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Agricultural Biotechnology and Environmental Research
Agriculture and the very environments we exist within are undeniably important and there is an interplay between them. Ecosystems not only provide vital life resources such as clean air and water, they also provide healthy land upon which to grow crops or farm livestock – about half of the ‘useable’ land across the globe is currently pastoral or employed in intensive agriculture. Research to support better understanding and management of agricultural practices, in addition to understanding and maintaining the wide variety of global ecosystems, is critical to a sustainable future for our growing population. There are a range of molecular tools available from Bioline to assist investigations within agricultural or environmental systems, whether through PCR, real-time PCR, or sequencing. Bioline also offers sample-to-answer workflows specially formulated for plant samples, streamlining and simplifying your laboratory time and maximizing result quality from these often difficult sample types.
Agriculture is a broad term encompassing the production, processing and distribution of crops, livestock, feed and other products that support humanity. In addition to traditional crop farming, many other industries fall under the agricultural banner including dairy, livestock, fisheries and forestry. Agriculture is often linked to definitions of civilization and there is evidence of plant breeding and animal domestication going back tens of thousands of years in many cultures.
From very early on, farmers recognized that desirable traits could be enhanced through breeding strategies – improving yields, disease resistance and other characteristics. It was through the study of plant breeding that Mendel identified the genotype-phenotype connection and described the fundamental laws of inheritance, often described as the founding of genetic science. While conventional breeding techniques have proven effective, results can often take multiple generations to manifest and sometimes it is difficult to identify a desired trait within existing populations. With a growing understanding of the genetic sequences and make-up of selected species, a more targeted approach looks at total genomics - linking complex phenotypic traits to the genetic architecture and underlying molecular mechanisms to better instruct cross breeding and back breeding efforts.
Genomics Assisted Breeding
Genomics assisted breeding programs greatly enhance selection efficiency through the construction of comprehensive genetic maps and establishment of marker-trait associations, directing molecular breeding programs for more precise outcomes. Molecular markers such as single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) or microsatellites are used to genotype segregating populations. With ongoing advances and applications of next generation sequencing (NGS), understanding of the genetic diversity within breeding material is increasing at a rapid pace, helping to improve knowledge of so-called ‘orphan’ crops and integrate the various core components of molecular breeding programs. Bioline offers a full range of sample preparation, cloning, sequencing and PCR reagents to support molecular breeding research and applications in all areas of agricultural science.
Breeding and Genetically Modified Organisms
The latest approach to breeding and agricultural crop improvement uses recombinant DNA techniques to genetically modify plants and animals. Individual genes that confer a desired trait, such as insect resistance or herbicide tolerance, are selected and transferred into the desired organism. Unlike conventional breeding, genetic modification (GM) can occur between completely unrelated species, thus opening access to a broader range of traits. GM breeding holds promise to support the future demands of a growing population and a changing climate, using traits such as drought and frost resistance, elevated iron or vitamin levels, or faster growth and maturation periods.
In order for transgenic breeding to be successful, it is necessary to have full understanding of the stability of the introduced gene across generations, as well as potential intergenic, intragenic or other interactions within the genome and within the environment where the crop or organism is being introduced. Nations across the Americas, Europe and Asia have embraced GM technology for many core crops. The US has granted approval for use of over 70 different GM varieties, with over 90% of cotton and 80% of corn across the US planted with GM seed. Approval has also been granted for the first use of GM fish – a salmon requiring half the usual time to grow to market size. Controversies surrounding GM technology mainly focus on the broader potential impacts of GM organisms on the surrounding ecosystems. Unknown future stability of engineered traits, the ability of organisms to transfer genes and breed naturally outside of agricultural constraints, competition with native plants and animals and other unintended and poorly understood outcomes, have seen some governments and organisations show caution towards the technology.
Our global environment and the individual ecosystems within are a complex interplay of a multitude of species and ecological processes and our understanding of all of that is limited at best. Our very existence relies on the continuation of healthy biological and ecological global and local systems and much of today’s research looks at how to define a ‘healthy’ system, how to monitor, measure and manage these systems, in order to maximize sustainable interaction. Apart from the intrinsic value of a healthy environment, essentials such as functional water cycles, soil formation and climate regulation, we also gain value from environmental services such as food, raw materials and fuels. Yet our agricultural and harvesting practices reduce the ability of ecosystems to continue to provide these services, thus effective and sustainable management practices and policies are key to our continued survival and sustainable future.
