A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

Low-cost CRISPR diagnostics for resource-limited settings.

Next-generation sequencing (NGS) has identified disease hallmarks and catalogued a vast reservoir of genetic information from humans and other species. Precise nucleotide-interrogation properties of clustered regularly interspaced short palindromic repeats (CRISPR) proteins have been harnessed to rapidly identify DNA-RNA signatures for diverse applications, bypassing the cost and turnaround times associated with diagnostic NGS.

Increasing DNA Isolation Performance from Blood Cells Using a Modified Commercial Low-Cost Reagent.

BACKGROUND: Genomic studies facilitate comprehension of the pathophysiology, diagnosis, and treatment of chronic diseases. Such studies require sufficient and good quality DNA isolated from a large number of blood samples. This study attempts to obtain a high-quality genomic DNA isolated from a large number of blood samples using a simple and cheap method. METHODS: The EasyPure® Genomic DNA Kit (Transgen Biotech) was modified to increase the amount of DNA recovery: a few steps and two additional column elutions were added to the original manufacturer´s procedure. RESULTS: The amount of DNA isolated from frozen blood samples increased by an average of 56%. Its 260/280 ratio and electrophoretic mobility properties make it suitable for genomic studies. CONCLUSIONS: A relatively low-cost commercial column and a simple modification of the manufacturer´s protocol, provided a simple and cheap procedure to isolate high-quality DNA from a large number of blood samples suitable for genomic studies.

Toxic reagents and expensive equipment: are they really necessary for the extraction of good quality fungal DNA?

The aim of this work was to evaluate a fungal DNA extraction procedure with the lowest inputs in terms of time as well as of expensive and toxic chemicals, but able to consistently produce genomic DNA of good quality for PCR purposes. Two types of fungal biological material were tested - mycelium and conidia - combined with two protocols for DNA extraction using Sodium Dodecyl Sulphate (SDS) and Cetyl Trimethyl Ammonium Bromide as extraction buffers and glass beads for mechanical disruption of cell walls. Our results showed that conidia and SDS buffer was the combination that lead to the best DNA quality and yield, with the lowest variation between samples. This study clearly demonstrates that it is possible to obtain high yield and pure DNA from pigmented conidia without the use of strong cell disrupting procedures and of toxic reagents. SIGNIFICANCE AND IMPACT OF THE STUDY: There are numerous methods for DNA extraction from fungi. Some rely on expensive commercial kits and/or equipments, unavailable for many laboratories, or make use of toxic chemicals such as chloroform, phenol and mercaptoethanol. This study clearly demonstrates that it is possible to obtain high yields of pure DNA from pigmented conidia without the use of strong and expensive cell disrupting procedures and of toxic reagents. The method herein described is simultaneously inexpensive and adequate to DNA extraction from several different types of fungi.

Ex-post assessment of genetically modified, low level presence in Canadian flax.

Canada is the world's largest producer and exporter of flaxseed. In 2009, DNA from deregistered genetically modified (GM) CDC Triffid was detected in a shipment of Canadian flaxseed exported to Europe, causing a large decrease in the amount of flax planted in Canada and a major shift in export markets. The flax industry in Canada undertook major changes to ensure the removal of transgenic flax from the supply chain. To demonstrate compliance, Canada adopted a protocol involving testing grain samples (post-harvest) using an RT-PCR test for the construct found in CDC Triffid. Efforts to remove the presence of GM flax from the value chain included reconstituting major flax varieties from GM-free plants. The reconstituted varieties represented the majority of planting seed in 2014. This study re-evaluates GM flax presence in Canadian grain stocks for an updated dataset (2009-2015) using a previously described simulation model to estimate low-level GM presence. Additionally, losses to the Canadian economy resulting from the reduction in flax production and export opportunities, costs associated with reconstituting major flax varieties, and testing for the presence of GM flax along the flax value chain are estimated.

Minimizing the time and cost of production of transgenic alfalfa libraries using the highly efficient completely sequenced vector pPZP200BAR.

Alfalfa is the most important forage legume worldwide. However, similar to other minor forage crops, it is usually harvested along with weeds, which decrease its nutrient quality and thus reduce its high value in the market. In addition, weeds reduce alfalfa yield by about 50 %. Although weeds are the limiting factor for alfalfa production, little progress has been made in the incorporation of herbicide-tolerant traits into commercial alfalfa. This is partially due to the high times and costs needed for the production of vast numbers of transgenic alfalfa events as an empirical approach to bypass the random transgenic silencing and for the identification of an event with optimal transgene expression. In this focus article, we report the complete sequence of pPZP200BAR and the extremely high efficiency of this binary vector in alfalfa transformation, opening the way for rapid and inexpensive production of transgenic events for alfalfa improvement public programs.

