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BACKGROUND: The on‐site molecular detection of plant pathogens is particularly important for the development of sustainable agriculture. Extracting DNA from plant tissues, microbes or coexisting environments is complex, labor‐intensive and time‐consuming. To facilitate this process, we propose a DNA purification strategy based on graphene oxide (GO). RESULTS: The excellent adsorption ability of GO was verified by visualizing changes in its microscopic surface and macroscopic mixture. To further optimize the DNA purification, we determined the optimal GO concentration and treatment time at 95 °C (2 mg mL−1 and 2 min, respectively). We confirmed that our strategy is effective on plant tissues and various microorganisms, and that the obtained DNA can be directly used for polymerase chain reaction amplification. Combining the proposed GO‐based DNA purification method with the loop‐mediated isothermal amplification method is superior, in terms of the required steps, time, cost and detection effect, to the cetyltrimethylammonium bromide method and a commercial kit for detecting plant pathogens. CONCLUSION: We present a feasible, rapid, simple and low‐cost DNA purification method with high practical value for scientific applications in plant pathogen detection. This strategy can also provide important technical support for future research on plant–microbial microenvironments. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Copyright of Baghdad Science Journal is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0–3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 μg/μL or 0.4 μg/μL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR's capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA. [ABSTRACT FROM AUTHOR]

An automated flow chemistry platform for DNA-encoded library (DEL) technologies requires the integration of a purification process for DNA-tagged substrates. It facilitates the development of further DEL reactions, building block rehearsal, and library synthesis. Therefore, a recently developed, manual affinity-based batch purification process for DNA-tagged substrates based on dispersive solid-phase extraction (DSPE) was transferred to automated flow chemistry using tailored 3D-printed microfluidic devices and open-source lab automation equipment. The immobilization and purification steps use Watson–Crick base pairing for a compound-encoding single-stranded DNA, which allows for the thorough removal of impurities and contaminations by washing steps and operationally simple recovery of the purified DNA-encoded compounds. This work optimized the annealing step for flow incubation and DNA purification was accomplished by flow DSPE washing/elution steps. The manually performed batch affinity-based purification process was compared with the microfluidic process by determining qualitative and quantitative DNA recovery parameters. It aimed at comparing batch and flow purification processes with regard to DNA recovery and purity to benefit from the high potential for automation, precise process control, and higher information density of the microfluidic purification process for DNA-tagged substrates. Manual operations were minimized by applying an automation strategy to demonstrate the potential for integrating the microfluidic affinity-based purification process for DNA-tagged substrates into an automated DNA-encoded flow chemistry platform. [ABSTRACT FROM AUTHOR]

In this study, one approach for synthesizing γ-Fe2O3@SiO2 nanoparticles using sodium silicate (Na2SiO3) as a precursor is investigated. To prepare activation sites for grafting the SiO2 network onto the magnetic particles, a presurface treatment involving an acid or base is utilized. The effects of pH on the surface charge and thickness of the coating layer on the γ-Fe2O3@SiO2 nanoparticles are analyzed. The optimal pH for achieving the best surface charge properties and dispersibility is identified and used for subsequent experiments. The resulting γ-Fe2O3@SiO2 nanoparticles exhibit improved dispersibility and are evaluated for plasmid deoxyribonucleic acid (DNA) purification. The result shows that their performance is comparable to that of nanoparticles prepared using conventional tetraethyl orthosilicate (TEOS). Residual Na ions on the particle surface can reduce the plasmid DNA purification efficiency. Therefore, additional acid treatment is performed to remove residual Na ions. The correlation between the surface charge and interparticle forces is examined by considering the presence or absence of residual Na ions. The zeta potential and particle size distribution of the γ-Fe2O3@SiO2 nanoparticles increase as the purification efficiency improves. In conclusion, γ-Fe2O3@Na2SiO3 nanoparticles are successfully fabricated by using Na2SiO3 by removing Na ions via an additional acid treatment. This approach is a promising alternative to conventional TEOS-based methods for synthesizing γ-Fe2O3@SiO2 nanoparticles with improved dispersibility and purification efficiency. [ABSTRACT FROM AUTHOR]

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0–3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 μg/μL or 0.4 μg/μL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR’s capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA.

Copyright of Biology Teaching is the property of East China Normal University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

DNA extraction is a crucial step in all nucleic acid-based protocols to identify microorganisms. Lactic acid bacteria are a significant part of healthy microbiota in the human gastrointestinal tract. These gram-positive bacteria have several layers of peptidoglycan in the cell walls. These structures cause difficulties in the cell lysis and obtaining reliable protocols for DNA isolations. The purpose of this study was to assess the autoclave and lysozyme-based DNA purification approaches for achieving the high-quality genomic DNAs of Lactobacillus acidophilus bacteria. DNA concentrations and qualities were also compared with the commercial kit. The results showed that the proper DNA isolation methods were various, according to the downstream applications. Protocols that included lysozyme produced a higher amount of DNA than the autoclave method. Lysozyme treatment combined with silica-guanidinethiocyanate procedure was the efficient protocol with affordable cost for routine lysis of L. acidophilus bacteria. Appropriate DNA concentration and quality were obtained through this method comparable to those of the commercial kit. Inversely, autoclave treatment had little effect on the breakage of the cell walls indicating low concentrations of extracted DNAs. This method could not completely break down all the bacterial cell walls. However, the breakage of low numbers of cell walls was microscopically observed in the supernatant of the autoclaved cell suspension. The quality of this protocol was found to be adequate for performing direct polymerase chain reaction (PCR) assay on samples with large amounts of lactobacilli. These conclusions suggest attentively selecting the DNA extraction method based on the planned downstream analysis of PCR products.

BackgroundCharacterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples. While sample preparation from other microbiomes (e.g., gut) has been the subject of extensive optimization, procedures for skin and wound microbiomes have received relatively little attention. Here we describe an improved method for obtaining both phage and microbial DNA from a single skin or wound swab, characterize the yield of DNA in model samples, and demonstrate the utility of this approach with samples collected from a wound clinic.ResultsWe find a substantial improvement when processing wound samples in particular; while only one-quarter of wound samples processed by a traditional method yielded sufficient DNA for downstream analysis, all samples processed using the improved method yielded sufficient DNA. Moreover, for both skin and wound samples, community analysis and viral reads obtained through deep sequencing of clinical swab samples showed significant improvement with the use of the improved method.ConclusionUse of this method may increase the efficiency and data quality of microbiome studies from low-biomass samples.

High-quality DNA extraction plays an important role to make sharp bands in the gel electrophoresis and also produces clean chromatograms. Usually, DNA extract is delivered using the modified CTAB method but this method cannot obtain high-quality DNA for molecular analysis from old dried leaves of Bellevalia due to having different chemical compounds which inhibit to obtain a clear DNA extraction. To solve the problem, in different phases of a modified CTAB protocol, activated carbon, phenol, potassium acetate, RNase and deionized water (instead of TE) were applied to eliminate contaminants and improve obtained high-quality DNA. The DNA absorption data from the modified method were compared with CTAB method in 260 and 280 nm wavelengths. The results showed a 15-80-fold change in the DNA concentration. This study suggests the modified method of extraction is more efficient in the quality of DNA obtained from the little amount of herbarium old leaves of Bellevalia than that of the CTAB method.

