High-throughput DNA Extraction Method Research Articles

Biopolymeric surface modified 96-well plates for equipment-free high-throughput DNA extraction and direct ACK-LAMP colorimetric detection of foodborne pathogen

Ensuring the safety and high quality of food relies on promptly and clearly identifying foodborne diseases, including Salmonella. Nucleic acid testing plays an important role in ensuring food safety. Commercially available high-throughput molecular extraction technologies in-field application is limited as they are reliant on specialized and expensive equipment. The 96-well microplate recycling through biopolymer inner surface coating, was confirmed with FTIR, XRD, and XPS. Once optimized the DNA extraction protocol was equipment-free, user and eco-friendly while reducing sample contamination. Using the biopolymer microplate, the extraction and direct LAMP amplification of Salmonella spp. from milk samples were assessed. The gDNA had an average 260/280 ratio of 1.97, with a DNA yield of 16.88 ± 5.40 μg mL−1 from 9 × 1010 CFU mL−1Salmonella. Subsequently, using acid chrome K we visually detected the presence of Salmonella. The quantity of bacteria was correlated to the increase in the blue intensity of the color indicator. A detection limit of 1.54 CFU mL−1 was achieved with good specificity and sensitivity in an equipment free setting. This study successfully established a 96-well plate-based high-throughput equipment free DNA extraction method. The methods enable the high-throughput extraction and amplification of 96 samples concomitantly within 1hr 30 min corresponding respectively to a total of 1536 and 768 samples within a working day treated by a researcher without the use of specialized and expensive equipment. The polymeric modified microplate was used for high-throughput DNA extraction and direct molecular amplification for pathogen detection. High-throughput DNA extraction no longer requires cumbersome equipment.

High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing.

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

New polymer brush-coated monodisperse magnetic nanoparticles prepared via interface-mediated RAFT polymerization for high-throughput DNA extraction from pathogen bacteria

Polymerase chain reaction (PCR) is gold-standard method for molecular detection of pathogens. DNA extraction from a complex biological medium is a crucial process for PCR based detection. Recently, magnetic nanoparticles-based methods are of great interest for genetic material isolation. In our work, we offer new magnetic nanoparticles based high-throughput DNA extraction method for pathogen detection in biofluids. For this purpose, first time polymer brush coated monodisperse magnetic nanoparticles were prepared with interface-mediated RAFT polymerization to be used in DNA extraction process. After synthesizing Fe3O4 nanoparticles, silica shell was coated on the magnetic nanoparticle core. Polymer brushes was immobilized on the amine functionalized silica layer using RAFT agent. Subsequently, the resulting particles with poly(vinylbenzyltrimethylammonium chloride) (PVBTAC) grafted were finalized through interface-mediated RAFT polymerization. The functional groups on the nanoparticles were characterized using FTIR spectroscopy. Size distribution and zeta potential analysis of the particles were performed by TEM and Zeta-sizer respectively. Adsorption and recovery studies of plasmid DNA were carried out on the polymer-coated magnetic nanoparticles. The developed particle provided both the rapid extraction of DNA and high-yield DNA recovery without requiring extended incubation and elution time. The maximum adsorption capacity was determined as 238.1 μg/mg. About 90 % of the input DNA was recovered. The total time of DNA extraction took just 20 min. We have successfully demonstrated the extraction of dsDNA of Enterococcus faecalis spiked to reference blood sample using the developed magnetic particles. The extracted plasmid DNA and dsDNA were confirmed by gel electrophoresis and qPCR methods respectively. The results showed that the proposed magnetic iron oxide nanoparticles (MIONp) will be a strong candidate for DNA extraction process from biological matrices.

Evaluation of 96-well high-throughput DNA extraction methods for 16S rRNA gene metabarcoding.

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.

Comparison of various methods for DNA extraction from human isolated paraffin-embedded hydatid cyst samples.

Successful molecular research with reliable results depends on achieving significant and uniform amounts of genomic DNA from the parasite as the first and most basic step. Therefore, selection of an appropriate method that minimizes damage to the DNA of the parasite, is very important. In this study, we are going to describe a method that can extract DNA from human isolated paraffin-embedded hydatid cysts with a high quality and quantity. Formalin fixed and Paraffin-embedded hydatid cyst samples isolated from human lung and archived in the pathology laboratory were used for this purpose. Several sections of the paraffin blocks were prepared with 5 micron thickness and DNA were extracted by three different methods including; modified boiling, commercial kit and the method described by Larissa A. Pikor et al. The obtained DNA were evaluated by Nanodrop in terms of the yield of DNA and possible contaminations. To compare the quality of DNA prepared, cox1 region was amplified using specific primers. It was found that the DNA extracted by modified boiling had the lowest rate of contamination and the best electrophoretic band on the gel, compared to other two performed methods. Considering the findings of this study, this simple and high throughput DNA extraction method with high yield and quality can be recommended for extraction of DNA from formalin fixed and paraffin-embedded hydatid cysts.

