scholarly journals MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

2019 ◽  
Author(s):  
Cassidy Petree ◽  
Gaurav K Varshney
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Fold Increase ◽  
Targeted Mutagenesis ◽  
Model Organisms ◽  
Multiple Alleles ◽  
Cost Effective Method ◽  
Single Reaction

AbstractThe powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis based sequencing equipment.

An International Campaign for Agricultural and Livestock Genomics (CALG)

2002 ◽  
Vol 06 (24) ◽  
pp. 958-965
Author(s):  
Jun Yu ◽  
Jian Wang ◽  
Huanming Yang
Keyword(s):  
Large Scale ◽  
Cost Effective ◽  
Model Organisms ◽  
Environmental Biology ◽  
Cdna Sequences ◽  
Governmental Agencies ◽  
Technology Innovations ◽  
A Genome ◽  
Starting Point ◽  
The Cost

A coordinated international effort to sequence agricultural and livestock genomes has come to its time. While human genome and genomes of many model organisms (related to human health and basic biological interests) have been sequenced or plugged in the sequencing pipelines, agronomically important crop and livestock genomes have not been given high enough priority. Although we are facing many challenges in policy-making, grant funding, regional task emphasis, research community consensus and technology innovations, many initiatives are being announced and formulated based on the cost-effective and large-scale sequencing procedure, known as whole genome shotgun (WGS) sequencing that produces draft sequences covering a genome from 95 percent to 99 percent. Identified genes from such draft sequences, coupled with other resources, such as molecular markers, large-insert clones and cDNA sequences, provide ample information and tools to further our knowledge in agricultural and environmental biology in the genome era that just comes to its accelerated period. If the campaign succeeds, molecular biologists, geneticists and field biologists from all countries, rich or poor, would be brought to the same starting point and expect another astronomical increase of basic genomic information, ready to convert effectively into knowledge that will ultimately change our lives and environment into a greater and better future. We call upon national and international governmental agencies and organizations as well as research foundations to support this unprecedented movement.

scholarly journals Fabrication and Characterization of Humidity Sensors Based on Graphene Oxide–PEDOT:PSS Composites on a Flexible Substrate

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 148 ◽  
Author(s):  
Francisco J. Romero ◽  
Almudena Rivadeneyra ◽  
Markus Becherer ◽  
Diego P. Morales ◽  
Noel Rodríguez
Keyword(s):  
Graphene Oxide ◽  
Large Scale ◽  
Flexible Substrate ◽  
High Sensitivity ◽  
Cost Effective ◽  
Humidity Sensors ◽  
Good Thermal Stability ◽  
Cost Effective Method ◽  
And Performance ◽  
Stability Time

In this paper, we present a simple, fast, and cost-effective method for the large-scale fabrication of high-sensitivity humidity sensors on flexible substrates. These sensors consist of a micro screen-printed capacitive structure upon which a sensitive layer is deposited. We studied two different structures and three different sensing materials by modifying the concentration of poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT:PSS) in a graphene oxide (GO) solution. The results show that the aggregation of the PEDOT:PSS to the GO can modify its electrical properties, boosting the performance of the capacitive sensors in terms of both resistive losses and sensitivity to relative humidity (RH) changes. Thus, in an area less than 30 mm2, the GO/PEDOT:PSS-based sensors can achieve a sensitivity much higher (1.22 nF/%RH at 1 kHz) than other similar sensors presented in the literature which, together with their good thermal stability, time response, and performance over bending, demonstrates that the manufacturing approach described in this work paves the way for the mass production of flexible humidity sensors in an inexpensive way.

scholarly journals 230 Genome editing applications in plants: high-throughput CRISPR/Cas editing for crop improvement

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 56-56
Author(s):  
Michael Thomson
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Positional Cloning ◽  
Gene Editing ◽  
Crop Improvement ◽  
Cost Effective ◽  
Gene Families ◽  
Ease Of Use ◽  
Plant Genome ◽  
Wide Range

