Rapid DNA Isolation Research Articles

A rapid, inexpensive and effective method for the efficient isolation of genomic DNA from Gram-negative bacteria.

Currently, there are several protocols to extract bacterial DNA based on different principles. However, the quantity and the quality of the DNA obtained by each method are highly variable and microorganism dependent. In most of these classical crude methods, highly toxic and hazardous organic solvents such as phenol and chloroform are used for deproteinization, whereas in certain protocols, expensive enzymes including RNases and Proteinases are used. This study was designed to introduce a simple, rapid, inexpensive and effective genomic DNA isolation procedure for Gram-negative bacteria, without the usage of toxic chemicals and costly enzymes. This novel method was compared with another classical method known as the salting-out method, which uses proteinase-K. Concentration and yield of the extracted DNA were determined by gel electrophoresis by comparing the gel band intensity of the sample DNA to that of a DNA quantitation standard and by the Quantus™ fluorometer. According to the results, the yield of extracted DNA was higher in the novel method compared to the salting-out method. Moreover, the entire process was accomplished in less than 2h with the novel method. Purity and integrity of extracted genomic DNA by both methods were similar. In addition, the quality of DNA was determined using Multicopy Associated Filamentation (MAF) gene amplification by polymerase chain reaction (PCR). Thus, the described technique is non-toxic, less time and fund consuming, efficient and a well-suited method for routine DNA isolation from Gram negative bacteria.

Development of a high-throughput DNA isolation method for livestock and poultry meat based on mesoporous metal-organic framework-coated solid-phase microextraction device

A rapid, portable, simple, low-cost, and high-throughput DNA isolation method was developed for the quantitative identification of meat adulteration based on mesoporous UiO-66 coated solid phase microextraction (SPME) devices. The high-throughput DNA extraction system consists of 96 UiO-66 SPME devices and a polyformaldehyde plate, which can extract DNA from 96 livestock and poultry meat samples simultaneously. At the same time, several parameters of the high throughput DNA isolation method were optimized, including lysis buffer volume, elution buffer volume, lysis time and elution time, etc. Compared with the single DNA isolation device, the 96-SPME high-throughput extraction system maintains the original good universality, reproducibility, chemical stability, and reusability. On this basis, it obtained higher DNA concentration (80 ± 34 ng μL−1), satisfactory DNA purity (A260/A280=2.4) and shorter extraction time (less than 7.2 s per sample), which are much more convenient and efficient than the conventional kit method and the single SPME DNA isolation method. In addition, the conventional multiplex polymerase chain reaction and real-time polymerase chain reaction techniques were used for the qualitative and quantitative detection of meat adulteration. The DNA obtained from this extraction method is highly reproducible and sensitive for the identification of duck ingredients in beef, with a detection range of 1–100 % and a minimum detection limit of 1 % (w/w).

A rapid, efficient and cost-effective DNA isolation protocol for various herbal products which is appropriate for different downstream genomics applications

A rapid, efficient and cost-effective DNA isolation protocol for various herbal products which is appropriate for different downstream genomics applications

Two-step method for rapid isolation of genomic DNA and validation of R81T insecticide resistance mutation in Myzus persicae

Isolation and amplification of nucleic acid (DNA) is considered a vital and potent instrument in molecular biological research. However, its functioning outside of a laboratory setting is difficult because of complex procedures that demand expert personnel and expensive equipment in addition to the fulfillment of several additional requirements. DNA isolation from minute insects is sometimes difficult, making diagnostic and genotyping procedures problematic. Thus, the current work offers a high-throughput, cost-effective, straightforward, and faster approach for isolating DNA from the aphid Myzus persicae. Intriguingly, two-step DNA extraction process yielded a high yield of extremely pure genomic DNA and required only 10 s to complete. PCR investigation aiming at amplifying the non-synonymous R81T region on the loop D site of the nAChR gene of M. persicae was subsequently utilized to successfully validate the recovered DNA. Moreover, the proposed method was compared in terms of yield and purity with conventionally used DNA isolation methods including, phenol:chloroform, salt out, and commercially available kits. In conclusion, this newly developed method would enable researchers to quickly process many biological samples used to analyze genetic diversity, mutant screening, and large spectrum diagnosis both in laboratory and field conditions.

Simple and Rapid DNA Isolation from Shrimps by Using Single tube extraction buffer with Single step

Simple and Rapid DNA Isolation from Shrimps by Using Single tube extraction buffer with Single step

Rapid solid phase microextraction of DNA using mesoporous metal-organic framework coating for PCR-based identification of meat adulteration.