A new, harmless, high-throughput endosperm-based DNA extraction method for wheat

ABSTRACT: In this study, a non-destructive, high-throughput, endosperm-based DNA extraction method was developed. To verify the non-destructive nature of this method, a germination test was performed on 288 seeds after sampling their endosperm, which gave a seedling emergence rate that was higher (97.6%) than that of the control group (92%). To confirm the feasibility of the new method, DNA was extracted from plants of a BC1F2 population by two different methods, namely, from endosperm using our rapid, high-throughput method (ER-DNA) and from young leaves emerging from the same sampled seed using the CTAB method (LC-DNA). The ER-DNA was undetectable by agarose gel electrophoresis, but was found to be an adequate replacement for LC-DNA for the amplification and detection of simple sequence repeats (SSRs). Further analysis revealed that ER-DNA was generally suitable for the generation of specific 500-750-bp fragments, but not for the amplification of 1,000-2,000-bp fragments. Our rapid, high-throughput method therefore has no deleterious effects on wheat seeds and yields DNA for SSR genotyping that is a suitable alternative to traditionally obtained DNA.

Assessing high-throughput environmental DNA extraction methods for meta-barcode characterization of aquatic microbial communities

The characterization of microbial community dynamics using genomic methods is rapidly expanding, impacting many fields including medical, ecological, and environmental research and applications. One of the biggest challenges for such studies is the isolation of environmental DNA (eDNA) from a variety of samples, diverse microbes, and widely variable community compositions. The current study developed environmentally friendly, user safe, economical, and high throughput eDNA extraction methods for mixed aquatic microbial communities and tested them using 16 s rRNA gene meta-barcoding. Five different lysis buffers including (1) cetyltrimethylammonium bromide (CTAB), (2) digestion buffer (DB), (3) guanidinium isothiocyanate (GITC), (4) sucrose lysis (SL), and (5) SL-CTAB, coupled with four different purification methods: (1) phenol-chloroform-isoamyl alcohol (PCI), (2) magnetic Bead-Robotic, (3) magnetic Bead-Manual, and (4) membrane-filtration were tested for their efficacy in extracting eDNA from recreational freshwater samples. Results indicated that the CTAB-PCI and SL-Bead-Robotic methods yielded the highest genomic eDNA concentrations and succeeded in detecting the core microbial community including the rare microbes. However, our study recommends the SL-Bead-Robotic eDNA extraction protocol because this method is safe, environmentally friendly, rapid, high-throughput and inexpensive.

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing

BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6–$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1–13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384–576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Application of a simple and high-throughput DNA extraction method to real-time PCR quantification of target plant-parasitic nematodes in nematode communities

Molecular techniques are prevailing in nematode identification and quantification, for which DNA extraction from nematodes is essential. However, the published DNA extraction methods are laborious and often require various expensive consumables and high-end equipment. In order to prepare DNA templates for a high-throughput real-time PCR assay, the present study modified a conventional DNA extraction method of Naklha et al. (2010) from nematode suspensions with ordinary lab equipment and achieved results and advantages comparable with two other conventional methods. The results of real-time PCR assays for quantifying Pratylenchus zeae, Tylenchorhynchus leviterminalis and Hoplolaimus sp. using the new protocol were highly correlated with those obtained by morphological counts and comparable to or more sensitive than those obtained by two conventional methods. Although the new protocol took over 100 min for DNA extraction, the manual processing took less than 10 min, i.e., half to one-fourth of the other methods. The running cost was less than half to one-tenth of the other methods.

Modeling microfluidic DNA extraction using superparamagnetic bead particles in COMSOL multiphysics simulation

High throughput DNA extraction method by superparamagnetic beads particles in microfluidic platform was studied using COMSOL multiphysics simulation software. The separation of DNA is based on solid phase microextraction method to isolate, fractionate and concentrate the analyte from complex sample matrices as well as implemented on microfluidic device with minimal procedures. The conceptual of the efficient flow design of microfluidic channel and the separation of DNA with superparamagnetic beads has shown in the COMSOL multiphysics simulation with complete calculation for every study steps. The purity of extracted DNA yield also has presented in the simulation and proved the sample can be used for further downstream process such as polymerase chain reaction analysis. Based on the simulation, 83.26 % of DNA yield able to be extracted using superparamagnetic beads and the rest of molecules are beads with absorbed DNA onto it. Even so, further DNA process typically, polymerase chain reaction possibly efficacious in the presence of superparamagnetic beads due to the addition of elution buffers in the following analysis.

