Method For Isolation Of DNA Research Articles

A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR

Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable faecal host DNA studies in animal disease models as well.

Comparison of Three Different DNA Isolation Methods To Degradate The Trichoderma Fungi Cell Wall

Trichoderma fungi cell walls are composed of chitin compounds that are very sturdy and resistant to enzyme activity. The specific techniques and methods are needed to degradate of fungi cell wall. This study aims to analyze the best DNA isolation method in lysing fungal cell walls by comparing several isolation methods. The methods are using DNeasy Plant Mini Kit, DNeasy Plant Mini Kit combination with heating, and DNeasy Plant Mini Kit combination in a physical way (grinding the sample using mortar and pestle in liquid nitrogen). This research was conducted in March to September 2018 at the Microbiology Laboratory, Laboratory of Biotechnology and Genetics, as well as the Integrated Research Laboratory, FMIPA, UNP. Data were analyzed qualitatively by agarose gel electrophoresis and quantitatively by calculating the purity and concentration of DNA using Nanodrop. The results showed that the DNeasy Plant Mini Kit combination method by physical means gave the highest concentration of DNA isolation of 2.4 μg / ml with a purity value of 1.857. In the DNeasy Plant Mini Kit method and the DNeasy Plant Mini Kit combination method with heating, the DNA concentrations obtained were 1.211 µg / ml and 0.933 µg / ml respectively with a purity value of 1.728 and 1.708. The results of the electrophoresis test showed a thin band of DNA bands in samples isolated by a combination of kit methods and physical methods, whereas in the other two samples no DNA bands were found. It can be concluded that the method of DNA isolation that can be used as a reference to degrade Trichoderma mushroom cell walls is a combination of kit and liquid nitrogen method with a note that additional levels of isolates are needed.

Gelatin-coated magnetic nanoparticles-based DNA isolation method: A comparison with commercial DNA isolation kits from whole blood

DNA isolation is the first step for most molecular tests, thus indicating the importance of using a reliable and suitable method for this procedure. The use of magnetic nanoparticles for blood DNA isolation offers several advantages including subjecting little mechanical stress to your sample, obtaining higher quality and quantity of DNA, and non-laborious procedures. In this study, we isolated genomic DNA using gelatin-coated magnetic nanoparticles from blood samples and test its use compare with three commercial blood DNA isolation kits. DNA purity and yield were assessed by measuring absorbance at A260/A280 and by agarose gel electrophoresis. The suitability of the isolated DNA for downstream applications was analyzed by end-point PCR and Sanger sequencing. In this study, gelatin-coated magnetite particles for genomic DNA isolation provide an efficient, simple, and inexpensive method that does not require the use of commercial blood DNA Isolation Kit.

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.

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (≥8) as needed for transcriptional profiling experiments.

Species-level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization.

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.

Developing a protocol for the isolation of high quality DNA from carbohydrate rich medicinal plant for molecular identification

There is an increasing international market for medicinal plants, which are used both for herbal medicine and for pharmaceutical products. For rapid and accurate identification of plants DNA barcoding is most authentic tool. The aim of this study is first optimize a method for the genomic DNA isolation from non-identified, carbohydrate rich, plant leaf tissues. Than do the DNA barcoding by using the ITS 2 as a potential molecular marker. In this study we first optimize a method for the genomic DNA isolation from non identified, carbohydrate rich, plant leaf tissues. For the optimization of DNA isolation method different combination and permutation were done at different stages of the DNA isolation. Further, the genomic DNA was utilized as a template for the amplification and sequencing of ITS2 region. BLAST search was performed against GenBank database by using annoted ITS2 region as sample query. We found very high yield around 123 µg per gram of leaf samples and the ratio at 260/280 nm found to be 1.82. Hence the yield as well as quality of the DNA was very good. Two PCR products of 419 and 390 bp were amplified by using ITS2F1, ITS2F2 as forward and ITS2R as reverse primers respectively. In annotation out of 419, 228 bp sequences were annotated as ITS2 for this plant in blast search it shows close resemblance to the plants of plantaginaceae in the order lamiales. In herbarium identification the plant was identified as Anisochilus carnosus (Lf) which belongs to Lamiaceae family in the order lamiales.

Diagnosis of Meningococcal Infection Using Internally Controlled Multiplex Real-Time PCR.

