lateral flow device (MCDA-LFD) was developed and validated, targeting the specific sdaA gene. The whole detection procedure, including rapid genomic DNA extraction (15min), amplification (30min) and result reporting (2min), was completed within 50min. No cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was observed. The sensitivity of sdaA-MCDA-LFD, Xpert MTB/RIF assay and culture results was 81·6, 48·3 and 37·9%, respectively, in TB patients. Among positive culture samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 93·9% (31/33) and 81·8% (27/33), respectively. Among culture-negative samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 74·1% (40/54) and 27·8% (15/54), respectively. The specificity of sdaA-MCDA-LFD and Xpert MTB/RIF was 95·4% (62/65) and 100% (65/65) in clinical samples from non-TB patients. The sdaA-MCDA-LFD assay was a rapid, simple, specific and sensitive TB diagnostic test. The sdaA-MCDA-LFD assay holds promise for application as a useful point-of-care test to detect MTB, and will play an important role in controlling and preventing TB.

The methylation status of TNF-α and SOCS3 promoters and the regulation of these gene expressions in patients with Behçet’s disease

Introduction: The aim of this study was to evaluate the methylation status of TNF-α and SOCS3 promoters in patients with BD and compare them with a healthy group.Method: This was a case–control study, in which 47 subjects with BD and 61 individuals as the control participated. Blood samples were collected from all the participants. Then, PBMCs were isolated using the Ficoll method and methylation of considered sites was investigated using the qMS-PCR technique after DNA extraction by the rapid genomic DNA extraction method and its analysis with Nano-drop.Results: The methylation and expression of TNF-α showed that the methylation level significantly declined in the patient in comparison with the healthy (p < 0.05). Moreover, the results on the mean expression showed that it significantly increased in the patient group, as compared with the healthy group (p < 0.05). In addition, the expression of the SOCS3 gene was not significantly different between the patients and healthy subjects while the level of SOCS3 methylation was significantly higher in the patient group than that in the healthy group (p < 0.05).Discussion: The present study revealed that the gene expression of TNF-alpha increased in BD patients, suggesting that TNF-alpha likely has a role in the pathogenesis of BD.

A Modified Method for Rapid Genomic DNA Extraction from Tuberose

The isolation of high yield and quality genomic DNA is crucial for studying the molecular genetics of plants. However, high contents of secondary metabolites, especially in medicinal and aromatic plants, interfere with the extraction of clean DNA, thereby rendering it useless for downstream analyses such as DNA amplification, restriction, sequencing and cloning. The chemotypic heterogeneity among plant species may not permit high quality DNA isolation with a single protocol, thus a species-specific extraction method is required for quality extractions. Here, we present a modified cetyltrimethylammonium bromide (CTAB) protocol for good quality DNA extraction from tuberose, which is an important plant for perfume and pharmaceutical industry due to its pleasant fragrance and essential oil content. In contrast to other CTAB methods, the modified procedure is rapid, omits the use of liquid nitrogen and phenol, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use ofPVP-10 to get rid of problems associated with polysaccharides and polyphenols, respectively. The yield and quality of extracted DNA were fairly good and amenable for downstream analyses. Moreover, to our knowledge, the described method is the first report of a modified DNA extraction protocol for tuberose.

Determination of mir-155 and mir-146a expression rates and its association with expression level of TNF-α and CTLA4 genes in patients with Behcet’s disease

IntroductionMicroRNAs (miRNAs) are involved in the pathogenesis of inflammatory diseases. MiR-146 and miR-155 emerged as key regulators of the immune response. This study designed to analyze the miR-146a and miR-155 expression in patients with Behcet’s disease (BD) and investigated their association with the expression of tumor necrosis factor-alpha (TNF-α) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) genes. MethodologyIn a case-control study, 47 Iranian Azeri BD patients and 61 age- and sex matched healthy controls recruited to the study. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood tubes by Ficoll density-gradient centrifugation. Genomic DNA samples of BD and healthy controls were extracted using the rapid genomic DNA extraction method from the peripheral blood collected in tubes containing EDTA. Total RNA was extracted from the PBMCs according to the TRIzol protocol. MiR-146a, miR-155, TNF-α and CTLA-4 expression were studied using real-time PCR. ResultsMiR-155 and TNF-α expression was significantly increased, whilst CTLA-4 expression was significantly decreased in the PBMCs of BD patients. There was no significant difference in the miR-146a expression rate between BD patients and controls. A positive correlation between miR-155 and TNF-α expression and negative correlation between miR-155 and CTLA-4 expression were observed. No significant association was observed between the expression of miR-155, miR-146a, TNF-α and CTLA-4 genes with BD activity. MiR-155 and miR-146a expression rate were significantly higher in patients with uveitis and phlebitis, respectively. Discussion and conclusionThe expression of miR-155 increased in BD and associated with upregulation of TNF-α and downregulation of CTLA-4 genes.