Although any measurable parameter of an ecosystem will offer valuable information, it is impossible to assess every aspect of complex natural systems, thus researchers look to define ‘indicators’ – biologically relevant measurements or surrogate organisms, that can represent or quantify key measures of an environment and offer insight into potential stress and or resilience of an environment. While typically a small group of key indicator organisms are studied in depth in order to assess ecosystem health, it is well recognized that it is the overall biodiversity within an ecosystem that offers the best opportunity for long-term stability, productivity and maximum functionality. This current epoch has been defined as the Anthropocene, a time when humans are a persistent and driving force of extinction – we are losing species 1,000 times faster than at any other point in history. The true impact of such events is yet to be seen and a sustainable relationship between humans and the global environment is not yet secure. Research efforts continue in an attempt to better define our current and future ecological state, using a range of methods including molecular characterisations of extant diversity and Bioline’s extensive range of molecular tools are well placed to support.
Methods for Assessing Biodiversity
Biodiversity can be defined on many different levels: from macro-level ecosystem diversity; species-level diversity of plants, animals or microorganisms; right down to the genetic diversity within individual populations. While species diversity is the most commonly used method for defining or measuring biodiversity in a given ecosystem, it is, in practice, a difficult variable to measure both in terms of appropriate sample size, spatial scale and method of identification, as well as in the very definition of diversity, be it richness, evenness, differentiation, or abundance. While many studies rely on traditional taxonomic identification methods, molecular-based methods are gaining favor representing rapid, less-subjective pathways for assessment of biodiversity. Total DNA from soil, water or invertebrate samples can be isolated and analysed using PCR with restriction enzyme digestion (PCR-RFLP), cloning and pyro-sequencing, or deep sequencing techniques, to establish DNA-based metrics that can then be linked to extant diversity. Whatever the chosen method, Bioline offers a suite of molecular tools to support the rapid characterisation of biodiversity across a range of environmental samples.
Population Growth and Climate Change– Managing the Future
One of the major concerns for agricultural and environmental scientist’s alike surrounds the management and support of the growing global population and the resulting impact on the environment and availability and health of environmental resources. Population has more than quadrupled over the past century and continues to rise, with a doubling in global food demand projected for the next 50 years. In addition to the basic challenges of increasing food production, it is the impact on the terrestrial and aquatic ecosystems that is critical to manage to ensure a sustainable future. For too long, humanity has demanded more than the regeneration capacity of the Earth’s biosphere and we are now witnessing the result of this overshoot – carbon-induced climate change, large-scale deforestation, collapsing fisheries, soil erosion and salinization, to name a few.
Environmental research efforts ultimately aim to integrate ecological studies with socioeconomic policies in order to affect the much needed changes in global and local policies. Meanwhile agricultural scientists are busy identifying ways to breed crops and livestock that are better placed to weather potential increases in temperature – introducing drought tolerance, salinity tolerance and other relevant characteristics to key crops and food supplies.
Working with a new qPCR target
When looking at a new target of unknown expression level, the amount of RNA required to detect the target of interest depends on the abundance of the target in each sample. For high-copy-number transcripts you may be able to detect in as little as 10 pg, while for low-copy-number transcripts you may require more than 100 ng.
With all new targets (new sets of primers or new qPCR kits), a 10 fold serial dilution should always be run, to validate both the slope and the limit of detection (LOD) (the highest Ct value observed for a truly positive sample, as verified by melt-curve analysis). Only a single peak, which represents the specific PCR product, should be observed on the melt curve, the presence of other peaks indicates the presence of primer–dimers and/or nonspecific PCR products which can contribute to a stronger fluorescence signal and an earlier Ct, but will not be as sensitive.
The best reward for us is hearing achievements through Bioline products, Kristina from West Virginia University speaks about why she gives Accuzyme Mix the “Thumbs Up”
Well done Jesse for working so closely and showing true commitment to his customers!
It’s that time of year again!!!! We are proud to announce Bioline is supporting iGEM teams in 2015. Our already competitive pricing drops 40% for the young and enthusiastic iGEM researchers in the United Kingdom.
‘Sponsorship is very difficult, particularly for small projects such as ours. Bioline are very kindly sponsoring us. A big thank you to them.’ - York iGEM 2014 Team
Popular tools used by successful iGEM teams around the world include:
- Bioline Competent Cells and Cloning Reagents - Transform plasmids from the iGEM kit plates with extremely efficient alpha-select silver or gold efficiency cells.
- MyTaq™ HS - Use MyTaq HS to amplify DNA from your minipreps in as little as 30 minutes, or amplify directly from your colonies! See the MyTaq colony PCR application note or this SOP written by the Danish iGEM2013 team.
- ISOLATE II Plasmid Mini Kit - Pure and very high DNA yields from your transformed colonies.
- VELOCITY DNA Polymerase - Perfect for all your high-fidelity applications.
- HyperLadder™ DNA Markers - Very popular Bioline range of markers for DNA sizing, as used by Craig Venter's research team.
- Real-time PCR Kits - Trusted SensiFAST™ kits for gene expression analysis.
- Core Reagents - High quality buffers, agarose and other essentials.
Get in touch with your local UK Bioline Account Manager to enquire about an iGEM offer pricing plan.