Neisseria meningitidis (Nm) is a leading cause of invasive infections associated with high mortality and morbidity, notably meningitis and septicemia. Etiological rapid diagnosis is key for the preventive management of invasive meningococcal disease (IMD). However, conventional methods for diagnosis are time-consuming and could be hampered by the difficulties in culturing the isolates from clinical specimens especially due to early antibiotic treatment. Therefore, sensitive, specific and rapid non-culture-based methods are valuable for early diagnosis, effective therapy, and prevention. Here we describe a real-time PCR multiplex assays for the detection of Nm targeting the meningococcal-specific gene crgA, coding for a LysR-like transcriptional regulator, and six serogroup-specific (A, B, C, W, X, Y) Nm capsular genes, using a Qiagen column-based method for the optimum isolation of DNA from clinical specimens. Internal quality controls were included to monitor extraction of DNA, inhibition and the technical validation of the PCR as well.

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 quantitative comparison of two kits for DNA extraction from canned tuna

The most common methods that can be used for species identification of tuna include methods based on detection of species-specific DNA via the polymerase chain reaction (PCR) method. The problem with DNA detection in processed products is the possibility of DNA fragmentation during the technological process. The quantity and quality of extracted DNA is a crucial step for species identification based on the DNA analysis. In this study, two DNA extraction methods (DNeasy Blood & Tissue Kit and DNeasy mericon Food Kit) for tuna DNA isolation were compared. Eight food products of canned tuna (three of them were declared as Thunnus albacares and five products were declared as Katsuwonus pelamis) with a different addition of various ingredients were tested. Furthermore, three different times of proteolysis (30 min, 60 min, overnight) for each sample and each extraction kit were evaluated. The DNA concentration was determined by a Qubit dsDNA HS Assay Kit fluorescence method and quantified using a Qubit fluorometer. The DNA purity was evaluated using the A260/A280 ratio of absorbances measured on a spectrophotometer. The main indicator of DNA quality and quantity was its amplifiability in the subsequent real-time PCR for Thunnus species, Thunnus albacares and Katsuwonus pelamis. Based on the results, both kits can be used for tuna species determination in highly heat-treated products with different composition, nevertheless, the DNeasy mericon Food Kit provided better statistical values in some parameters. The effect of different times of proteolysis was significant in most of the samples with regard to the crossing point values determined by real-time PCR.

A cost-effective protocol for total DNA isolation from animal tissue

ABSTRACTLaboratories that perform PCR on a routine basis need to count on reliable DNA isolation methods. In locations where supply of DNA extraction kits depends on importation, having an in-house protocol is desirable. This is also important for laboratories limited by budget constraints. We present a low-cost DNA isolation protocol that incorporates well-known techniques, but that we have adapted to various animal tissues. We tested this protocol on animal blood and muscle, and on cell suspension from skin swabs. The results were comparable, in terms of amount and quality of DNA, to those obtained with two other commercially available methods. DNA retrieved with this protocol has been successfully employed for Sanger sequencing of gene PCR products from animal tissues and blood, as well as for PCR-based diagnosis of chytrid fungus in amphibians and blood parasites in birds.

Method for DNA Isolation From Sweetpotato Weevil (Coleoptera: Curculionidae) Collected in Pheromone-Baited Traps.

This study provides a protocol for the isolation of high-quality DNA from sweetpotato weevils (Cylas formicarius elegantulus (Summers)) collected from pheromone-baited aerial funnel traps. This study was based on our discovery that a 2-wk collection interval of sweetpotato weevils from pheromone traps did not permit isolation of intact high-quality genomic DNA. To test the effect of collection methods, i.e., sample collection interval and preservation method, on quality of isolated DNA, we placed freshly killed male sweetpotato weevils into aerial funnel traps in the field and removed subsamples at several times thereafter. DNA yield from freshly isolated (day = 0) samples was significantly greater than samples preserved in 70% ethanol or at -20°C, whereas there was no difference between 70% ethanol and -20°C storage. Likewise, DNA yield from freshly isolated (day = 0) samples was significantly greater than for later sampling times. Quality assessment of genomic DNA through gel electrophoresis and polymerase chain reaction (PCR) indicated isolation of high molecular weight DNA for all samples collected at t ≤ 7 d, but that DNA quality was degraded by 14 d. Our goal was to develop a reliable method for isolation of genomic DNA from field-collected sweetpotato weevil suitable for direct use in PCR. We discovered that it is critical to collect specimens from traps at an interval of 1 wk or less. Our findings allow for scheduling of sampling at reasonable intervals without the need for special materials. This has the added benefit of allowing individuals without special training to collect and prepare sweetpotato weevil specimens for genetic studies.