Investigation of the Connexin-30 gene Deletion Mutations Del (GJB6-D13s1830) and Del (GJB6-D13s1854) among Subjects with Non-Syndromic Sensorineural Hearing Loss in Khorasan Razavi Province

Introduction: Hearing loss (HL) is the most common sensorineural disorder with the frequency of 1 to 1000 neonates and it's seen in two syndromic and non-syndromic states. Given that the mutations of DFNB1 locus, containing Connexin 26 (Cx26) and 30 (Cx30) genes are the only responsible for 50% non-syndromic HL with autosomal recessive hereditary in different populations, the aim of this study is the screening of the connexin30 gene deletion mutations including Del (GJB6-D13S1830) and Del (GJB6-D13S1854) in HL subjects were negative to mutation in Cx26 gene. Materials & methods: In this descriptive study, 85 unrelated patients (44 males and 41 females) with autosomal recessive hearing loss of Khorasan Razavi province that were negative towards the Cx26 gene mutations, were studied. Genomic DNA was extracted from 1 ml of EDTA anticoagulated peripheral blood by rapid genomic DNA extraction (RGDE) method. For molecular analysis, multiplex PCR and direct sequencing methods were used. Findings: Out of 170 studied chromosomes, none of the Del (GJB6-D13S1830) and Del (GJB6-D13S1854) mutations found in the studied subjects. Discussion & conclusions: Lack of the deletion mutations in the Cx30 gene, indicates the involvement of other genes in causing disease. Thus, it is necessary to study other HL genes in these subjects.

A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis.

BackgroundThe CRISPR/Cas9 system has become a regularly used tool for editing the genome of many model organisms at specific sites. However, two limiting steps arise in the process of validating guide RNA target sites in larvae and adults: the time required to identify indels and the cost associated with identifying potential mutant animals.ResultsHere we have combined and optimized the HotSHOT genomic DNA extraction technique with a two-steps Evagreen PCR, followed by a high-resolution melting (HRM) assay, which facilitates rapid identification of CRISPR-induced indels. With this technique, we were able to genotype adult zebrafish using genomic DNA extracted from fin-clips in less than 2 h. We were also able to obtain a reliable and early read-out of the effectiveness of guide RNAs only 4 h after the embryos were injected with the constructs for the CRISPR/Cas9 mutagenic system. Furthermore, through mutagenesis kinetic assay, we identified that the 2-cell stage is the earliest time point at which indels can be observed.ConclusionsBy combining an inexpensive and rapid genomic DNA extraction method with an HRM-based assay, our approach allows for high-throughput genotyping of adult zebrafish and embryos, and is more sensitive than standard PCR approaches, permitting early identification of CRISPR-induced indels and with applications for other model organisms as well.

A rapid genomic DNA extraction method and its combination with helicase dependent amplification for the detection of genetically modified maize

A simple, solvent-free extraction method for DNA facilitates the analysis for genetically modified organisms.

An inexpensive and rapid genomic DNA extraction protocol for rosaceous species

SummaryThe development of high-throughput DNA genotyping, as part of marker-assisted selection and other DNA-based applications, has led to the need for a cheap, rapid and simple method for extracting genomic DNA (gDNA) from plant material. Commonly available gDNA extraction protocols do not provide consistently good quality gDNA from a wide range of plant species, particularly those in families such as the Rosaceae, which contain variable concentrations of polysaccharides and phenolic compounds. Furthermore, although commercial DNA extraction kits are widely available, they are expensive for the preparation of large numbers of DNA samples. Here, we present an improved gDNA extraction method which we have tested on 12 different species from the family Rosaceae: apple, apricot, blackberry, strawberry, peach, sweet cherry, sour cherry, pear, plum, red raspberry, quince, and almond. The method employs a novel lysis buffer which is a crucial step in obtaining high quality gDNA. It does not require expensive or toxic reagents and can be performed in the open laboratory, rather than in an air-flow hood. The method is reliable and produces good quality DNA that can be used in a variety of DNA-based studies.