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution. [ABSTRACT FROM AUTHOR]

Globally, yield losses associated with failed crop protection due to fungicide-resistant pathogens present an increasing problem. For stubble-borne pathogens, assessment of crop residues during the off-season could provide early fungicide resistance quantification for informed management decisions to mitigate yield losses. However, stubble assessment is hampered by assay inhibitors that are derived from decaying organic matter. To overcome assay inhibition from weathered stubble samples, we used a systems approach to quantify the frequency of resistance to demethylase inhibitor fungicides of the barley pathogen Pyrenophora teres f. teres. The system canvassed (i) 10 ball-milling conditions; (ii) four DNA extraction methodologies; and (iii) three column purification techniques for the provision of sufficient yield, quality, and purity of fungal DNA for a PCR-based fungicide resistance assay. Results show that DNA quantity and purity differed within each of the above three categories, with the optimized pipeline being (i) ball-milling samples in a 50-ml stainless steel canister for 5 min using a 20-mm ball at 30 revolutions s−1; (ii) a modified Brandfass method (extracted 64% more DNA than other methods assessed); and (iii) use of silica resin columns for the highest DNA concentration with optimal DNA purity. The chip-digital PCR assay, which quantified fungicide resistance from field samples, was unaffected by the DNA extraction method or purification technique, provided that thresholds of template quantity and purity were satisfied. In summary, this study has developed molecular pipeline options for pathogen fungicide resistance quantification from cereal stubbles, which can guide management for improved crop protection outcomes. [ABSTRACT FROM AUTHOR]

DNA extraction is a crucial step in all nucleic acid-based protocols to identify microorganisms. Lactic acid bacteria are a significant part of healthy microbiota in the human gastrointestinal tract. These gram-positive bacteria have several layers of peptidoglycan in the cell walls. These structures cause difficulties in the cell lysis and obtaining reliable protocols for DNA isolations . The purpose of this study was to assess the autoclave and lysozyme-based DNA purification approaches for achieving the high-quality genomic DNAs of Lactobacillus acidophilus bacteria. DNA concentrations and qualities were also compared with the commercial kit. The results showed that the proper DNA isolation methods were various, according to the downstream applications. Protocols that included lysozyme produced a higher amount of DNA than the autoclave method. Lysozyme treatment combined with silica -guanidinethiocyanate procedure was the efficient protocol with affordable cost for routine lysis of L. acidophilus bacteria. Appropriate DNA concentration and quality were obtained through this method comparable to those of the commercial kit. Inversely, autoclave treatment had little effect on the breakage of the cell walls indicating low concentrations of extracted DNAs. This method could not completely break down all the bacterial cell walls. However, the breakage of low numbers of cell walls was microscopically observed in the supernatant of the autoclaved cell suspension. The quality of this protocol was found to be adequate for performing direct polymerase chain reaction (PCR) assay on samples with large amounts of lactobacilli. These conclusions suggest attentively selecting the DNA extraction method based on the planned downstream analysis of PCR products. [ABSTRACT FROM AUTHOR]

Conventional and commercially available DNA extraction methods have several limitations regarding, for instance, contamination, and complex and slow precipitation and recovery processes. Herein, we report the synthesis of oxygen and phosphorus-doped Graphitic carbon nitride structures (g-POCN), via a novel Zinc-catalyzed one-pot solvothermal approach, and its application in the extraction of genomic DNA (gDNA) from a vegetal matrix (P. argentatum). Experimental and molecular modeling analyses demonstrate the high affinity of gDNA with g-POCN, which provided highly efficient gDNA extraction processes, with extraction yield, as well as integrity and quality of the extracted gDNA, comparable or superior to a commercial extraction kit and isopropanol extraction. Moreover, under suitable elution conditions, this method allows the easy removal of high concentrations of gDNA from g-POCN, rendering this method as a low-cost, simple, and fast approach for the extraction of even small amounts of gDNA. Remarkably, the extracted gDNA shows no degradation, and no inhibition of the polymerase chain reaction. Therefore, g-POCN represents a promising material for the highly efficient, cost-effective, and biocompatible extraction of DNA, which could stimulate research focused on broad DNA sources, e.g., RNA extraction, plasmids, ssDNA, etc. [ABSTRACT FROM AUTHOR]

DNA purification methods are indispensable tools of molecular biology, used for many decades. Nevertheless, for certain specialized applications, the currently employed techniques are not sufficiently effective. While examining a number of the existing methods to purify the genomic DNA of the thermophilic bacteriophage TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus), we have found out that the obtained DNA is contaminated with trace amounts of infectious TP-84 particles. This was detrimental for the bacteriophage genetic manipulation purposes, as finding the recombinant TP-84 clones was essentially impossible due to the appearance of a high background of native bacteriophage plaques. Thus, we have developed a method, which enables the fast and efficient isolation of a bacteriophage genomic DNA from concentrated phage preparations, obtained using CsCl gradient ultracentrifugation, without the need to remove concentrated CsCl solutions. The method employs silica columns and mini-scale isolation of microgram amounts of high quality DNA. It is universal—the silica mini-columns from various manufacturers can be used to conduct the procedure. The purified DNA, free from infectious bacteriophage particles, is ready for further manipulations. This is particularly important for such thermophilic bacteriophages that may partially survive standard isolation procedures and contaminate the final DNA product. [ABSTRACT FROM AUTHOR]

High-quality DNA is an important guarantee to start downstream experiments in many biological and medical research areas. Magnetic particle-based DNA extraction methods from blood mainly depend on electrostatic adsorption in a low-pH environment. However, the strong acidic environment can influence the DNA stability. Herein, a polydopamine-functionalized magnetic particle (PDA@Fe 3 O 4)-based protocol was developed for DNA extraction from whole blood samples. In the protocol, Mg2+ and Ca2+ were utilized to bridge the adsorption of DNA by PDA@Fe 3 O 4 via the metal-mediated coordination. Isopropanol was found to efficiently promote DNA adsorption by triggering the change of the conformation of DNA from B-form to more compact A-form. In 50 % isopropanol solution, the DNA adsorption efficiency was nearly 100 % in the presence of 0.5 mM Ca2+ or 1.5 mM Mg2+. The role of metal ions and isopropanol in DNA adsorption was explored. The protocol averts the strong acidic environment and PCR inhibitors, such as high concentrations of salt or polyethylene glycol. It demonstrates superiority in DNA yield (59.13 ± 3.63 ng μL−1) over the commercial kit (27.33 ± 4.98 ng μL−1) and phenol-chloroform methods (37.90 ± 0.47 ng μL−1). In addition, to simplify the operastion, an automated nucleic acid extraction device was designed and fabricated to extract whole genomic DNA from blood. The feasibility of the device was verified by extracting DNA from cattle and pig blood samples. The extracted DNA was successfully applied to discriminate the beef authenticity by a duplex PCR system. The results demonstrate that the DNA extraction protocol and the automated device have great potential in blood samples. [Display omitted] • PDA@Fe 3 O 4 was used to purify DNA from blood based on metal-mediated coordination. • Isopropanol promotes DNA adsorption by triggering the change of DNA conformation. • The protocol demonstrates absolute superiority in DNA yield over other methods. • An automated device was fabricated to simplify DNA extraction. • The platform combined with PCR was applied to identify beef authenticity. [ABSTRACT FROM AUTHOR]