Magnetofection and isolation of DNA using polyethyleneimine functionalized magnetic iron oxide nanoparticles.

This study was carried out to develop a simple and efficient method to isolate DNA directly from biological samples using iron oxide nanoparticles (IONPs) functionalized with polyethyleneimine (PEI). IONPs were synthesized via co-precipitation method followed with direct attachment of branched PEI. Nanoparticles were characterized using STEM, FT-IR spectroscopy and XRD analysis. The binding capacity of synthesized PEI-IONPs for plasmid and genomic DNA was assessed using purified DNA samples. In order to elute bound DNA, elution conditions were optimized, changing pH, salt concentration and temperature. Synthesized PEI-IONPs were subjected to isolation of DNA from bacterial cell culture and from human blood. PCR and magnetofection of the enhanced green fluorescence protein (EGFP) were carried out to verify the downstream applications of isolated DNA. The results indicated that the synthesized nanoparticles were of 5–10 nm. The binding capacity of PEI-IONPs for plasmid DNA and genomic DNA were 5.4 and 8.4 µg mg−1, respectively, which were even higher than the commercially available kits such as Mag-bind, MagJET and Magmax. The optimized condition for plasmid DNA elution was 0.1 M Tris HCl (pH 10.0), 1.5 M NaCl and 5% formamide, maintained at the temperature of 60°C. The optimized condition for genomic DNA elution was 0.1 M Tris HCl (pH 10.0), 1.5 M NaCl and 10% formamide, maintained at 60°C. PCR and magnetofection processes were successful. This study revealed that the magnetic separation of DNA using PEI-IONPs is a simple and efficient method for direct isolation of DNA from biological samples which can be then used in various downstream applications.

Optimization of DNA isolation method from Formalin-Fixed-Paraffin-Embedded tissues (FFPE) and comparative performance of four different Polymerase Chain Reaction (PCR) kits

The tissue sample may have important genetic information in diagnostic, prognostic and counselling issues. Formalin-Fixed-Paraffin-Embedded (FFPE) is a routine method for preserving tissues. However, DNA isolated from FFPE tissue is often difficult to be amplified in PCR due to fragmentation and DNA-protein crosslinks. This study aimed to optimize the DNA isolation method from FFPE tissue and compare the performance of four different PCR ready-to-use kits. Genomic DNA was isolated from FFPE tissue colon of Short-segment Hirschsprung (S-HSCR) patients and prostate cancer tissue using Quick-DNA™ FFPE Kit (Zymo Research) with and without pre-heating treatment in KOH/NOH solution. Primers for Androgen Receptor (AR) gene and four different PCR kits: MyTaq HS Red Mix 2X (BioLine), FastStart Taq DNA Polymerase (Roche), KAPA2G fast PCR Kit 2X (KAPA Biosystem) and KOD FX Neo (Toyobo) were used for amplification. DNA electrophoresis was performed to compare the PCR results. BioLine and Toyobo kits gave better PCR results than those of Roche and KAPA Biosystem. Increasing amount of Taq polymerase and dNTPs of Roche kit by two-fold could increase the quality of PCR results. Toyobo could amplify DNA up to 417 bp, however, none of these PCR kits could amplify DNA above 450 bp. Pre-heated treatment of FFPE tissue in NaOH/KOH did not improve the DNA quality and PCR results. Toyobo PCR ready-to-use kit gave the best result among the other three PCR kits used in this study in amplifying DNA isolated from FFPE tissue. Designing the primers producing amplicon not more than 450 bp is suggested.

Multiplex testing for Factor II and Factor V mutations in thrombophilia: technical verification and clinical validation of the cobas\xae Factor II and Factor V test

Laboratory testing for thrombophilia is complicated but essential for diagnosis. In 2017, the cobas® Factor II and Factor V Test (cobas F2F5 test) was launched for use with the cobas z 480 analyzer. This qualitative polymerase chain reaction test enables multiplex Factor II and Factor V testing with flexible reporting and workflow efficiency. Here, we report the results from studies investigating the performance of the cobas F2F5 test. Technical performance verification, clinical validation, external laboratory performance, and workflow comparison studies were performed. Fresh and frozen whole-blood and genomic DNA (gDNA) samples were tested, and several manual and automated DNA isolation methods were used. Bidirectional Sanger sequencing was used to verify genotypes identified by the cobas F2F5 test. One hundred percent agreement between the cobas F2F5 test and Sanger sequencing was observed for all genotypes. An external laboratory using remnant clinical samples also yielded 100% agreement between cobas F2F5 test results and their routine testing method. The cobas F2F5 test reduced the total sample processing time compared with the LightCycler® 1.2 platform (98.6 vs 420.2 min; 96 samples). Hemoglobin, extraction buffer, and ethanol contamination of the gDNA sample can lead to invalid results. The cobas F2F5 test has a high degree of accuracy for identification of Factor II and Factor V genotypes. This multiplex testing with short sample processing time can reduce handling errors and increase efficiency. Both manual and automated DNA isolation methods can be used with the cobas F2F5 test.