A rapid, convenient, low-cost, and selective DNA isolation method was developed for identifying meat adulteration. A mesoporous metal organic framework (Meso-UIO-66)-coated solid phase microextraction system was employed as an isolation device to simplify DNA isolation into three steps (lysis, washing, and elution). Meso-UIO-66 was utilized as the adsorbent because of its positively charged surface, high chemical stability, and mesoporous structure. Meso-UIO-66 was characterized by scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction, Fourier transform infrared spectroscopy, ultraviolet‒visible spectroscopy, X-ray photoelectron spectroscopy, and nitrogen adsorption-desorption tests. Parameters that affected DNA isolation were optimized. This method can be used to isolate and purify DNA from meat in 60s, and the DNA concentration and purity are comparable to those of samples isolated with a commercial kit. Multiple DNA detection was achieved by coupling this method with the multiplex polymerase chain reaction technique, and the detection limit was lower than 1% (w/w).

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva

BackgroundThe isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages – volume, time, cost, and yield are a few notable ones. ObjectiveWe aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. MethodsFive DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. ResultsOut of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. ConclusionIn the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Novel and PCR ready rapid DNA isolation from Drosophila

Introduction Isolation of genomic DNA is an initial step in molecular biology techniques. The quality of isolated DNA depends on procedures and chemicals, as well as source and types of the sample used. Several existing procedures are expensive and time consuming. In this study, we isolated high quality genomic DNA with an inexpensive and least time consuming procedure using Drosophila melanogaster flies, larvae, and pupae. Methods Drosophila melanogaster samples were collected from pre-cultured bottles, and genomic DNA was extracted using a proposed novel and PCR-ready method from three different pools of flies [PF1, PF2, and PF3], similarly from larvae and pupae [PL1, PL2, PL3, PP1, PP2, and PP3, respectively]. Isolated genomic DNA was subjected to PCR amplification with different dilutions using the COI gene and further amplicons were used for RAPD and DNA sequencing. Results The high quality of isolated genomic DNA was confirmed by 0.8% agarose gel electrophoresis and the purity and quantity of the DNA isolated from single fly, larva and pupa was similar to the purity and quantity of the DNA isolated using the NucleoSpinR Tissue kit method. Isolated genomic DNA was successfully amplified when the template was diluted in the ratio of 1:10. Further successful RAPD amplification and sequencing analysis of the COI gene confirms the efficiency of the downstream application of the proposed novel method. Conclusion The present Novel and PCR ready rapid DNA isolation method will be potentially beneficial, and it can be successfully used for quick isolation of high molecular weight DNA from Drosophila flies larvae and pupae for DNA barcoding, identification of new species, genotyping, RAPD analysis, etc. Moreover, it can also be easily scaled up for bulk preparations.

Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay, coupled with simplified sample preparation, for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples.

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.

Thin Film Microextraction Enables Rapid Isolation and Recovery of DNA for Downstream Amplification Assays.

Nucleic acid analysis has been at the forefront of the COVID-19 global health crisis where millions of diagnostic tests have been used to determine disease status as well as sequencing techniques that monitor the evolving genome of SARS-CoV-2. In this study, we report the development of a sample preparation method that decreases the time required for DNA isolation while significantly increasing the sensitivity of downstream analysis. Functionalized planar supports are modified with a polymeric ionic liquid sorbent coating to form thin film microextraction (TFME) devices. The extraction devices are shown to have a high affinity for DNA while also exhibiting high reproducibility and reusability. Using quantitative polymerase chain reaction (qPCR) analysis, the TFME devices are shown to require low equilibration times while achieving higher preconcentration factors than solid-phase microextraction (SPME) by extracting larger masses of DNA. Rapid desorption kinetics enable higher DNA recoveries using desorption solutions that are less inhibitory to qPCR and loop-mediated isothermal amplification (LAMP). To demonstrate the advantageous features of the TFME platform, a customized leuco crystal violet LAMP assay is used for visual detection of the ORF1ab DNA sequence from SARS-CoV-2 spiked into artificial oral fluid samples. When coupled to the TFME platform, 100% of LAMP reactions were positive for SARS-CoV-2 compared to 66.7% obtained by SPME for a clinically relevant concentration of 4.80 × 106 DNA copies/mL.

A rapid and efficient DNA isolation method for qPCR-based detection of pathogenic and spoilage bacteria in milk

The objective of this study was to find an efficient, rapid, simple, and cost-effective method of pretreating raw milk samples to produce PCR-ready DNA for subsequent microbial detection using the strains of eight bacterial species. A total of 17 in-house protocols and three commercial kits were evaluated in three steps from scientific, practical, and economic perspectives. The results showed that an in-house procedure involving Triton X-100-based pretreatment and an inhibitor removal resin was superior to all other methods tested in terms of DNA yield, sensitivity, ease of sample handling, time efficiency, and cost per sample. Overall, this simplified preanalytical protocol was shown to have a great potential for use in rapid detection of dairy-related bacterial species, thereby enabling early intervention in the food chain and thus reducing the risk of negative economic and health outcomes. • Methods of pretreating raw milk samples to yield PCR-ready bacterial DNA were tested. • Of 20 procedures, a Triton X-100-based in-house protocol showed the best results. • This approach has a great potential for use in detection of dairy-related bacteria. • With further optimization, the protocol may be included in milk quality guidelines.