Foodborne diseases represent a significant global health concern, with a substantial number of illnesses and fatalities reported annually owing to the consumption of contaminated food. Current methods for identifying foodborne pathogens cannot effectively distinguish live from dead bacteria or efficiently detect multiple targets. Therefore, we developed a selective detection method for three pathogenic bacteria in food samples that leverages bacteriophage-assisted live-cell lysis and exposed genomic DNA amplification. The antimicrobial activity and host specificity of these three bacteriophages were employed to confirm the target bacteria selectivity. Bacterial DNA purification using bacteriophage-assisted lysis demonstrated the superior activity of the selected phages in extracting DNA from Escherichia coli O157:H7 and Salmonella Typhimurium. The results from reactions involving the three strains, including Bacillus cereus , highlighted that a 1 h incubation period yielded the most efficient DNA extraction and purification. The polymerase chain reaction (PCR) products obtained were comparable in terms of concentration and purity with those generated using bacterial DNA prepared using a commercial kit. In the practical food sample application experiment, bacterial strains were successfully recovered and concentrated using a polyethersulfone filter, resulting in a concentration factor >3 for all samples. Furthermore, the concentration of purified bacterial DNA from contaminated kimchi cabbage exceeded 10 ng/μL. Notably, we could detect E. coli O157:H7, S. Typhimurium, and B. cereus spiked at 103 colony-forming units/mL using multiplex PCR. This innovative bacteriophage-based approach holds substantial promise for the selective and practical detection of live foodborne pathogens, thereby significantly advancing food safety standards. • A selective detection method for pathogenic bacteria in food samples is proposed. • Bacteriophage-assisted live-cell lysis and genomic DNA amplification are leveraged. • Selected phages exhibited potent ability to extract DNA from pathogenic bacteria. • E. coli , S. Typhimurium, and B. cereus strains were detected using multiplex PCR. • This novel bacteriophage-based approach can help advance food safety standards. [ABSTRACT FROM AUTHOR]

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

Conventional and commercially available DNA extraction methods have several limitations regarding, for instance, contamination, and complex and slow precipitation and recovery processes. Herein, we report the synthesis of oxygen and phosphorus-doped Graphitic carbon nitride structures (g-POCN), via a novel Zinc-catalyzed one-pot solvothermal approach, and its application in the extraction of genomic DNA (gDNA) from a vegetal matrix (P. argentatum). Experimental and molecular modeling analyses demonstrate the high affinity of gDNA with g-POCN, which provided highly efficient gDNA extraction processes, with extraction yield, as well as integrity and quality of the extracted gDNA, comparable or superior to a commercial extraction kit and isopropanol extraction. Moreover, under suitable elution conditions, this method allows the easy removal of high concentrations of gDNA from g-POCN, rendering this method as a low-cost, simple, and fast approach for the extraction of even small amounts of gDNA. Remarkably, the extracted gDNA shows no degradation, and no inhibition of the polymerase chain reaction. Therefore, g-POCN represents a promising material for the highly efficient, cost-effective, and biocompatible extraction of DNA, which could stimulate research focused on broad DNA sources, e.g., RNA extraction, plasmids, ssDNA, etc.

DNA purification methods are indispensable tools of molecular biology, used for many decades. Nevertheless, for certain specialized applications, the currently employed techniques are not sufficiently effective. While examining a number of the existing methods to purify the genomic DNA of the thermophilic bacteriophage TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus), we have found out that the obtained DNA is contaminated with trace amounts of infectious TP-84 particles. This was detrimental for the bacteriophage genetic manipulation purposes, as finding the recombinant TP-84 clones was essentially impossible due to the appearance of a high background of native bacteriophage plaques. Thus, we have developed a method, which enables the fast and efficient isolation of a bacteriophage genomic DNA from concentrated phage preparations, obtained using CsCl gradient ultracentrifugation, without the need to remove concentrated CsCl solutions. The method employs silica columns and mini-scale isolation of microgram amounts of high quality DNA. It is universal—the silica mini-columns from various manufacturers can be used to conduct the procedure. The purified DNA, free from infectious bacteriophage particles, is ready for further manipulations. This is particularly important for such thermophilic bacteriophages that may partially survive standard isolation procedures and contaminate the final DNA product.

Genomic analysis of circulating, cell-free DNA (cfDNA) is being used extensively for molecular diagnostics. Many approaches rely on the construction of cfDNA genomic libraries, targeted retrieval of specific genomic regions and analysis by next-generation DNA sequencing. Several steps during sample preparation require isolation of DNA fragments within a particular size range. In this Benchmark article, two related methods for size-selective DNA fragment enrichment are described.

Abstract Background Characterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples. While sample preparation from other microbiomes (e.g., gut) has been the subject of extensive optimization, procedures for skin and wound microbiomes have received relatively little attention. Here we describe an improved method for obtaining both phage and microbial DNA from a single skin or wound swab, characterize the yield of DNA in model samples, and demonstrate the utility of this approach with samples collected from a wound clinic. Results We find a substantial improvement when processing wound samples in particular; while only one-quarter of wound samples processed by a traditional method yielded sufficient DNA for downstream analysis, all samples processed using the improved method yielded sufficient DNA. Moreover, for both skin and wound samples, community analysis and viral reads obtained through deep sequencing of clinical swab samples showed significant improvement with the use of the improved method. Conclusion Use of this method may increase the efficiency and data quality of microbiome studies from low-biomass samples.

Current mitochondrial purification techniques are tedious and protracted due to their emphasis on recovering physiologically active mitochondria. However, for studies that are exclusively interested in isolating mitochondrial DNA (mtDNA) for applications such as PCR and sequencing, respiring mitochondria − and the complex procedures that stem from the need to retain their function − are unnecessary. Still, global DNA extraction methods have proven insufficient for mitochondrial DNA isolation because nuclear mitochondrial DNA segments (NUMTs) pose unique challenges to accurate mtDNA quantification and characterization. We present a rapid and simple extraction technique that maximizes recovery of mitochondrial DNA from plant cells, while minimizing the presence of nuclear DNA. Through real-time PCR, we show that this method provides a significant increase in the enrichment of mitochondrial DNA compared to that of nuclear DNA in both Arabidopsis thaliana and Brassica rapa. This method has important implications for future mitochondrial DNA analyses as it possesses few procedural limitations and minimizes the analytical problems typically associated with mtDNA purification by other techniques.