Replicating research in ecology and evolution: feasibility, incentives, and the cost-benefit conundrum.

We believe that replicating studies in ecology and evolution is extremely valuable, but replication within species and systems is troublingly rare, and even 'quasi-replications' in different systems are often insufficient. We make a case for supporting multiple types of replications and point out that the current incentive structure needs to change if ecologists and evolutionary biologist are to value scientific replication sufficiently.

Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.

The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and highlighted the key inadequacies of traditional diagnostic methods that make them unsuitable for use in resource-limited settings, from expensive equipment and infrastructure requirements to unacceptably long turnaround times. While rapid immunoassay diagnostic tests were much less costly and more context-appropriate, they suffered from drastically low sensitivities and high false negative rates. An accurate, sensitive, and specific molecular diagnostic that is also rapid, low-cost, and independent of laboratory infrastructure is needed for effective point-of-care detection and epidemiological control in these developing regions. We developed a paper-based assay that allows for the extraction and purification of RNA directly from human clinical nasopharyngeal specimens through a poly(ether sulfone) paper matrix, H1N1-specific in situ isothermal amplification directly within the same paper matrix, and immediate visual detection on lateral flow strips. The complete sample-to-answer assay can be performed at the point-of-care in just 45 min, without the need for expensive equipment or laboratory infrastructure, and it has a clinically relevant viral load detection limit of 10(6) copies/mL, offering a 10-fold improvement over current rapid immunoassays.

A Rapid and Cost-Effective Method for DNA Extraction from Archival Herbarium Specimens.

Here we report a rapid and cost-effective method for the extraction of total DNA from herbarium specimens up to 50-90-year-old. The method takes about 2 h, uses AMPure XP magnetic beads diluted by PEG-8000- containing buffer, and does not require use of traditional volatile components like chloroform, phenol, and liquid nitrogen. It yields up to 4 µg of total nucleic acid with high purity from about 30 mg of dry material. The quality of the extracted DNA was tested by PCR amplification of 5S rRNA and rbcL genes (nuclear and chloroplast DNA markers) and compared against the traditional chloroform/isoamyl alcohol method. Our results demonstrate that the use of the magnetic beads is crucial for extraction of DNA suitable for subsequent PCR from herbarium samples due to the decreasing inhibitor concentrations, reducing short fragments of degraded DNA, and increasing median DNA fragment sizes.

Epigenetics for anthropologists: An introduction to methods.

The study of epigenetics, or chemical modifications to the genome that may alter gene expression, is a growing area of interest for social scientists. Anthropologists and human biologists are interested in epigenetics specifically, as it provides a potential link between the environment and the genome, as well as a new layer of complexity for the study of human biological variation. In pace with the rapid increase in interest in epigenetic research, the range of methods has greatly expanded over the past decade. The primary objective of this article is to provide an overview of the current methods for assaying DNA methylation, the most commonly studied epigenetic modification. We will address considerations for all steps required to plan and conduct an analysis of DNA methylation, from appropriate sample collection, to the most commonly used methods for laboratory analyses of locus-specific and genome-wide approaches, and recommendations for statistical analyses. Key challenges in the study of DNA methylation are also discussed, including tissue specificity, the stability of measures, timing of sample collection, statistical considerations, batch effects, and challenges related to analysis and interpretation of data. Our hope is that this review serves as a primer for anthropologists and human biologists interested in incorporating epigenetic data into their research programs.

The interplay of ethics and genetic technologies in balancing the social valuation of the human genome in UNESCO declarations.

This study investigates changes in the social valuation of the human genome over the more than 30 years since the establishment of the Human Genome Project. It offers a descriptive sociological analysis of the three waves of this valuation, mainly by considering three key UNESCO declarations and a relevant report. These waves represent a shifting balance between collectivism and individualism, starting with a broadly constructed valuation of the human genome as common human heritage and moving toward a valuation of dynamic applications within various social and medical contexts (e.g., personalized genomic medicine and genome editing). We seek to broaden the analytical perspective by examining how the declarations' ethical foci are framed within the context of rapidly evolving genetic technologies and their social applications. We conclude by discussing continuity and change in value balancing vis-à-vis changing genomic technologies.

Genotyping technologies for genetic research.

The past few years have seen enormous advances in genotyping technology, including chips that accommodate in excess of 1 million SNP assays. In addition, the cost per genotype has been driven down to levels unimagined only a few years ago. These developments have resulted in an explosion of positive whole-genome association studies and the identification of many new genes for common diseases. Here I review high-throughput genotyping platforms as well as other approaches for lower numbers of assays but high sample throughput, which play an important role in genotype validation and study replication. Further, the utility of SNP arrays for detecting structural variation through the development of genotyping algorithms is reviewed and methods for long-range haplotyping are presented. It is anticipated that in the future, sample throughput and cost savings will be increased further through the combination of automation, microfluidics, and nanotechnologies.