Comparison of DNA Isolation Results with Simple Methods and Kits in Samples of Psidium guajava Leaves

DNA isolation is one of a series of methods that must be carried out on the basic techniques of Molecular Biology Analysis. Especially PCR-based molecular marking techniques. Many ways are done in DNA isolation. This study discusses the comparison of the results of DNA isolation using two methods. Simple DNA isolation methods and using Kit. Samples of Psidium guajava leaves used were taken from 15 different locations used. In general, DNA isolation methods include three steps, namely destruction, precipitation, and purification. Simple DNA isolation is done with detergents, alcohol groups, which are commonly available in the laboratory. Methods of DNA isolation with KIT are carried out according to the Promega Universal Wizard KIT protocol. The comparison results are seen from spectrophotometric absorption Å230 nm, Å260 nm, Å280 nm, Å320 nm, ratio Å260/Å230, ratio Å260/Å280 to see DNA purity, protein concentration before purification step, and DNA concentration produced. The results showed that there were no statistically significant differences in the results of DNA isolate spectrophotometry. However, the use of KIT with modified protocols is more recommended if researchers want to carry out DNA analysis more precisely and accurately.
 
 Keywords: DNA isolation, spectrophotometry, DNA concentration and purity

A simple and rapid method for isolation of DNA for molecular markers and transgene analysis in safflower

In spite of the possibility of analysing large samples and the rapidity of Polymerase Chain Reaction (PCR) technology, the usual protocols for DNA plant extraction remain time-consuming, slow and involves use of hazardous chemicals. To lessen labor, time or cost of DNA extraction, a simple and instant method for genomic DNA extraction from leaf tissue of safflower (Carthamus tinctorius L.) leaf is established. Small quantity of tissue material (typically3-5mg) was ground in a centrifuge tube using plastic pestle in extraction solution. ExtractedDNA was suitable for PCR analysis, without centrifugation. The feasibility of this method was confirmed by testing molecular markers and transgene detection. This method requires less than 1 mg of plant tissue stored frozen or used fresh and is useful for molecular marker analyses as well as transgene detection.

A Rapid Microwave Method for Isolation of Genomic DNA and Identification of White Rot Fungi

A Rapid Microwave Method for Isolation of Genomic DNA and Identification of White Rot Fungi

Isolation and Quantification of DNA.

Purifying DNA is the key to successful cloning. The cleaner the final preparation of DNA, the more efficient will be the enzymatic reactions that use the DNA as a template or a substrate. In the 1930s and 1940s, the scientific literature began to accumulate methods to release DNA from cells and to remove cellular constituents that inhibit or act as competitors on enzymatically catalyzed reactions. Since then, thousands of protocols for purification of DNA from a wide variety of organisms, tissues, and bodily fluids have been published. This introduction provides an overview of methods for isolation and quantification of DNA.

A quick and simple benchtop vortex egg-disruption approach for the molecular diagnosis of Fasciola hepatica from ruminant faecal samples.

Commonly employed diagnostic methods for Fasciola spp., such as a traditional sedimentation and faecal egg count, or a commercially available coprological ELISA, have limitations in their sensitivity or ability to differentiate species. A reliable DNA isolation method coupled with real-time PCR addresses these issues by providing highly sensitive and quantitative molecular diagnosis from faecal samples. The current study evaluated a standard benchtop vortex for F. hepatica egg disruption in sheep and cattle faecal samples and determined the minimum faecal egg load required for a positive result from un-concentrated (raw) faecal samples. The minimum faecal egg load for a positive real-time PCR result from 150mg raw faecal sample was 10 and 20 eggs per gram for sheep and cattle, respectively. No significant difference (P = 0.4467) between disruptions on a benchtop vortex for 5 or 10min was observed when compared to 40s of disruption at 6.0m/s in a benchtop homogeniser.