We present a disposable polymeric microfluidic device capable of reversibly binding and purifying Salmonella DNA through solid phase extraction (SPE). The microfluidic channels are first oxygen plasma treated and simultaneously micro-nanotextured, and then functionalized with amine groups via modification with L-histidine or poly-L-histidine. L-Histidine and poly-L-histidine bind on the plasma treated chip surface, and are not detached when rinsing with DNA purification protocol buffers. A pH-dependent protocol is applied on-chip to purify Salmonella DNA, which is first bound on the protonated amines at a pH (5.0) lower than their pKa of surface amine-groups which is 6.0 and then released at a pH higher than the pKa value (10.5). It was found that modification with poly-L-histidine resulted in higher surface density of amine groups onto microfluidic channel. Using the chip modified with poly-L-histidine, high recovery efficiency of at least 550 ng of isolated Salmonella DNA as well as DNA purification from Salmonella cell lysates corresponding to less than 5000 cells or 0.026 ng of Salmonella DNA was achieved. The protocol developed does not require ethanol or chaotropic solutions typically used in DNA purification, which are known inhibitors for downstream operations such as polymerase chain reactions (PCR) and which can also attack some polymeric microfluidic materials. Therefore, the microfluidic device and the related protocol hold promise for facile incorporation in microfluidics and Lab-on-a-chip (LOC) platforms for pathogen detection or in general for DNA purification. [ABSTRACT FROM AUTHOR]

In vivo DNA engineering such as recombineering (recombination‐mediated genetic engineering) and DNA gap repair typically involve growing Escherichia coli (E coli) containing plasmids, followed by plasmid DNA extraction and purification prior to downstream PCR‐mediated DNA modifications and DNA sequencing. We previously demonstrated that crude cell lysates could be used for some limited downstream DNA applications. Here, we show how live E coli cell PCR and one‐step LiCl‐isopropanol purification can streamline DNA engineering. In DNA gap repair, live‐cell PCR allowed the convenient elimination of clones containing background plasmids prior to DNA sequencing. Live‐cell PCR also enabled the generation of specific DNA sequences for DNA engineering up to 11 kilo base pairs in length and with up to 80 base pair terminal non‐homology. Using gel electrophoresis and DNA melting curve analysis, we showed that LiCl‐isopropanol DNA precipitation removed primers and small, nonspecific PCR products from live‐cell PCR products in only ~10‐minutes. DNA sequencing of purified products yielded Phred quality scores values of ~55%. These data indicate that live‐cell PCR and LiCl‐isopropanol DNA precipitation are ideal to prepare DNA for sequencing and other downstream DNA applications, and might therefore accelerate high‐throughput DNA engineering pipelines. [ABSTRACT FROM AUTHOR]

A novel functional micropipette tip for DNA purification, called DNAtip, is described based on a novel coating process. The functional layer is prepared by an internal coating with silica particles supported into a polyvinyl alcohol (PVA) film. The coating layer is stabilized by simultaneous radiation-induced crosslinking to avoid its dissolution. Three types of silica particles of different diameters: micron, sub-micron, and nano sizes are studied. In addition, pH of the coating solution and concentration of the silica particles were optimized. Tips coated with fumed silica showed the highest absorption capacity of a plasmid DNA with an acceptable purity. Different linear dsDNA fragments were used for evaluating the absorption capacity and size discrimination of fragments. The recovery in the range of 700 ng and 200 ng per tip was measured for dsDNA fragments in the range of 400 bp to 2 kbp and 50 bp to 500 bp respectively. DNA PCR fragments contaminated with BSA were successfully purified with sufficient quality to be sequenced or enzymatically digested. This disposable tip device, useful for plasmids or DNA fragments purification, is a novel lab-on-a-tip device, which can effectively improve automate genetic engineering procedures. [ABSTRACT FROM AUTHOR]

