Building science

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Building_science 

Small furnace capable of 600°C and of applying a static load for testing building materials

Building science is the science and technology-driven collection of knowledge to provide better indoor environmental quality (IEQ), energy-efficient built environments, and occupant comfort and satisfaction. Building physics, architectural science, and applied physics are terms used for the knowledge domain that overlaps with building science. In building science, the methods used in natural and hard sciences are widely applied, which may include controlled and quasi-experiments, randomized control, physical measurements, remote sensing, and simulations. On the other hand, methods from social and soft sciences, such as case study, interviews & focus group, observational method, surveys, and experience sampling, are also widely used in building science to understand occupant satisfaction, comfort, and experiences by acquiring qualitative data. One of the recent trends in building science is a combination of the two different methods. For instance, it is widely known that occupants' thermal sensation and comfort may vary depending on their sex, age, emotion, experiences, etc. even in the same indoor environment. Despite the advancement in data extraction and collection technology in building science, objective measurements alone can hardly represent occupants' state of mind such as comfort and preference. Therefore, researchers are trying to measure both physical contexts and understand human responses to figure out complex interrelationships.

Building science traditionally includes the study of indoor thermal environment, indoor acoustic environment, indoor light environment, indoor air quality, and building resource use, including energy and building material use. These areas are studied in terms of physical principles, relationship to building occupant health, comfort, and productivity, and how they can be controlled by the building envelope and electrical and mechanical systems. The National Institute of Building Sciences (NIBS) additionally includes the areas of building information modeling, building commissioning, fire protection engineering, seismic design and resilient design within its scope.

One of the applications of building science is to provide predictive capability to optimize the building performance and sustainability of new and existing buildings, understand or prevent building failures, and guide the design of new techniques and technologies.

Applications

During the architectural design process, building science knowledge is used to inform design decisions to optimize building performance. Design decisions can be made based on knowledge of building science principles and established guidelines, such as the NIBS Whole Building Design Guide (WBDG) and the collection of ASHRAE Standards related to building science.

Computational tools can be used during design to simulate building performance based on input information about the designed building envelope, lighting system, and mechanical system. Models can be used to predict operational energy use, solar heat and radiation distribution, air flow, and other physical phenomena within the building. These tools are valuable for evaluating a design and ensuring it will perform within an acceptable range before construction begins. Many of the available computational tools analyze building performance goals and perform design optimization. The accuracy of the models is influenced by the modeler's knowledge of building science principles and by the amount of validation performed for the specific program.

When existing buildings are being evaluated, measurements and computational tools can be used to evaluate performance based on measured existing conditions. An array of in-field testing equipment can be used to measure temperature, moisture, sound levels, air pollutants, or other criteria. Standardized procedures for taking these measurements are provided in the Performance Measurement Protocols for Commercial Buildings. For example, thermal infrared (IR) imaging devices can be used to measure temperatures of building components while the building is in use. These measurements can be used to evaluate how the mechanical system is operating and if there are areas of anomalous heat gain or heat loss through the building envelope.

Measurements of conditions in existing buildings are used as part of post occupancy evaluations. Post occupancy evaluations may also include surveys of building occupants to gather data on occupant satisfaction and well-being and to gather qualitative data on building performance that may not have been captured by measurement devices.

Many aspects of building science are the responsibility of the architect (in Canada, many architectural firms employ an architectural technologist for this purpose), often in collaboration with the engineering disciplines that have evolved to handle 'non-building envelope' building science concerns: Civil engineering, Structural engineering, Earthquake engineering, Geotechnical engineering, Mechanical engineering, Electrical engineering, Acoustic engineering, & fire code engineering. Even the interior designer will inevitably generate a few building science issues.

Topics

Daylighting and visual comfort

Daylighting is the controlled admission of natural light, direct sunlight, and diffused skylight into a building to reduce electric lighting and save energy. A daylighting system comprises of not just daylight apertures, such as skylights and windows, but is coupled with a daylight-responsive lighting control system. Daylight positively impacts the psychological and physiological health of a human being by stimulating the human circadian rhythm, which can lower depression, improve sleep quality, reduce lethargy, and prevent illness.

However, studies do not always lead to a positive correlation between maximizing daylighting availability and human comfort and health. When large windows exist within the buildings, we need to control the quantity and the quality of the visual environment. A lack of attention to visual comfort issues often makes the best daylighting intentions ineffective due to excessive brightness and high contrast luminance ratios within the space which result in glare. Illuminating Engineering Society (IES)’s Lighting Handbook defines glare as the sensation produced by luminance levels in the visual field, sufficiently greater than those that our eyes can adapt to, that causes discomfort or loss in visual performance or visibility. Glare interferes with visual perception caused by an uncomfortably bright light source or reflection. If the occupants experience visual discomfort from excessive sunlight penetration through the windows of the buildings, they may wish to close the shading devices which would decrease the daylight availability and increase the electric lighting energy consumption.

Daylighting and visual comfort is an extensively studied topic in building science that allows for successful harvesting of daylighting and energy savings. It is critical that architects, engineers, and building owners use daylight and glare metrics to evaluate lighting conditions in daylit spaces for occupant health and comfort.