Rapid Detection of Transposon-Induced Genome Rearrangements.

This chapter details the techniques used to detect transposon-induced genome rearrangements. Here, we describe a rapid DNA isolation technique, PCR amplification, and a novel High Efficiency Agarose Gel Electrophoresis Method (HEA-GEM).

Comparative detection of banana bunchy top virus in banana cv. Safed velchi utilizing DNA/RNA by PCR technique

The present study was carried out to describe the ideal PCR protocol to find viral infection in plantlets of banana. 50 plantlets selected from Plant Biotechnology Center, Dapoli from which DNA (50 samples bulked into 10) as well as RNA (20 samples) were extracted along with BBTV infected samples. The priviously standard rapid DNA isolation protocol was used. A protocol using 1.80 g ml-1 Tris-base, 3.75 g ml-1 EDTA, 1.5 g ml-1 SDS and 100µl ml-1 βMercaptoethanol produced the highest yields and best quantity RNA. cDNA synthesis (M-MuLV kit) containing Oligo-dT, dNTP’s, Molecular Biology Grade Water and isolated RNA template were used to prepare reaction mixture. The optimum PCR procedure utilizes to synthesized cDNA from template RNA. 21 samples get assayed at RNA level by preparing cDNA. Strong reactions were obtained from the sample no 10, 11, 22 and 23 of BBTV infected samples and a positive control plant at no 12 and 24 confirms the presence of BBTV with amplicon size of 389 bp with primer BBTV F & R. All 50 DNA samples were tested for BBTV gene specific primers (CP, NSP and FPCR’4+30MER gene) all the tested regenerated plantlets were found to be negative and the lot no 11 of BBTV infected samples and a positive control plant at no 12 confirms the presence of BBTV with amplicon size of 511bp, 349 bp and 1,349 bp respectively. But, RNA level mechanism was effective in which the actual coding genome was transcribed. So it is easy and useful to detect the genome at RNA level than DNA which contains large genome size.

A Simple and Rapid Fungal DNA Isolation Assay Based on ZnO Nanoparticles for the Diagnosis of Invasive Aspergillosis.

Invasive aspergillosis (IA) is an important cause of morbidity and mortality among immunocompromised people. Imaging and specimen tests used in the clinical diagnosis of aspergillosis with weak and indistinct defects leads to delay in the treatment of early aspergillosis patients. The developing molecular techniques provide a new method for the aspergillosis diagnosis. However, the existing methods are complex, time-consuming and may even be potentially hazardous. In this study, we developed a simple and rapid Aspergillus fumigatus spores DNA isolation assay using synthesized zinc oxide (ZnO). ZnO nanoparticles were used to take the place of the traditional commercial lysis buffer. The quality and quantity of the extracted DNA were sufficient for further diagnostics with polymerase chain reaction (PCR) analysis. This method offers easy, green, and economic alternative DNA isolation for the diagnosis of invasive aspergillosis.

Poly (2-hydroxypropylene imines) functionalized magnetic polydopamine nanoparticles for high-efficiency DNA isolation

A rapid and efficient DNA isolation method without toxic agents and centrifugation is essential. In this study, we synthesized a novel kind of magnetic nanocomposite (Fe3O4@PDA@PHP) readily by coating polydopamine (PDA) and grafting poly (2-hydroxypropylene imines) (PHP) onto the surface of Fe3O4 magnetic nanoparticles for DNA isolation. TEM, FTIR, VSM and XPS characterization results indicated the prepared Fe3O4@PDA@PHP nanocomposite possessed core-shell structure and PHP was successfully grafted onto magnetic polydopamine nanoparticles. The isolation behaviors of Fe3O4@PDA@PHP for DNA were also evaluated taking into factors such as DNA concentration, initial binding solution pH and extraction time. Results showed that pseudo-second-order model and Freundlich isotherm model well fitted the kinetic results and the equilibrium extraction data, respectively. Meanwhile, the resultant magnetic nanocomposite performed excellent isolation properties toward DNA higher than other studied materials at same conditions, and had excellent specificity in presence of some proteins and metal ions. Moreover, Fe3O4@PDA@PHP could retain high isolation capacity after five using-cycles and have high biocompatibility. Therefore, the resultant Fe3O4@PDA@PHP would be a promising device for the isolation of DNA in biological application.