[Ｏｂｊｅｃｔｉｖｅｓ]Ｔｈｉｓ ｓｔｕｄｙ ａｉｍｅｄ ｔｏ ｏｐｔｉｍｉｚｅ ｔｈｅ ｍｅｔｈｏｄ ｔｏ ｐｕｒｉｆｙ ＤＮＡ ｆｒｏｍ ｌｉｖｅｓｔｏｃｋ ｆｒｏｚｅｎ ｓｅｍｅｎ ｕｓｉｎｇ ａ ＤＮＡ ｋｉｔ ｄｅｓｉｇｎｅｄ ｆｏｒ ｈｕｍａｎ ｓｅｍｅｎ, ａｎｄ ｔｏ ｅｘｐｌｏｒｅ ｔｈｅ ｃｏｍｐｏｎｅｎｔｓ ｉｎｆｌｕｅｎｃｉｎｇ ｔｈｅ ｑｕａｌｉｔｙ ｏｆ ＤＮＡ ｅｘｔｒａｃｔｉｏｎ ｉｎ ｆｒｏｚｅｎ ｓｅｍｅｎ. [Ｍｅｔｈｏｄｓ]Ｆｒｏｚｅｎ ｓｅｍｅｎ ＤＮＡ ｆｒｏｍ Ｍｕｒｒａｈ ｂｕｆｆａｌｏ ｗａｓ ｅｘｔｒａｃｔｅｄ ｗｉｔｈ ａ ｋｉｔ, ａｃｃｏｒｄｉｎｇ ｔｏ １００ μＬ(ｏｒ ２００ μＬ)ｏｆ ｓｔａｒｔｉｎｇ ｓａｍｐｌｅ ｖｏｌｕｍｅ ａｓ ｗｅｌｌ ａｓ ０.５, １, １.５, ２, ３ ａｎｄ ５ ｈ ｐｒｏｔｅａｓｅ ｉｎｃｕｂａｔｉｏｎ ｔｉｍｅ. ＤＮＡ ｐｒｏｄｕｃｔｓ ｗｅｒｅ ａｎａｌｙｚｅｄ ｂｙ ｃｏｎｃｅｎｔｒａｔｉｏｎ, Ａ２６０ / Ａ２８０ ａｎｄ Ａ２６０ / Ａ２３０ ｉｎ ｃｏｍｂｉｎａｔｉｏｎ ｗｉｔｈ ａｇａｒｏｓｅ ｇｅｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ ａｎｄ ＰＣＲ ａｍｐｌｉｆｉｃａｔｉｏｎ ｔｏ ｄｅｔｅｒｍｉｎｅ ｔｈｅ ｂｅｓｔ ｓｔａｒｔｉｎｇ ｖｏｌｕｍｅ ａｎｄ ｐｒｏｔｅａｓｅ ｄｉｇｅｓｔｉｏｎ ｔｉｍｅ ｆｏｒ ＤＮＡ ｐｕｒｉｆｉｃａｔｉｏｎ. Ｔｈｅｎ ｆｒｏｚｅｎ ｓｅｍｅｎ ｆｒｏｍ ６７ ｄｉｆｆｅｒｅｎｔ ｂｏｖｉｎｅｓ, ｈｏｒｓｅｓ ａｎｄ ｄｏｎｋｅｙｓ ｂｒｅｅｄｓ ｗｅｒｅ ａｃｃｏｒｄｉｎｇｌｙ ｕｓｅｄ ｆｏｒ ＤＮＡ ｐｕｒｉｆｉｃａｔｉｏｎ. Ｔｈｅ ａｍｏｕｎｔｓ ｏｆ ｐｒｏｔｅｉｎ, ｇｌｙｃｅｒｏｌ/ ｔｒｉｇｌｙｃｅｒｉｄｅ ａｎｄ ｆｒｕｃｔｏｓｅ ｗｅｒｅ ｍｅａｓｕｒｅｄ, ａｎｄ ｆｒｏｚｅｎ ｔｉｍｅ ｗａｓ ｒｅｃｏｒｄｅｄ ｓｉｍｕｌｔａｎｅｏｕｓｌｙ ｆｏｒ ｅａｃｈ ｓａｍｐｌｅ ｔｏ ｉｎｖｅｓｔｉｇａｔｅ ｔｈｅ ｒｅｌａ- ｔｉｏｎｓｈｉｐｓ ｂｅｔｗｅｅｎ ｔｈｅｓｅ ｆａｃｔｏｒｓ ａｎｄ ＤＮＡ ｐｒｏｄｕｃｔｓ. [Ｒｅｓｕｌｔｓ] Ｔｈｅ ｒｅｓｕｌｔｓ ｓｈｏｗｅｄ ｔｈａｔ ＤＮＡ ｐｕｒｉｆｉｃａｔｉｏｎ ｓｔａｒｔｉｎｇ ｗｉｔｈ １００ μＬ ｆｒｏｚｅｎ ｓｅｍｅｎ ａｆｔｅｒ ３ ｈ ｐｒｏｔｅａｓｅ ｄｉｇｅｓｔｉｏｎ ｒｅｐｒｏｄｕｃｉｂｌｙ ｐｒｏｖｉｄｅｄ ｔｈｅ ｂｅｓｔ ｑｕａｌｉｔｙ ｏｆ ＤＮＡ, ａｎｄ ｌｏｎｇｅｒ ｐｒｏｔｅａｓｅ ｉｎｃｕｂａｔｉｏｎ ｓｈｏｗｅｄ ｎｏ ｓｉｇｎｉｆｉｃａｎｔ ｉｍｐｒｏｖｅｍｅｎｔ. Ｂｏｔｈ ｔｈｅ ＤＮＡ ｐｒｏｄｕｃｔｓ ａｔ ２ ａｎｄ ３ ｈ ｃｏｕｌｄ ｂｅ ｕｓｅｄ ｆｏｒ ｓｕｂｓｅｑｕｅｎｔ ｒｅｇｕｌａｒ ｎｕｃｌｅａｒ ｇｅｎｏｍｅ ａｎｄ ｍｉｔｏｃｈｏｎｄｒｉａｌ ｇｅｎｏｍｅ ｓｔｕｄｉｅｓ. Ｉｎ ａｄｄｉｔｉｏｎ, ｔｈｅ ａｍｏｕｎｔｓ ｏｆ ｐｒｏｔｅｉｎ, ｇｌｙｃｅｒｏｌ/ ｔｒｉｇｌｙｃｅｒｉｄｅ ａｎｄ ｆｒｕｃｔｏｓｅ ｆｒｏｍ ｌｉｖｅｓｔｏｃｋ ｆｒｏｚｅｎ ｓｅｍｅｎ ｗｅｒｅ ｎｅｇａｔｉｖｅｌｙ ｃｏｒｒｅｌａｔｅｄ&395;&395;ｗｉｔｈ ｔｈｅ ｃｏｎｃｅｎｔｒａｔｉｏｎ ａｓ ｗｅｌｌ ａｓ Ａ２６０ / Ａ２８０ ａｎｄ Ａ２６０ / Ａ２３０ ｐｕｒｉｔｉｅｓ ｏｆ ｅｘｔｒａｃｔｅｄ ＤＮＡ ｔｏ ｄｉｆｆｅｒｅｎｔ ｅｘｔｅｎｔｓ ｂｙ ａ Ｓｐｅａｒｍａｎ’ｓ ｃｏｒｒｅｌａｔｉｏｎ ａｎａｌｙｓｉｓ. Ａｍｏｎｇ ｔｈｅｓｅ ｆａｃｔｏｒｓ, ｆｒｕｃｔｏｓｅ ｗａｓ ａ ｋｅｙ ｆａｃｔｏｒ ｔｈａｔ ｉｎｔｅｒｆｅｒｅｄ ｗｉｔｈ ＤＮＡ ｐｕｒｉｆｉｃａｔｉｏｎ. Ｔｈｅｒｅ ｗａｓ ａｌｓｏ ａ ｓｉｇｎｉｆｉｃａｎｔ ｐｏｓｉｔｉｖｅ ｃｏｒｒｅｌａｔｉｏｎ ｂｅｔｗｅｅｎ ｆｒｏｚｅｎ ｔｉｍｅ ａｎｄ ＤＮＡ ｑｕａｌｉｔｙ. Ａｌｔｈｏｕｇｈ ｐｒｏｔｅｉｎ, ｇｌｙｃｅｒｏｌ/ ｔｒｉｇｌｙｃｅｒｉｄｅ, ａｎｄ ｆｒｕｃｔｏｓｅ ｐｒｅｖｅｎｔ ｓｐｅｒｍ ｆｒｏｍ ｄａｍａｇｅ ｄｕｒｉｎｇ ｆｒｅｅｚｉｎｇ ａｎｄ ｔｈａｗｉｎｇ, ｅｌｅｖａｔｉｎｇ ｔｈｅ ａｍｏｕｎｔ ｏｆ ｔｈｅｓｅ ｃｏｍｐｏｎｅｎｔｓ ｃｏｕｌｄ ｈａｖｅ ａ ｎｅｇａｔｉｖｅ ｉｍｐａｃｔ ｏｎ ＤＮＡ ｐｕｒｉｆｉｃａｔｉｏｎ, ｅｖｅｎ ｉｆ ｔｈｅｓｅ ｆａｃｔｏｒｓ ｃｏｕｌｄ ｂｅ ａｍｅｌｉｏｒａｔｅｄ ｂｙ ｐｒｏｌｏｎｇａｔｉｏｎ ｏｆ ｆｒｏｚｅｎ ｔｉｍｅ ａｎｄ ｄｉｇｅｓｔｉｏｎ ｔｉｍｅ ｄｕｒｉｎｇ ＤＮＡ ｐｕｒｉｆｉｃａｔｉｏｎ. [Ｃｏｎｃｌｕｓｉｏｎｓ] Ｔｈｅ ｈｕｍａｎ ｓｅｍｅｎ ＤＮＡ ｋｉｔ ｍｅｔｈｏｄ ｃａｎ ｂｅ ｆｕｌｌｙ ａｐｐｌｉｅｄ ｔｏ ｐｕｒｉｆｙｉｎｇ ＤＮＡ ｆｒｏｍ ｌｉｖｅｓｔｏｃｋ ｆｒｏｚｅｎ ｓｅｍｅｎ ａｆｔｅｒ ｏｐｔｉｍｉｚａｔｉｏｎ. [ABSTRACT FROM AUTHOR]