Abstract The precision and ease of use of CRISPR nucleases, such as Cas9 and Cpf1, for plant genome editing has the potential to accelerate a wide range of applications for crop improvement. For upstream research on gene discovery and validation, rapid gene knock-outs can enable testing of single genes and multi-gene families for functional effects. Large chromosomal deletions can test the function of tandem gene arrays and assist with positional cloning of QTLs by helping to narrow down the target region. Nuclease-deactivated Cas9 fusion proteins with transcriptional activators and repressors can be used to up and down-regulate gene expression. Even more promising, gene insertions and allele replacements can provide the opportunity to rapidly test the effects of different alleles at key loci in the same genetic background, providing a more precise alternative to marker-assisted backcrossing. Recently, Texas A&M AgriLife Research has supported the development of a Crop Genome Editing Lab at Texas A&M working towards optimizing a high-throughput gene editing pipeline and providing an efficient and cost-effective gene editing service for research and breeding groups. The lab is using rice as a model to test and optimize new approaches aimed towards overcoming current bottlenecks. For example, a wealth of genomics data from the rice community enables the development of novel approaches to predict which genes and target modifications may be most beneficial for crop improvement, taking advantage of known major genes, high-resolution GWAS data, multiple high-quality reference genomes, transcriptomics data, and resequencing data from the 3,000 Rice Genomes Project. Current projects have now expanded to work across multiple crops to provide breeding and research groups with a rapid gene editing pipeline to test candidate genes in their programs, with the ultimate goal of developing nutritious, high-yielding, stress-tolerant crops for the future.

scholarly journals Simple, Fast, and Cost-Effective Fabrication of Wafer-Scale Nanohole Arrays on Silicon for Antireflection

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Di Di ◽  
Xuezhong Wu ◽  
Peitao Dong ◽  
Chaoguang Wang ◽  
Jian Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
Crystalline Silicon ◽  
Colloidal Crystal ◽  
Cost Effective ◽  
Wafer Scale ◽  
Crystalline Silicon Solar Cells ◽  
Cost Effective Method ◽  
Nanohole Arrays ◽  
Coating Method ◽  
Hole Arrays

A simple, fast, and cost-effective method was developed in this paper for the high-throughput fabrication of nanohole arrays on silicon (Si), which is utilized for antireflection. Wafer-scale polystyrene (PS) monolayer colloidal crystal was developed as templates by spin-coating method. Metallic shadow mask was prepared by lifting off the oxygen etched PS beads from the deposited chromium film. Nanohole arrays were fabricated by Si dry etching. A series of nanohole arrays were fabricated with the similar diameter but with different depth. It is found that the maximum depth of the Si-hole was determined by the diameter of the Cr-mask. The antireflection ability of these Si-hole arrays was investigated. The results show that the reflection decreases with the depth of the Si-hole. The deepest Si-hole arrays show the best antireflection ability (reflection < 9%) at long wavelengths (>600 nm), which was about 28 percent of the nonpatterned silicon wafer’s reflection. The proposed method has the potential for high-throughput fabrication of patterned Si wafer, and the low reflectivity allows the application of these wafers in crystalline silicon solar cells.

Cost-effectiveness of two monitoring strategies for the great crested newt (Triturus cristatus)

2010 ◽  
Vol 31 (3) ◽  
pp. 403-410 ◽  
Author(s):  
Jérôme Pellet ◽  
Madeleine Kröpfli ◽  
Patrick Heer
Keyword(s):  
Large Scale ◽  
Cost Effective ◽  
Amphibian Conservation ◽  
Submerged Vegetation ◽  
Vegetation Density ◽  
Triturus Cristatus ◽  
Monitoring Strategies ◽  
Cost Effective Method ◽  
Crested Newt ◽  
Monitoring Protocols

AbstractDesigning cost-effective monitoring protocols is a fundamental prerequisite for amphibian conservation. Here, we report a comparison of flashlight survey and trapping (with and without light sticks as trap baits) in order to determine flashlight detectability and trap detectability of great crested newts (Triturus cristatus). Twelve ponds were surveyed in Switzerland where T. cristatus had been known to occur. We measured covariates affecting both flashlight detectability and trap detectability. Newt flashlight detectability using 20 min long flashlight surveys was on average ± SE = 39% ± 10%). Flashlight detectability was mostly influenced by surface and submerged vegetation density, as well as by water temperature. Newt trap detectability during one night using six funnel traps per pond was on average±SE = 41%±10%. Trap detectability was mainly affected by trap position in the pond, with traps lying on the pond floor being more likely to attract newts. The use of light sticks did not enhance the trap detectability. Estimates of flashlight detectability and trap detectability were used to define how many times the sites have to be visited to be 95% certain of not missing T. cristatus in ponds where they are present. In both cases multiple visits (7 flashlight surveys or 6 trapping sessions) have to be performed. Flashlight surveys are the most easily applied and most cost-effective method to use in large scale programs.

scholarly journals STAMP: a multiplex sequencing method for simultaneous evaluation of mitochondrial DNA heteroplasmies and content

2020 ◽  
Author(s):  
Xiaoxian Guo ◽  
Yiqin Wang ◽  
Ruoyu Zhang ◽  
Zhenglong Gu
Keyword(s):  
Large Scale ◽  
Nuclear Dna ◽  
Large Population ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Sequencing Method ◽  
Scale Population ◽  
Mtdna Variants ◽  
Multiplex Sequencing