Molecular characterization of covalently closed circular plasmid DNA in local Streptomyces strains isolated from soil with detection of antibiotic production and antibacterial activity

Various of Streptomyces species have two kinds of plasmids, circular plasmids (8 to 31 kb) and linear plasmids (12 to 1700 kb). Covalently closed circular (CCC) plasmids are profuse in the genus of Streptomyces and involved in production and resistance of antibiotics by genetic controlling. We collected fifty clinical soil samples from different regions in Al-Najaf Al-Ashraf province/Iraq. The samples included five from Al-Ghadeer Quarter, five from Al-Karama Quarter, 10 from Kufa University, five from Al-Ameer Quarter, four from Al-Forat Quarter, 10 from North Quarters and eleven from desert roads in Al-Najaf. Diluted samples were cultured on Yeast extract Malt extract (YEME) agar medium as a selective medium; then the presumptive Streptomyces colonies were subcultured on Tryptone Yeast extract (TYE) agar, then incubation at 37ᵒC for 7 days. Seven biochemical tests for identification of Streptomyces isolates these are: Catalase test, Oxidase test, Urase test, Kligler Iron Agar test (KIA), Simmon᾽s Citrate test, addition to MacConkey agar test and Mannitol Salt agar test. Five antibiotic discs were used for detection of antibiotic sensitivity of the Streptomyces isolates; these are: Tetracycline, Gentamycin, Vancomycin, Ampicillin, Erythromycin. The sensitivity of the antibiotics was observed by recorded the diameter of inhibition zone around the discs. Two test bacteria (Staphylococcus aureus and E. coli) were used for the determination of antibacterial activity. Plasmid isolation was done by the alkaline lysis method. This method is characterized by the rapid isolation of DNA from Streptomyces. Then, detection of Plasmid DNA occurred by using agarose gel electrophoresis.

Development of an innovative and sustainable one-step method for rapid plant DNA isolation for targeted PCR using magnetic ionic liquids

BackgroundNowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bottleneck in the bioanalytical workflow. New opportunities have been created by the use of magnetic ionic liquids (MILs) thanks to their affinity for nucleic acids.ResultsIn the present study, a MIL-based magnet-assisted dispersive liquid–liquid microextraction (maDLLME) method was optimized for the extraction of genomic DNA from Arabidopsis thaliana (L.) Heynh leaves. MILs containing different metal centers were tested and the extraction method was optimized in terms of MIL volume and extraction time for purified DNA and crude lysates. The proposed approach yielded good extraction efficiency and is compatible with both quantitative analysis through fluorimetric-based detection and qualitative analysis as PCR amplification of multi and single locus genes. The protocol was successfully applied to a set of plant species and tissues.ConclusionsThe developed MIL-based maDLLME approach exhibits good enrichment of nucleic acids for extraction of template suitable for targeted PCR; it is very fast, sustainable and potentially automatable thereby representing a powerful tool for screening plants rapidly using DNA-based methods.

An improved method for rapid isolation of DNA and RNA from leaves, flowers and roots of blackgram [Vigna mungo (L.) Hepper] for detection of begomovirus infection and RT-PCR.

Blackgram is an important pulse crop grown for its protein rich seeds. Its tissues are rich in polyphenols which hampered pure nucleic acid extraction and thus use of molecular techniques in breeding programme. In this study, a well established DNA extraction method for legumes, Gem-CTAB method has been modified to isolate PCR compatible DNA from blackgram in a quick and economical way from a very small tissue. The amplification rate of DNA extracted through modified Gem-CTAB method was found to be high compared to Gem-CTAB method for begomovirus specific genes. While for RNA isolation, among different protocols, column based kit method followed by lithium chloride precipitation yielded high quality RNA for leaves compatible with RT-PCR. For flowers and roots, kit method alone or with its modifications yielded pure RNA. The above modified nucleic acid extraction method yielded PCR compatible DNA and RNA from phenolics rich blackgram tissues.

A modified protocol for rapid DNA isolation from cotton (Gossypium spp.)

Extraction of high-quality DNA from Gossypium (Cotton) species is notoriously difficult due to high contents of polysaccharides, quinones and polyphenols other secondary metabolites. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from cotton, which is amenable for downstream analyses. In contrast to other CTAB methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, CsCl gradient ultracentrifugation, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use of PVP-10 to rid the problems associated with polysaccharides and polyphenols, respectively. The average yield was approximately 10–15 μg of good quality DNA from 100 mg of tissue weight, which is adequate for projects, like genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation.•Buffers A and B were redesigned from Paterson et al. (1993) and Porebski et al. (1997), respectively.•Ribonuclease A was added before chloroform extraction.•A simple, rapid and inexpensive DNA extraction method is described.

Rapid Isolation of Yeast DNA.

This method is used to isolate yeast genomic DNA or shuttle plasmids that replicated both in Saccharomyces cerevisiae and in E. coli.