Abstract Background Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. Results We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. Conclusions This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Agarose gel electrophoresis is performed routinely by molecular biologists as both an analytical and a preparative method for characterization of nucleic acids. Gel analysis of highly dilute DNA solutions is challenging because of the limited sensitivity of detection available with conventional methods. In this study a new approach is described for concentrating samples directly within gels called SURE (su ccessive re loading) electrophoresis. The approach involves loading of dilute samples multiple times into a single well, with each loading followed by a brief pulse of electrical current before the next sample is loaded. The procedure generates single bands created by molecular stacking that exhibit strongly enhanced signal intensities and minimal band broadening. Using optimized voltages and time intervals as many as 20 successive loadings could be performed and up to 800 μL could be loaded into a single well. Gel extraction and fluorescent quantitation demonstrated that approximately 97 % of the DNA from each loading was incorporated into the resultant band. Highly dilute DNA samples (<0.0007 ng per microliter) could be readily detected after six loadings. The method produced good results with either TAE or TBE as electrophoresis buffers, using loading dyes with or without SDS, and in both minigels and large gels. [Display omitted] • Conventional methods for DNA analysis using gel electrophoresis have limited sample loading and detection capabilities. • A new approach involving successive reloading produces strong signal amplification with minimal band broadening. • Up to twenty successive loadings are possible using optimized voltages and time intervals. • Band quality is high using either of the two most common electrophoresis buffers, TAE or TBE. • Previously undetectable DNAs in highly dilute samples are readily detectable with the new method. [ABSTRACT FROM AUTHOR]

Bridging-induced aggregation of individual nanozyme particles by long-chain biomacromolecules causes loss of peroxidase mimetic activity for various nanozymes. When a common nanozyme (Fe 3 O 4 nanoparticles) was used for colorimetric assay of a long-chain biomacromolecule, human genomic DNA (hg DNA, 14.000 kDa, containing 22 kilobases), peroxidase-like activity was absent owing to the irreversible aggregation of Fe 3 O 4 nanoparticles in the presence of hg DNA. We synthesized an aggregation-resistant nanozyme containing Fe 3 O 4 nanoparticles immobilized in 5.1 μm monodisperse-porous silica microspheres. Fe 3 O 4 nanoparticles were immobilized in a form accessible to the substrate and large biomolecules in the solution within the monodispersed silica microspheres including both mesopores and macropores. An appreciable and stable spectrophotometric response was obtained, originating from the satisfactorily high peroxidase-like activity of the synthesized nanozyme. No aggregation was observed in the aqueous dispersion of the nanozyme in the presence of hg DNA. Large particle size, low particle number density, high surface area, and the presence of macropores were evaluated as factors contributing to the adsorption of hg DNA chains onto the synthesized nanozyme without interparticle bridge formation between the individual microspheres causing aggregation. Here, for the first time a colorimetric assay was developed based on the enhancement of peroxidase-mimetic activity of non-aggregated porous silica microspheres to determine hg DNA concentration up to 300 ng/μL. hg DNA could be also isolated with 47% yield and an equilibrium hg DNA adsorption of 19.000 ng/mg, using the same nanozyme. Hence, a material acting as a nanozyme for colorimetric determination of hg DNA was also evaluated as a magnetic, solid phase extraction sorbent for the first time. [ABSTRACT FROM AUTHOR]

Destructive maceration, a wide host range, and longevity in non-plant substrates has established Dickeya dianthicola (blackleg of potato) as a significant threat to potato industries worldwide. To protect these businesses, a specific and sensitive point-of-care D. dianthicola detection tool is necessary. We have developed a loop-mediated isothermal amplification (LAMP) assay for specific, sensitive, and rapid detection of D. dianthicola, which can be streamlined for point-of-care use. The developed LAMP assay targets a unique gene, alcohol dehydrogenase, of D. dianthicola. Assay specificity was assessed using strains present in inclusivity (16 D. dianthicola strains) and exclusivity panels (56 closely related, potato pathogenic, and other bacterial strains). Amplification with strains of inclusivity panel occurred, and cross-reactivity with non-target DNA was not observed. The limit of detection (LOD) was 10 CFU/ml when dilutions were made before isolating the genomic DNA; however, LOD was determined as 1 pg using 10-fold serially diluted D. dianthicola genomic DNA. Similar LOD of 1 pg was observed when serially diluted target genomic DNA was mixed with host genomic DNA. LOD (1 pg) was also calculated with 10-fold serially diluted synthetic DNA fragments containing primer target sites. Naturally and artificially inoculated plant samples were used for field adaptability tests with the field-deployable Optigene Plant Material Lysis Kit and a heat block (65°C); the results were obtained within 20 minutes. Despite the lack of method precision, no false positives or false negatives were observed. Therefore, with prepared reactions and a steady heat source, this assay can be used for rapid point-of-care detection, which is imperative for quarantine, eradication, disease management, and border protection. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is the most common curable sexually transmitted infection (STI) worldwide. Although predominately asymptomatic, the disease spectrum of trichomoniasis in women is characterized primarily by signs and symptoms of vaginitis, including purulent discharge and localized vulvar pruritus and erythema. Several FDA-cleared nucleic acid amplification tests (NAATs) are available for the diagnosis of T. vaginalis infections, but laboratory developed tests (LDTs) are widely utilized and cost-effective solutions in both the research and clinical diagnostic settings. LDT diagnosis of T. vaginalis is particularly appealing since it can be performed using remnant specimens collected for other STI testing. Using a LDT implemented as part of this study, T. vaginalis was detected in 7% of participating Louisiana women (14/199). The mean T. vaginalis organism burden was 1.0x106 ± 4.5x105 organisms per mL of ThinPrep PreservCyt. Using DNA eluates obtained after HPV testing on the cobas 4800 system, the T. vaginalis LDT was characterized by excellent intra- and interassay reproducibility (coefficient of variation values all <3.5%). Compared with two commercially available NAATs from TIB MOLBIOL, the sensitivity and specificity of the LDT was 92.9 and 99.5%, respectively. Collectively, this study details the diagnostic and quantitative utility of a LDT for T. vaginalis. When applied in the clinical research setting, we confirmed the high prevalence of T. vaginalis, but also observed extraordinarily high organism burdens in the cervix. These findings highlight the unique host-pathogen relationship of T. vaginalis with lower reproductive tract tissues, and substantiate the need for continued investigation of this highly prevalent STI. [ABSTRACT FROM AUTHOR]

