Thread-based isotachophoresis for DNA extraction and purification from biological samples

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Liang Chen ◽  
Joan M Cabot ◽  
Brett Paull

A rapid, low-cost, and disposable microfluidic thread-based isotachophoresis method was developed for the purification and preconcentration of nucleic acids from biological samples, prior to their extraction and successful analysis using...

2014 ◽  
Vol 955-959 ◽  
pp. 306-309 ◽  
Author(s):  
Lin Hui Wu ◽  
Jian Li Liu ◽  
Jing Zeng ◽  
Ji Zhao

There is an increased interest in the extraction of nucleic acids from various environmental samples, since only a minority of naturally occurring microbes can be cultured using standard techniques. Nucleic acids extraction and purification from soils are extremely challenging due to the low biomass, high organic contents and high variability of soil types. This has been regarded as one of the major difficulties that hamper the development of soil microbial ecology study. No commercial nucleic acids kits currently available are capable of preparing the DNAs without modifications. The cost can be very high for DNA extraction from extreme environmental soil samples, such as soils that have extreme high or low pHs. In this work, we developed and optimized soil DNA extraction and purification methods on different soils and compared the impact of three different DNA extraction protocols on DNA yield and purity. For the three different types of soil we used, direct extraction obtained the highest DNA recover rate, but required more cleanup steps. MoBio PowerSoil® DNA Isolation Kit yields less but do not require as many downstream cleaning steps. Both of the two methods obtained a more abundant microbial community than Meta-G-NomeTMDNA Isolation Kit.


Retrovirology ◽  
2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Catherine M. Moore ◽  
Melanie Grandits ◽  
Clemens Grünwald-Gruber ◽  
Friedrich Altmann ◽  
Maria Kotouckova ◽  
...  

Abstract Background HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such ‘broadly neutralising’ antibody is ‘N6’. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. Results N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. Conclusions The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries. Graphic abstract


mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.


The Analyst ◽  
2012 ◽  
Vol 137 (17) ◽  
pp. 4023 ◽  
Author(s):  
Lindsay N. Strotman ◽  
Guangyun Lin ◽  
Scott M. Berry ◽  
Eric A. Johnson ◽  
David J. Beebe

2011 ◽  
Vol 47 (2) ◽  
pp. 211-220 ◽  
Author(s):  
D. D. Mamaev ◽  
D. A. Khodakov ◽  
E. I. Dementieva ◽  
I. V. Filatov ◽  
D. A. Yurasov ◽  
...  

2011 ◽  
Vol 340 ◽  
pp. 318-323
Author(s):  
Wen Yi Zhang ◽  
Ning Han ◽  
Li Rong Yao ◽  
Xiao Lan Qiu ◽  
Xiao Liang Chen

The MC-LR from the the blue-green algae of Taihu Lake was extracted, at the same time, a set of microcystins extraction method with methanol as extraction solvent and purification method with C18-SPE as purification workstations were established. The extraction solvent concentration, extraction time, extraction solvent amount, leacheate concentration and eluent concentration were used to research the extraction efficiency of MC-LR. Finally, 80% methanol was used to wash microcytins to make MC-LR high purity and the purity was over 85%. This research presented a method of low cost and high efficiency. It provided the foundation for the further research of microcytins.


Revista Vitae ◽  
2021 ◽  
Vol 28 (3) ◽  
Author(s):  
Laura Carvajal Barbosa ◽  
Diego Insuasty Cepeda ◽  
Andrés Felipe León Torres ◽  
Maria Mercedes Arias Cortes ◽  
Zuly Jenny Rivera Monroy ◽  
...  

BACKGROUND : Biosensing techniques have been the subject of exponentially increasing interest due to their performance advantages such as high selectivity and sensitivity, easy operation, low cost, short analysis time, simple sample preparation, and real-time detection. Biosensors have been developed by integrating the unique specificity of biological reactions and the high sensitivity of physical sensors. Therefore, there has been a broad scope of applications for biosensing techniques, and nowadays, they are ubiquitous in different areas of environmental, healthcare, and food safety. Biosensors have been used for environmental studies, detecting and quantifying pollutants in water, air, and soil. Biosensors also showed great potential for developing analytical tools with countless applications in diagnosing, preventing, and treating diseases, mainly by detecting biomarkers. Biosensors as a medical device can identify nucleic acids, proteins, peptides, metabolites, etc.; these analytes may be biomarkers associated with the disease status. Bacterial food contamination is considered a worldwide public health issue; biosensor-based analytical techniques can identify the presence or absence of pathogenic agents in food. OBJECTIVES: The present review aims to establish state-of-the-art, comprising the recent advances in the use of nucleic acid-based biosensors and their novel application for the detection of nucleic acids. Emphasis will be given to the performance characteristics, advantages, and challenges. Additionally, food safety applications of nucleic acid-based biosensors will be discussed. METHODS: Recent research articles related to nucleic acid-based biosensors, biosensors for detecting nucleic acids, biosensors and food safety, and biosensors in environmental monitoring were reviewed. Also, biosensing platforms associated with the clinical diagnosis and food industry were included. RESULTS: It is possible to appreciate that multiple applications of nucleic acid-based biosensors have been reported in the diagnosis, prevention, and treatment of diseases, as well as to identify foodborne pathogenic bacteria. The use of PNA and aptamers opens the possibility of developing new biometric tools with better analytical properties. CONCLUSIONS: Biosensors could be considered the most important tool for preventing, treating, and monitoring diseases that significantly impact human health. The aptamers have advantages as biorecognition elements due to the structural conformation, hybridization capacity, robustness, stability, and lower costs. It is necessary to implement biosensors in situ to identify analytes with high selectivity and lower detection limits.


protocols.io ◽  
2021 ◽  
Author(s):  
Julien Serret ◽  
marie.couderc not provided ◽  
Cedric Mariac ◽  
Laurencealbar not provided ◽  
Francois Sabot
Keyword(s):  
Low Cost ◽  

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