A high-throughput DNA extraction method from rice seeds

ABSTRACTRapid and inexpensive preparation of genomic DNA from rice seeds for marker-assisted selection and seed purity estimation is a major bottleneck for plant breeders. Here, we describe a high-throughput method that provides DNA at sufficient quantity and quality for these applications. Optimization of buffer composition and individual protocol stages allow processing of 384 samples within 2 h, yielding templates that reliably support downstream polymerase chain reaction of single copy amplicons up to 1.2 kb.

Non-destructive high-throughput DNA extraction and genotyping methods for cotton seeds and seedlings.

Extensive use of targeted PCR-based genotyping is precluded for many plant research laboratories by the cost and time required for DNA extraction. Using cotton (Gossypium hirsutum) as a model for plants with medium-sized seeds, we report here manual procedures for inexpensive non-destructive high-throughput extraction of DNA suitable for PCR-based genotyping of large numbers of individual seeds and seedlings. By sampling only small amounts of cotyledon tissue of ungerminated seed or young seedlings, damage is minimized, and viability is not discernibly affected. The yield of DNA from each seed or seedling is typically sufficient for 1000 or 500 PCR reactions, respectively. For seeds, the tissue sampling procedure relies on a modified 96-well plate that is used subsequently for seed storage. For seeds and seedlings, the DNA is extracted in a strongly basic DNA buffer that is later neutralized and diluted. Extracts can be used directly for high-throughput PCR-based genotyping. Any laboratory can thus extract DNA from thousands of individual seeds/seedlings per person-day at a very modest cost for consumables (~$0.05 per sample). Being non-destructive, our approach enables a wide variety of time- and resource-saving applications, such as marker-assisted selection (MAS), before planting, transplanting, and flowering.

A high-throughput and cost-efficient DNA extraction protocol for the tree fruit crops of apple, sweet cherry, and peach relying on silica beads during tissue sampling

The basis of any marker-assisted seedling selection (MASS) scheme is having a high-throughput method of DNA extraction. To date, few high-throughput DNA extraction methods have been described for tree fruit. We describe a method that uses silica gel beads, which serve to both desiccate and grind leaf samples. This method provides a logistically flexible and time-efficient method for sample collection and routine production of PCR-quality DNA from apple, cherry, and peach. With a quick, non-laborious method of DNA extraction now being available, high-throughput MASS can be routinely conducted in breeding programs of apple, cherry, and peach to facilitate the development of superior cultivars.

Comparison of High-throughput and Manual DNA Extraction Methods for the Qualitative and Quantitative Analysis of MON810 Maize

PCR-based methods are widely used in the European Union and in other countries for the detection, identification, and quantification of genetically modified organisms (GMOs). The preparation of good-quality DNA from plant samples for GMO detection can be a challenging task, particularly if the DNA will be used for quantitative analysis. Two DNA extraction methods, namely manual (NucleoSpin Food kit from Machery-Nagel) and high-throughput partially automated (NucleoMag Plant kit from Machery-Nagel) methods, which utilize different DNA separation principles, were used for the isolation of DNA from maize flour samples. Despite the higher DNA recovery obtained using the high-throughput isolation method, a lower PCR efficiency was achieved, most likely due to the presence of PCR inhibitors in the extracts. We found both DNA extraction methods suitable for GMO analysis.

Cost-Effective and Scalable DNA Extraction Method from Dried Blood Spots

Dried blood spot (DBS) samples have been widely used in newborn screening (NBS) for the early identification of disease to facilitate the presymptomatic treatment of congenital diseases in newborns. As molecular genetics knowledge and technology progresses, there is an increased demand on NBS programs for molecular testing and a need to establish reliable, low-cost methods to perform those analyses. Here we report a flexible, cost-efficient, high-throughput DNA extraction method from DBS adaptable to small- and large-scale screening settings. Genomic DNA (g.DNA) was extracted from single 3-mm diameter DBS by the sequential use of red cell lysis, detergent-alkaline, and acid-neutralizing buffers routinely used in whole blood and plant tissue DNA extractions. We performed PCR amplification of several genomic regions using standard PCR conditions and detection methods (agarose gel, melting-curve analysis, TaqMan-based assays). Amplicons were confirmed by BigDye® Terminator cycle sequencing and compared with reference sequences. High-quality g.DNA was extracted from hundreds of DBS, as proven by mutation detection of several human genes on multiple platforms. Manual and automated extraction protocols were validated. Quantification of g.DNA by Oligreen® fluorescent nucleic acid stain demonstrated a normal population distribution closely corresponding with white blood cell counts detected in newborn populations. High-quality, amplifiable g.DNA is extractable from DBSs. Our method is adaptable, reliable, and scalable to low- and high-throughput NBS at low cost ($0.10/sample). This method is routinely used for molecular testing in the New York State NBS program.