Assessment of alternative genotyping strategies to maximize imputation accuracy at minimal cost.

BACKGROUND: Commercial breeding programs seek to maximise the rate of genetic gain while minimizing the costs of attaining that gain. Genomic information offers great potential to increase rates of genetic gain but it is expensive to generate. Low-cost genotyping strategies combined with genotype imputation offer dramatically reduced costs. However, both the costs and accuracy of imputation of these strategies are highly sensitive to several factors. The objective of this paper was to explore the cost and imputation accuracy of several alternative genotyping strategies in pedigreed populations. METHODS: Pedigree and genotype data from a commercial pig population were used. Several alternative genotyping strategies were explored. The strategies differed in the density of genotypes used for the ancestors and the individuals to be imputed. Parents, grandparents, and other relatives that were not descendants, were genotyped at high-density, low-density, or extremely low-density, and associated costs and imputation accuracies were evaluated. RESULTS: Imputation accuracy and cost were influenced by the alternative genotyping strategies. Given the mating ratios and the numbers of offspring produced by males and females, an optimized low-cost genotyping strategy for a commercial pig population could involve genotyping male parents at high-density, female parents at low-density (e.g. 3000 SNP), and selection candidates at very low-density (384 SNP). CONCLUSIONS: Among the selection candidates, 95.5% and 93.5% of the genotype variation contained in the high-density SNP panels were recovered using a genotyping strategy that costs respectively, $24.74 and $20.58 per candidate.

In-house human immunodeficiency virus-1 genotype resistance testing to determine highly active antiretroviral therapy resistance mutations in Hong Kong.

OBJECTIVE: To determine the frequency of highly active antiretroviral therapy resistance mutations in the viral pol gene of human immunodeficiency virus-1 (HIV-1) genotypes that circulate in Hong Kong, by means of an in-house HIV-1 genotyping system. DESIGN: Retrospective study. SETTING: Two HIV clinics in Hong Kong. PATIENTS: A modified in-house genotyping resistance test was used to sequence the partial pol gene in 1165 plasma samples from 965 patients. The performance of our test was cross-compared with the US Food and Drug Administration-approved ViroSeq HIV-1 genotyping system. The results of genotyping were submitted to the Stanford HIV-1 drug resistance database for analysis. RESULTS: The cost-effective in-house genotypic resistance test (US$40) demonstrated comparable performance to the US Food and Drug Administration-approved ViroSeq system. The detection limit of this in-house genotypic resistance test could reach 400 copies/mL for both HIV-1 subtype B and CRF01_AE, which were the predominant genotypes in Hong Kong. Drug resistance mutations were detected only in post-treatment samples from treatment-failure patients. However, there was no significant difference in the frequency of drug resistance mutations between subtype B and CRF01_AE. CONCLUSION: Our cost-effective in-house genotypic resistance test detected no significant difference in drug resistance-related mutations frequencies between HIV-1 subtype B and CRF01_AE in Hong Kong. A drug resistance-related mutations database for different HIV-1 genotypes should be established in Hong Kong to augment guidance for HIV treatment.

Rapid and cost-effective screening of newly identified microsatellite loci by high-resolution melting analysis.

This study describes a new method for identifying microsatellite loci that will reliably amplify and show high degree of polymorphism in a given species. Microsatellites are the most powerful codominant markers available today, but the development of novel loci remains a labour-intensive and expensive process. In de novo isolation, approaches using next generation sequencing (NGS) are gradually replacing ones using Escherichia coli libraries, resulting in unparalleled numbers of candidate loci available. We present a systematic review of published microsatellite primer notes and show that, on average, about half of all candidate loci are lost due to insufficient PCR amplification, monomorphism or multicopy status in the genome, no matter what isolation strategy is used. Thus, the screening of candidate loci remains a major step in marker development. Re-assessing capillary-electrophoresis genotyped loci via high-resolution melting analysis (HRM), we evaluate the usefulness of HRM for this step. We demonstrate its applicability in a genotyping case study and introduce a fast, HRM-based workflow for the screening of microsatellite loci. This workflow may readily be applied to NGS-based marker development and has the potential to cut the costs of traditional testing by half to three quarters.

Development and characterization of microsatellite loci in Pericopsis elata (Fabaceae) using a cost-efficient approach.