Then, why not let your fellow iGEM researchers know about this great offer using the sharing links below? Also feel free to Tweet or email us if you have any further questions www.bioline.com/social We look forward to hearing from you soon and we wish all iGEM2015 entrants every success in this year's competition!
Terms and Conditions
This offer is valid on any product in our range for any iGEM team in the United Kingdom. No minimum or maximum spend to qualify for the discount. Expires on September 30, 2015. Not valid with any other promo codes or special offers. Please contact us for details of discounts available to iGEM teams in the United States, Germany, France, Singapore and Australia.
Recently the polymerase chain reaction (PCR) turned 30, marking an important birthday for molecular biologists. PCR has transformed molecular research and diagnostics, both in a recognisable form such as single end-point PCR or real-time PCR reactions but also present at the heart of the latest technologies. Digital PCR, molecular diagnostic and even Next Generation Sequencing sample preparation all rely on PCR. The principle behind all these applications is always the same and depends on the original three thermal steps and their repetition: DNA denaturation, primer annealing and primer extension.
The extraordinary longevity of the PCR technique can be explained by its ability to continuously absorb new technologies in its equipment (hardware) and chemistry (DNA polymerase). For example, technologies such as thermoelectric cooling or microfluidics have given a major boost in improving the specificity of the reaction as well as its speed. Simultaneously, the scope and performance of PCR has been improved by the development of new DNA polymerases, with the original enzyme from Thermus aquaticus (Taq) being supplemented by Pyrococcus furiosus family B polymerase (Pfu) as well as many other natural variants or mutants.
The growth in the diversity of applications has generated a demand for PCR reagents with additional properties and better performance such as high-fidelity, higher yield or higher specificity. As a consequence, new thermostable DNA polymerases with different properties have been introduced. The improvement of the PCR fidelity was mainly achieved by the discovery that Pfu could be used in PCR, and possessed a valuable proofreading 3’-5’ exonuclease activity. This was followed by the discovery of other high-fidelity enzymes such as that from Thermococcus kodakaraensis (KOD) and Thermococcus litoralis (Vent DNA polymerase) all aiming at increasing the yield of the PCR up to the level observed with Taq DNA polymerase. In addition to the search of potentially interesting enzymes in natural environments, new variants of thermostable DNA polymerases have been engineered using rational protein engineering or directed molecular evolution approaches. Another major improvement in PCR performance has been achieved by the development of “hot-start” polymerases, where enzyme function is inhibited either by a thermolabile element (such as an antibody or aptamer) or by chemically crosslinking the enzyme. In both cases, the polymerase only becomes fully activated during an initial denaturation step of the PCR, carried out at relatively high temperature (usually above 94°C).The inhibition of the polymerase at room temperature avoids modifications of the primers by the polymerase, which not only facilitates the experimental set-up, but also increases the specificity of the PCR.
Today, the challenges for PCR are even bigger: a higher level of sensitivity is required across a vast range of applications ranging from single cell analysis to fast real-time PCR directly from crude material. A very low amount of available template per PCR, the presence of PCR inhibitors or the introduction of ultra-fast thermocyclers, makes the stakes even higher and some limitations in the technology abilities may soon be reached. So, in order to overcome these current challenges and deliver higher sensitivity or faster cycling abilities it is necessary to improve performance of PCR further.
In order to address these challenges, Bioline has adopted a very simple but global approach based on a careful optimization of all the components of PCR. This strategy is centred on harnessing the best performance of ultra-pure components and placing them in a finely optimized physico-chemical environment. The purity of the components does not stop at the DNA polymerase protein itself but extends to the dNTP, additives and other chemicals involved, including, where possible, the template. Highly pure components allow a fine control of the enzyme/buffer system properties which enables the PCR reagent to be tailored for a particular application. This strategy by Bioline benefits PCR performance and minimizes contamination issues, non-specific amplification and false positives as well as decreases the batch to batch variation. In combination this increases the robustness and reliability of all Bioline's kits.
To achieve higher PCR performance levels, the particularities of specific applications are considered and the enzyme/buffer system is totally reformulated and adapted to suit assay requirements. Each and every component of the PCR reaction is carefully selected and its concentration optimized. Furthermore, the interactions between components and their effects on the PCR performance are also considered and the best concentrations and ratios are selected. This tailored approach to PCR also takes into account the preparation of the template. A finely tuned PCR would not deliver maximum sensitivity or yield without a template of good quality. Removal of any contaminants from the sample or carry-over of the extraction and purification process is very important as it avoids any uncontrolled effects of the template addition on the physico-chemical environment of the DNA polymerase, keeping the system perfectly optimized for the application.
In conclusion, through the careful combination of pure enzymes and chemicals, ultra-high purity nucleotides and strict quality standards, Bioline has pushed the PCR performance further, delivering a reliable and powerful amplification platform at the service of today’s molecular biologists.