ABSTRACTHuman mitochondrial genome (mtDNA) variations, such as mtDNA heteroplasmies (the co-existence of mutated and wild-type mtDNA), have received increasing attention in recent years for their clinical relevance to numerous diseases. But large-scale population studies of mtDNA heteroplasmies have been lagging due to the lack of a labor- and cost-effective method. Here, we present a novel human mtDNA sequencing method called STAMP (sequencing by targeted amplification of multiplex probes) for measuring mtDNA heteroplasmies and content in a streamlined workflow. We show that STAMP has high mapping rates to mtDNA, deep coverage of unique reads, and high tolerance to sequencing and PCR errors when applied to human samples. STAMP also has high sensitivity and low false positive rates in identifying artificial mtDNA variants at fractions as low as 0.5% in genomic DNA samples. We further extend STAMP, by including nuclear DNA-targeting probes, to enable assessment of relative mtDNA content in the same assay. The high cost-effectiveness of STAMP, along with the flexibility of using it for measuring various aspects of mtDNA variations, will accelerate the research of mtDNA heteroplasmies and content in large population cohorts, and in the context of human diseases and aging.

scholarly journals High efficient extracellular production of recombinant Streptomyces PMF phospholipase D in Escherichia coli

2020 ◽  
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Large Scale ◽  
Expression System ◽  
Cost Effective ◽  
Scale Production ◽  
Secretory Expression ◽  
E Coli ◽  
Cost Effective Method ◽  
High Efficient

Abstract Background Currently, Streptomyces is widely used in the preparation of phospholipase D (PLD) with high transphosphatidylation activity. However, the yield of PLD from Streptomyces was low and the culture period was long. Therefore, an efficient and cost-effective method is needed urgently.Results Firstly, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E. coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLDPMF gene was expressed together with its native signal peptide (Nat) and the signal peptide PelB from E. coli. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.Conclusions We succeeded in over-expressing PLD from Streptomyces PMF in E. coli with high transphosphatidylation activity and enhanced the yield by secretory expression. The secreted PLD was successfully used in the production of PS. Our work makes the large-scale production of PLD and PS feasible.

scholarly journals Development of High-Throughput Multiplex Serology to Detect Serum Antibodies against Coxiella burnetii

2021 ◽  
Vol 9 (11) ◽  
pp. 2373
Author(s):  
Rima Jeske ◽  
Larissa Dangel ◽  
Leander Sauerbrey ◽  
Dimitrios Frangoulidis ◽  
Lauren R. Teras ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
High Throughput ◽  
Large Scale ◽  
Q Fever ◽  
Reference Group ◽  
Clinical Manifestations ◽  
Cost Effective ◽  
Population Monitoring ◽  
Increased Risk ◽  
Multiplex Serology

The causative agent of Q fever, the bacterium Coxiella burnetii (C. burnetii), has gained increasing interest due to outbreak events and reports about it being a potential risk factor for the development of lymphomas. In order to conduct large-scale studies for population monitoring and to investigate possible associations more closely, accurate and cost-effective high-throughput assays are highly desired. To address this need, nine C. burnetii proteins were expressed as recombinant antigens for multiplex serology. This technique enables the quantitative high-throughput detection of antibodies to multiple antigens simultaneously in a single reaction. Based on a reference group of 76 seropositive and 91 seronegative sera, three antigens were able to detect C. burnetii infections. Com1, GroEL, and DnaK achieved specificities of 93%, 69%, and 77% and sensitivities of 64%, 72%, and 47%, respectively. Double positivity to Com1 and GroEL led to a combined specificity of 90% and a sensitivity of 71%. In a subgroup of seropositives with an increased risk for chronic Q fever, the double positivity to these markers reached a specificity of 90% and a sensitivity of 86%. Multiplex serology enables the detection of antibodies against C. burnetii and appears well-suited to investigate associations between C. burnetii infections and the clinical manifestations in large-scale studies.

scholarly journals Efficient Homology-directed Repair with Circular ssDNA Donors

2019 ◽  
Author(s):  
Sukanya Iyer ◽  
Aamir Mir ◽  
Joel Vega-Badillo ◽  
Benjamin P. Roscoe ◽  
Raed Ibraheim ◽  
...  
Keyword(s):  
Genome Editing ◽  
K562 Cells ◽  
Cost Effective ◽  
Cell Types ◽  
Traffic Light ◽  
Homology Directed Repair ◽  
Mammalian Cell Lines ◽  
Cost Effective Method ◽  
Circular Ssdna ◽  
Fluorescent Tag

AbstractWhile genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

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