PREMISE OF THE STUDY: Microsatellite loci were developed in the endangered Pericopsis elata using a combination of low-cost procedures. METHODS AND RESULTS: Microsatellite isolation was performed simultaneously on three distinct species through a newly available procedure that associates multiplex microsatellite enrichment and next-generation sequencing, allowing the rapid and low-cost development of microsatellite-enriched libraries through the use of a 1/32nd GS-FLX plate. Genotyping using M13-like labeling in multiplexed reactions allowed additional cost savings. From 72 primers selected for initial screening, 21 positively amplified P. elata, and 11 showed polymorphism with two to 11 alleles per locus and a mean value of 5.4 alleles per locus. CONCLUSIONS: These microsatellite loci will be useful to further investigate the level of genetic variation within and between natural populations of P. elata in Africa.

Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis.

PURPOSE OF WORK: A simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities of DNA are usually obtained, and with large scale analysis multiple isolations are required. A protocol for DNA isolation from Phytophthora and Pythium suitable for the evaluation of a large set of molecular markers was modified from one previously developed for soybean seed. There was a one to three fold increase in the amount of DNA that was extracted using the modified protocol compared to a commercial kit. The DNA obtained using the modified protocol was suitable for the amplification of microsatellite markers as well as the ITS region. This protocol is inexpensive, easy, quick, and efficient in terms of the volume of reagents and the number of steps involved in the procedure. The method may be applicable to other oomycetes and effectively implemented in other laboratories.

Fast, Scalable, and Practical: An Alkaline DNA Extraction Pipeline for Emergency Microbiomics Biosurveillance.

Pandemics and environmental crises evident from the first two decades of the 21st century call for methods innovation in biosurveillance and early detection of risk signals in planetary ecosystems. In crises conditions, conventional methods in public health, biosecurity, and environmental surveillance do not work well. In addition, the standard laboratory amenities and procedures may become unavailable, irrelevant, or simply not feasible, for example, owing to disruptions in logistics and process supply chains. The COVID-19 pandemic has been a wakeup call in this sense to reintroduce point-of-need diagnostics with an eye to limited resource settings and biosurveillance solutions. We report here a methodology innovation, a fast, scalable, and alkaline DNA extraction pipeline for emergency microbiomics biosurveillance. We believe that the presented methodology is well poised for effective, resilient, and anticipatory responses to future pandemics and ecological crises while contributing to microbiome science and point-of-need diagnostics in nonelective emergency contexts. The alkaline DNA extraction pipeline can usefully expand the throughput in emergencies by deployment or to allow backup in case of instrumentation failure in vital facilities. The need for distributed public health genomics surveillance is increasingly evident in the 21st century. This study makes a contribution to these ends broadly, and for future pandemic preparedness in particular. We call for innovation in biosurveillance methods that remain important existentially on a planet under pressure from unchecked human growth and breach of the boundaries between human and nonhuman animal habitats.

Cost-effective analysis of genotyping using oral cells in the geriatric population.

We evaluated the cost-effectiveness of using buccal swab brushes in comparison with blood samples for obtaining DNA for large epidemiological studies of the elderly population. The data reported here are from the third phase of the Integral Study of Depression among the Elderly in Mexico City's Mexican Institute of Social Security, conducted in 2007. The total cost of the two procedures was determined. The measurement of effectiveness was the quality and quantity of DNA measured in ng/µL and the use of this DNA for the determination of apolipoprotein E (APO E) polymorphism by PCR. Similar rates of amplification were obtained with the two techniques. The cost of the buccal swab brushes, including sample collection and DNA extraction, was US$16.63, compared to the cost per blood sample of US$23.35. Using the buccal swab, the savings was US$6.72 per patient (P < 0.05). The effectiveness was similar. Quantity and quality of DNA obtained were similar for the oral and blood procedures, demonstrating that the swab brush technique offers a feasible alternative for large-scale epidemiological studies.

Dictyostelium discoideum--a promising expression system for the production of eukaryotic proteins.

In general, four different expression systems, namely, bacterial, yeast, baculovirus, and mammalian, are widely used for the overproduction of biochemical enzymes and therapeutic proteins. Clearly, bacterial expression systems offer ease of maneuverability with respect to large-scale production of recombinant proteins, while, a baculovirus expression system ensures proper protein modifications, processing, and refolding of complex proteins. Despite these advantages, mammalian cells remain the preferred host for many eukaryotic proteins of pharmaceutical importance, particularly, those requiring post-translational modifications. Recently, the single-celled slime mold, Dictyostelium discoideum (Dd), has emerged as a promising eukaryotic host for the expression of a variety of heterologous recombinant eukaryotic proteins. This organism possesses the complex cellular machinery required for orchestrating post-translational modifications similar to the one observed in higher eukaryotes. This review summarizes the advantages and disadvantages of Dictyostelium as an alternate system compared to other well-established expression systems. The key lessons learned from the expression of human recombinant proteins in this system are reviewed. Also, the strengths, weaknesses, and challenges associated with industrial-scale production of proteins in Dd expression system are discussed.