A high throughput DNA extraction method from chemotypically heterogeneous plant species

A high throughput DNA extraction method from chemotypically heterogeneous plant species

Assessment of a fully automated high-throughput DNA extraction method from formalin-fixed, paraffin-embedded tissue for KRAS, and BRAF somatic mutation analysis

Preoperative biopsies or imbedded cytological cells will become more and more a primary source of tissue for molecular diagnostic analyses as a result of novel neo-adjuvant treatment regimens for several cancer types. Furthermore there is a growing need to examine metastatic cancer tissue. Hence, nucleic acids need to be reliably isolated and analyzed from small amounts of formalin-fixed and paraffin-embedded (FFPE) tissue. The limited numbers of (tumor) cells in these samples make high quality and sensitive DNA isolation challenging. Also demands for faster turnaround times are growing. Therefore, we evaluated a fully automated DNA/RNA isolation system and compared this with a manual, classical routine molecular pathology method. We compared the quality of the isolates from both tissue cores and micro-dissection for detection of hotspot mutations in KRAS, BRAF applying hydrolysis probe assays. In addition we determined whether the automated method decreases the hands-on-time and turnaround times in routine molecular pathology workflow.In conclusion, the automated method delivers high quality DNA from both small FFPE tissue cores and micro-dissected tissue material. In comparison to classical methods, less than 50% of starting tissue was sufficient as input for micro-dissection. Turnaround times decreased significantly and 50% less hands-on time was needed.

A high throughput DNA extraction method with high yield and quality

BackgroundPreparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome), and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L.) Moench] leaves and dry seeds with high yield, high quality, and affordable cost.ResultsWe developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles.ConclusionA high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

Assessment of DNA extraction methods for PCR testing of discontinued or unapproved biotech events in single seeds of canola, flax and soybean

The purpose of the study was to assess the suitability of three DNA extraction methods (Fast ID, SDS-based and relatively high-throughput methods) for single seeds (kernels) of canola, flax and broken pieces of soybean seeds. The extracted DNA was used for conventional and real-time qualitative PCR detection of the biotech events OXY235 canola, FP967 flax and DP305423 soybean. The mean weight of single canola and flaxseeds ranged from 2.3 to 3.2 mg and 5.5 to 5.8 mg, respectively. For canola, mean total DNA yields of 216, 796 and 377 ng per seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. For flax, mean total DNA yields of 329, 826 and 957 ng per seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. For soybean, mean DNA yields of 58, 102 and 175 ng per mg of broken seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. Comparison of mean DNA yields indicated statistically significant differences among the three DNA extraction methods for all the three grain types. There was either weak or no correlation between seed weight and DNA yield for all the three DNA extraction methods. Abs260/280 ratio of ≥1.8 was obtained for DNA extracted with Fast ID and SDS-based methods. DNA isolated with all three extraction methods had low Abs260/230 ratios indicating the presence of contaminants that absorb at 230 nm. Consistent and repeatable PCR amplification was obtained for DNA extracted with the Fast ID and SDS-based extraction methods. Inhibition of PCR was observed for flax DNA extracted using relatively high-throughput method; however, reducing the amount of DNA to 10 ng in the PCR resulted in consistent amplification. Overall, all three DNA extraction methods can be used for fast DNA-based detection of unapproved or discontinued biotech events in single seeds of canola and flax as well as broken pieces of soybean seeds.

Detection of Spirorchiid Trematodes in Gastropod Tissues by Polymerase Chain Reaction: Preliminary Identification of an Intermediate Host of Learedius learedi

Marine spirorchiid trematodes are associated with morbidity and mortality in sea turtles worldwide. The intermediate hosts remain unknown, and discovery efforts are hindered by the large number and great diversity of potential hosts within sea turtle habitats, as well the potential for low prevalence and overdispersion. A high-throughput DNA extraction and polymerase chain reaction-based method was developed to detect the internal transcribed spacer 2 (ITS2) region of the ribosomal gene of 2 spirorchiid genera, Learedius and Hapalotrema , within pooled samples of gastropod tissues. A model system consisting of freshwater snail ( Pomacea bridgesii ) tissues and DNA extracts spiked with adult Learedius learedi and known quantities of spirorchiid DNA was used to develop and test the technique. Threshold of detection was found to be equivalent to an early prepatent infection within 1.5 g of gastropod tissue. This technique was used to screen approximately 25 species of marine gastropods at a captive facility where green turtles ( Chelonia mydas ) become infected by L. learedi . The parasite was detected in a sample of knobby keyhole limpet ( Fissurella nodosa ), thus providing the first evidence of an intermediate host for a marine spirorchiid trematode. This technique has many potential applications in trematode life cycle discovery studies.