In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Fabrication, and evaluation of an integrated Lab-on-Chip platform. • Bacteria capture, thermal lysis, DNA purification, and isothermal DNA amplification on chip. • Pathogen screening in milk samples for cell lysates corresponding to <500 cells. Abstract Strict regulations govern food safety as the latter is of extremely high importance and a major public issue, while it is demanding in respect of analytical effort. The duration of common food analysis practices is typically 2–5 days. Microfluidics can reduce labor and analysis time to a few hours, proving its beneficial use and rendering the response of food industry as well as public safety agents faster. The aim of the present work is the fabrication and evaluation of a modular integrated Lab-on-Chip platform for sample preparation, accommodating bacteria capture, thermal lysis, DNA purification, and isothermal DNA amplification, addressing pathogen screening in milk samples for cell lysates corresponding to <500 cells. We note that individual modules have been tested to work down to 10 cells. [ABSTRACT FROM AUTHOR]

Aims: This study evaluated the effects of DNA extraction method, DNA purification and pooling of PCR amplification products on the description of bacterial and archaeal diversity. Methods and Results: Soil DNA was extracted by the Power Soil DNA extraction kit and a customized Griffiths' protocol. Both methods are based on cell disruption by bead beating. In total, we used three soils and six independent extractions from each soil obtained by each of the two methods. Then, three of the six extracts of each treatment were further purified by spin columns filled with Sepharose 2B and polyvinylpolypyrrolidone (PVPP). The V4 hypervariable region of the 16S rRNA gene was amplified from each extract using the 515F/806R primer pair in four independent reactions. Three amplification products were combined and sequenced as a pooled sample, while the additional amplification product was sequenced individually. The resulting 72 amplification products were sequenced by Illumina MiSeq platform. DNA extraction method had a statistically significant effect on the estimation of the composition of microbial communities that might overwhelm differences in microbial communities from distinct soils. On the other hand, a further DNA purification step or pooling of PCR amplification products had a minor effect on the description of bacterial and archaeal communities. Conclusions: DNA extraction had the strongest effect on the description of bacterial and archaeal communities; low concentration of impurities, which allow PCR amplification, can still generate a minor additional bias, while PCR stochastic variability had the lowest effect. Significance and Impact of the Study: Although it is well known that methodological factors affect the description of microbial communities, the relative importance of each step is still unknown. The present study determined that of the factors tested, the DNA extraction method had the strongest effects on the description of bacterial and archaeal communities. [ABSTRACT FROM AUTHOR]

Telomerase-independent ALT (alternative lengthening of telomeres) cells are characterized by high frequency of telomeric homologous recombination (HR), C-rich extrachromosomal circles (C-circles) and C-rich terminal 5' overhangs (C-overhangs). However, underlying mechanism is poorly understood. Here, we show that both C-circle and C-overhang form when replication fork collapse is induced by strand break at telomeres. We find that endogenous DNA break predominantly occur on C-rich strand of telomeres in ALT cells, resulting in high frequency of replication fork collapse. While collapsed forks could be rescued by replication fork regression leading to telomeric homologous recombination, those unresolved are converted to C-circles and C-overhang at lagging and leading synthesized strand, respectively. Meanwhile, multiple hallmarks of ALT are provoked, suggesting that strand break-induced replication stress underlies ALT. These findings provide a molecular basis underlying telomeric HR and biogenesis of C-circle and C-overhang, thus implicating the specific mechanism to resolve strand break-induced replication defect at telomeres in ALT cells. [ABSTRACT FROM AUTHOR]

Urine holds great promise as a non-invasive sampling method for molecular diagnostics. The cell-free nucleic acids of urine however are small, labile, and difficult to purify. Here an efficient method for the purification of these nucleic acids is presented. An empirically derived protocol was devised by first identifying conditions that allowed recovery of a 100 base pair (bp) DNA, followed by optimization using a quantitative polymerase chain reaction (qPCR) assay. The resulting method efficiently purifies both small sized DNAs and RNAs from urine, which when combined with quantitative reverse transcription PCR (qRTPCR), demonstrably improves detection sensitivity. Fractionation experiments reveal that nucleic acids in urine exist both in the cell-free and cellular fraction, roughly in equal proportion. Consistent with previous studies, amplicons > 180bp show a marked loss in PCR sensitivity for cell-free nucleic acids. Finally, the lysis buffer developed here also doubles as an effective preservative, protecting against nucleic acid degradation for at least two weeks under simulated field conditions. With this method, volumes of up to 25ml of whole urine can be purified in a high-throughput and cost-effective manner. Coupled with its ability to purify both DNA and RNA, the described method may have broad applicability for improving the diagnostic utility of urine, particularly for the detection of low abundant targets. [ABSTRACT FROM AUTHOR]

Abstract In this study, we demonstrate a simple and facile method to enable DNA purification and amplification in a continuous step inside a thermoplastic microdevice. The innate property of thermoplastics was adopted to simplify DNA purification because negatively charged DNA can electrostatically interact with the induced positively charged surfaces of thermoplastics as a sample flows inside a microchannel; thus, the DNA purification step can be eliminated. We report the use of natural plastics for the selective adsorption of DNA and conduct a subsequent flow-through polymerase chain reaction (PCR) using a thermoplastic microdevice. Four thermoplastics—poly(methyl methacrylate) (PMMA), polycarbonates (PC), polystyrene (PS), and polypropylene (PP)—were selected as analytical models, and DNA adsorption phenomena on their pristine surfaces were examined. DNA was successfully immobilized onto the pristine thermoplastic surfaces, and the results were confirmed by fluorescent measurement and contact angle measurement and by performing PCR using a thermocycler. The electrostatically attached DNA was subsequently released from the pristine thermoplastic surface using a PCR reagent, which contained a low ionic strength salt as a component that could be used for DNA elution. The eluted DNA was then seamlessly amplified in the subsequent microchannel designed to perform flow-through PCR. The PCR products were collected by a microchamber connected at the end of the microchannel for on-site fluorescence detection. The PMMA microdevice was used to successfully purify and amplify a 210 bp target amplicon from a foodborne pathogen, Escherichia coli O157:H7, within 35 min. This study is expected to pave the way for developing an integrated monolithic thermoplastic microdevice suitable for rapidly identifying foodborne pathogens with reduced operation, decreased manufacturing cost, and enhanced device disposability. Graphical abstract We fabricate a fully integrated monolithic PMMA microdevice and demonstrate DNA purification, amplification, and on-site detection of major foodborne pathogens in one continuous flow. Image 1 Highlights • An integrated monolithic PMMA device was fabricated for pathogens identification. • Plastic surface itself was utilized for DNA attachment for sample purification. • The eluted DNA by PCR reagents was successfully amplified inside microchannel. • DNA purification, amplification, and detection were realized in a seamless manner. • The overall process for foodborne pathogen detection was realized within 35 min. [ABSTRACT FROM AUTHOR]