Molecular Identification of Mediterranean Mutation in Patients with Deficiency of Glucose-6-Phosphate Dehydrogenase (G6pd) in North West of Iran

Glucose-6-phosphate dehydrogenase (G6PD) is a highly polymorphic enzyme that is encoded by human Xlinked gene.G6PD deficiency is the most common enzymopathy in human with approximately 400 million people affected worldwide. According to the previous studies, Mediterranean mutation (C563T) is one of the most common G6PD enzyme mutations in Iranian population with a prevalence range of %63 to %86/4.This study was conducted in order to determine Mediterranean mutation, through molecular method ,in the population of the North-west of Iran. In this study, by Rapid Genomic DNA Extraction (RGDE) method, from 77 blood samples of unrelated male and female patients with genetic deficiency of G6PD, DNA was extracted and after digestion by MboII enzymes, in order to search for Mediterranean mutation, they were analyzed by means of PCR-RFLP and sequencing methods. Of the 77 patients, 48 patients (37 male, 11 female) had a Mediterranean mutation frequency of 62/3 %. 29 patients (37/7%) had mutations other than Mediterranean type. This study revealed that G6PD is the most common mutation in Iranian North-west population. Further studies are recommended to identify the mutation type of other varieties.

Templated synthesis of monodisperse mesoporous maghemite/silica microspheres for magnetic separation of genomic DNA

A novel method is described for the preparation of superparamagnetic mesoporous maghemite (γ-Fe 2O 3)/silica (SiO 2) composite microspheres to allow rapid magnetic separation of DNA from biological samples. With magnetite (Fe 3O 4) and silica nanoparticles as starting materials, such microspheres were synthesized by the following two consecutive steps: (1) formation of monodispersed organic/inorganic hybrid microspheres through urea–formaldedyde (UF) polymerization and (2) removal of the organic template and phase transformation of Fe 3O 4 to γ-Fe 2O 3 by calcination at elevated temperatures. The as-synthesized particles obtained by heating at temperature 300 °C feature spherical shape and uniform particle size ( d particle=1.72 μm), high saturation magnetization ( M s=17.22 emu/g), superparamagnetism ( M r/ M s=0.023), high surface area ( S BET=240 m 2/g), and mesoporosity ( d pore=6.62 nm). The composite microsphere consists of interlocked amorphous SiO 2 nanoparticles, in which cubic γ-Fe 2O 3 nanocrystals are homogeneously dispersed and thermally stable against γ- to α-phase transformation at temperatures up to 600 °C. With the exposed iron oxide nanoparticles coated with a thin layer of silica shell, the magnetic microspheres were used as a solid-phase adsorbent for rapid extraction of genomic DNA from plant samples. The results show that the DNA templates isolated from pea and green pepper displayed single bands with molecular weights greater than 8 kb and A 260/ A 280 values of 1.60–1.72. The PCR amplification of a fragment encoding the endogenous chloroplast ndhB gene confirmed that the DNA templates obtained were inhibitor-free and amenable to sensitive amplification-based DNA technologies.

Preparation of magnetite-loaded silica microspheres for solid-phase extraction of genomic DNA from soy-based foodstuffs

Solid-phase extraction has been widely employed for the preparation of DNA templates for polymerase chain reaction (PCR)-based analytical methods. Among the variety of adsorbents studied, magnetically responsive silica particles are particularly attractive due to their potential to simplify, expedite, and automate the extraction process. Here we report a facile method for the preparation of such magnetic particles, which entails impregnation of porous silica microspheres with iron salts, followed by calcination and reduction treatments. The samples were characterized using powder X-ray diffractometry (XRD), scanning electron microscopy (SEM), nitrogen adsorption/desorption isotherms, and vibrating sample magnetometry (VSM). XRD data show that magnetite nanocrystals of about 27.2 nm are produced within the pore channels of the silica support after reduction. SEM images show that the as-synthesized particles exhibit spherical shape and uniform particle size of about 3 μm as determined by the silica support. Nitrogen sorption data confirm that the magnetite-loaded silica particles possess typical mesopore structure with BET surface area of about 183 m 2/g. VSM data show that the particles display paramagnetic behavior with saturation magnetization of 11.37 emu/g. The magnetic silica microspheres coated with silica shells were tested as adsorbents for rapid extraction of genomic DNA from soybean-derived products. The purified DNA templates were amplified by PCR for screening of genetically modified organisms (GMOs). The preliminary results confirm that the DNA extraction protocols using magnetite-loaded silica microspheres are capable of producing DNA templates which are inhibitor-free and ready for downstream analysis.

Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

A series of simplified protocols are developed for extracting genomic DNA from saliva by using the magnetic nanobeads as absorbents. In these protocols, both the enrichment of the target cells and the adsorption of DNA can be achieved simultaneously by our functionally modified magnetic beads in one step, and the DNA–nanobeads complex can be used as PCR templates. HLA typing based on an oligonucleotide array was conducted by hybridization with the PCR products. The result shows that the protocols are robust and sensitive.