We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. The efficiency of filter paper-based spin columns was evaluated for purification of nucleic acids from various sources. Following protocols of commercial kits, we found filter paper to be a useful binding material for purification of nucleic acids, including plant genomic DNA, plant total RNA, PCR products, and DNA from agarose gels. However, filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Protocols for the use of filter paper recharged spin columns or homemade spin columns for low throughput purification of plant genomic DNA and total RNA with unused commercial kit buffers or less expensive homemade buffers are presented. [ABSTRACT FROM AUTHOR]

Previously we developed and tested the Salmonella GenoSerotyping Array (SGSA), which utilized oligonucleotide probes for O- and H- antigen biomarkers to perform accurate molecular serotyping of 57 Salmonella serotypes. Here we describe the development and validation of the ISO 17025 accredited second version of the SGSA (SGSA v. 2) with reliable and unambiguous molecular serotyping results for 112 serotypes of Salmonella which were verified both in silico and in vitro. Improvements included an expansion of the probe sets along with a new classifier tool for prediction of individual antigens and overall serotype from the array probe intensity results. The array classifier and probe sequences were validated in silico to high concordance using 36,153 draft genomes of diverse Salmonella serotypes assembled from public repositories. We obtained correct and unambiguous serotype assignments for 31,924 (88.30%) of the tested samples and a further 3,916 (10.83%) had fully concordant antigen predictions but could not be assigned to a single serotype. The SGSA v. 2 can directly use bacterial colonies with a limit of detection of 860 CFU/mL or purified DNA template at a concentration of 1.0 x 10−1 ng/μl. The SGSA v. 2 was also validated in the wet laboratory and certified using panel of 406 samples representing 185 different serotypes with correct antigen and serotype determinations for 60.89% of the panel and 18.31% correctly identified but an ambiguous overall serotype determination. [ABSTRACT FROM AUTHOR]

[Display omitted] • A preparation method for direct real-time qPCR of Coxiella burnetii was designed to replace previous kits. • A meat preparation method was designed to extract and disrupt C. burnetii from meat. • Sixteen protocols involving protein precipitation and DNA binding steps were assessed. • Among various protein precipitants, 5.0 M NaCl induced the lowest A 280 (P < 0.05). • Stability of the most effective protocols was verified with low-spiked or spore-inoculated loin, rib, and hind leg meat. Coxiella burnetii is a zoonotic pathogen that has been associated with foodborne outbreaks in products with ruminant origins. However, a method to detect C. burnetii in meat has been merely studied, and commercial kits cannot efficiently fulfill this purpose. In this study, an in-house preparation method for direct real-time qPCR of C. burnetii in beef, goat, and lamb meat was designed. In the sample preparation step (step 1), trypsin digestion and cell disruption techniques were introduced to target C. burnetii in an obligate intracellular or spore-like form. Afterward, 16 DNA purification protocols involving the following steps (steps 2–3) were assessed: the precipitation of meat proteins (step 2; using 2.5, 5.0 M NaCl or 1:1, 2:1 ethanol as the precipitant) and binding of DNA to silicon dioxide particles with chaotropic salts (step 3; using 2.5, 5.0 M NaCl or 2.5, 5.0 M guanidine thiocyanate as the salt). The protocols with superior performance in high-spiked loins (estimated 4–5 log cells/g) were verified in low-spiked (1–2 log cells/g) or Bacillus thuringiensis spore-inoculated (1–2 log CFU/g) loins, ribs, and hind legs. During the protein precipitation, 5.0 M NaCl induced significantly lower protein level as demonstrated by A 280 , when compared to 2.5 M NaCl or ethanol (P < 0.05). For the DNA binding step, Ct values were lowered in high-spiked goat or lamb loins (3.5–6.0▾; P < 0.05) when the concentration of NaCl was doubled or guanidine thiocyanate was introduced instead of NaCl as a chaotropic salt. Based on these results, two protocols using 5.0 M NaCl as the protein precipitant and 5.0 M NaCl (N2 + N2) or guanidine thiocyanate (N2 + G2) as the chaotropic salt were selected, which demonstrated successful detection in low-spiked (Ct values of N2 + N2, 32.9–35.6; N2 + G2, 32.3–36.4) or spore-inoculated meat (N2 + N2, 30.9–37.5; N2 + G2, 29.7–32.7). Verification in low-spiked meat showed that meat type/part significantly impacted the Ct values of N2 + G2 but not those of N2 + N2. To our knowledge, this is the first study that developed a highly accessible method for detecting C. burnetii in meat which could reveal the possibility of meat-borne Q fever in humans. [ABSTRACT FROM AUTHOR]

Molecular diagnostics for sepsis is still a challenge due to the presence of compounds that interfere with gene amplification and bacteria at concentrations lower than the limit of detection (LOD). Here, we report on the development of a 3D printed modular microfluidic device (3DpmμFD) that preconcentrates bacteria of interest in whole blood and purifies their genomic DNA (gDNA). It is composed of a W-shaped microchannel and a conical microchamber. Bacteria of interest are magnetically captured from blood in the device with antibody conjugated magnetic nanoparticles (Ab-MNPs) at 5 mL/min in the W-shaped microchannel, while purified gDNA of the preconcentrated bacteria is obtained with magnetic silica beads (MSBs) at 2 mL/min in the conical microchamber. The conical microchamber was designed to be connected to the microchannel after the capturing process using a 3D-printed rotary valve to minimize the exposure of the MSBs to interfering compounds in blood. The pretreatment process of spiked blood (2.5 mL) can be effectively completed within about 50 min. With the 3DpmμFD, the LOD for the target microorganism Escherichia coli O157:H7 measured by both polymerase chain reaction (PCR) with electrophoresis and quantitative PCR was 10 colony forming unit (CFU) per mL of whole blood. The results suggest that our method lowers the LOD of molecular diagnostics for pathogens in blood by providing bacterial gDNA at high purity and concentration.

The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist.

DNA extraction is a routine procedure in most plant laboratories. Plant DNA isolation requires the use of several hazardous chemicals. The use of Proteinase K and RNase A contributes major isolation cost. Also the use of liquid nitrogen does not only increase the cost but also opens new ways of introduction of contamination and injury risks. In the present study a CTAB-based cotton DNA extraction and purification method suitable for single cotton seed is reported. It is an inexpensive, rapid and simple procedure, especially when hundreds of cotton seed samples need to be analyzed. The main advantages of the method are high yield, high quality, and cost effectiveness. This method could satisfy demand when high amount plant genomic DNA required from large numbers of cotton plant samples. The quality (integrity, originality, and purity) and quantity (amount) of the extracted DNA are suitable for most molecular biology research including restriction enzyme digestion studies, polymerase chain reaction, next generation sequencing, and next generation marker technologies. [ABSTRACT FROM AUTHOR]

Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-basedDNApurification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1-1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample. [ABSTRACT FROM AUTHOR]

Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin ® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content. [ABSTRACT FROM AUTHOR]