Indoor environmental quality (IEQ)

Indoor environmental quality (IEQ) refers to the quality of a building's environment in relation to the health and wellbeing of those who occupy space within it. IEQ is determined by many factors, including lighting, air quality, and temperature. Workers are often concerned that they have symptoms or health conditions from exposures to contaminants in the buildings where they work. One reason for this concern is that their symptoms often get better when they are not in the building. While research has shown that some respiratory symptoms and illnesses can be associated with damp buildings, it is still unclear what measurements of indoor contaminants show that workers are at risk for disease. In most instances where a worker and his or her physician suspect that the building environment is causing a specific health condition, the information available from medical tests and tests of the environment is not sufficient to establish which contaminants are responsible. Despite uncertainty about what to measure and how to interpret what is measured, research shows that building-related symptoms are associated with building characteristics, including dampness, cleanliness, and ventilation characteristics.

Indoor environments are highly complex and building occupants may be exposed to a variety of contaminants (in the form of gases and particles) from office machines, cleaning products, construction activities, carpets and furnishings, perfumes, cigarette smoke, water-damaged building materials, microbial growth (fungal, mold, and bacterial), insects, and outdoor pollutants. Other factors such as indoor temperatures, relative humidity, and ventilation levels can also affect how individuals respond to the indoor environment. Understanding the sources of indoor environmental contaminants and controlling them can often help prevent or resolve building-related worker symptoms. Practical guidance for improving and maintaining the indoor environment is available.

Building indoor environment covers the environmental aspects in the design, analysis, and operation of energy-efficient, healthy, and comfortable buildings. Fields of specialization include architecture, HVAC design, thermal comfort, indoor air quality (IAQ), lighting, acoustics, and control systems.

HVAC systems

The mechanical systems, usually a sub-set of the broader Building Services, used to control the temperature, humidity, pressure and other select aspects of the indoor environment are often described as the Heating, Ventilating, and Air-Conditioning (HVAC) systems. These systems have grown in complexity and importance (often consuming around 20% of the total budget in commercial buildings) as occupants demand tighter control of conditions, buildings become larger, and enclosures and passive measures became less important as a means of providing comfort.

Building science includes the analysis of HVAC systems for both physical impacts (heat distribution, air velocities, relative humidities, etc.) and for effect on the comfort of the building's occupants. Because occupants' perceived comfort is dependent on factors such as current weather and the type of climate the building is located in, the needs for HVAC systems to provide comfortable conditions will vary across projects. In addition, various HVAC control strategies have been implemented and studied to better contribute to occupants' comfort. In the U.S., ASHRAE has published standards to help building managers and engineers design and operate the system. In the UK, a similar guideline was published by CIBSE. Apart from industry practice, advanced control strategies are widely discussed in research as well. For example, closed-loop feedback control can compare air temperature set-point with sensor measurements; demand response control can help prevent electric power-grid from having peak load by reducing or shifting their usage based on time-varying rate. With the improvement from computational performance and machine learning algorithms, model prediction on cooling and heating load with optimal control can further improve occupants comfort by pre-operating the HVAC system. It is recognized that advanced control strategies implementation is under the scope of developing Building Automation System (BMS) with integrated smart communication technologies, such as Internet of Things (IoT). However, one of the major obstacles identified by practitioners is the scalability of control logics and building data mapping due to the unique nature of building designs. It was estimated that due to inadequate interoperability, building industry loses $15.8 billion annually in the U.S. Recent research projects like Haystack and Brick intend to address the problem by utilizing metadata schema, which could provide more accurate and convenient ways of capturing data points and connection hierarchies in building mechanical systems. With the support of semantic models, automated configuration can further benefit HVAC control commissioning and software upgrades.

Enclosure (envelope) systems

The building enclosure is the part of the building that separates the indoors from the outdoors. This includes the wall, roof, windows, slabs on grade, and joints between all of these. The comfort, productivity, and even health of building occupants in areas near the building enclosure (i.e., perimeter zones) are affected by outdoor influences such as noise, temperature, and solar radiation, and by their ability to control these influences. As part of its function, the enclosure must control (not necessarily block or stop) the flow of moisture, heat, air, vapor, solar radiation, insects, or noise, while resisting the loads imposed on the structure (wind, seismic). Daylight transmittance through glazed components of the facade can be analyzed to evaluate the reduced need for electric lighting.

Building sustainability

Building sustainability, often referred to as sustainable design, integrates strategies to lower building environmental impacts, including lowering both operational carbon, which is the emissions from energy use during a building's life, and embodied carbon, which accounts for the emissions from material production and construction. Building sustainability practices aim to design with consideration for future resources and environmental realities.

Buildings are responsible for approximately 40% of global energy consumption and 13% carbon emissions, primarily related to building HVAC systems operation. Reducing operational carbon is critical to mitigate climate change. To address these emissions, renewable energy sources, such as solar and wind energy, are adopted by the building industry to support electricity generation. However, the electricity demand profile shows imbalance between supply and demand, which is known as the 'duck curve'. This could impact on maintaining grid system stability. Therefore, other strategies such as thermal energy storage systems are developed to achieve higher levels of sustainability by reducing grid peak power.

A push towards zero-energy building also known as Net-Zero Energy Building has been present in the Building Science field. The qualifications for Net Zero Energy Building Certification can be found on the Living Building Challenge website.

Embodied Carbon and Decarbonization

Embodied carbon refers to the total carbon emissions associated with the entire life cycle of a building material (i.e. material extraction, manufacturing and production, transportation, construction, and end of life). As building performance research has decreased operational carbon, there has been an increase in embodied carbon within the building sector, partly due to the higher material demands of energy-efficient designs. This shift has underscored the need to address embodied carbon alongside operational emissions to achieve holistic decarbonization.

Building decarbonization is most impactful during early-stage design, where materials, systems, and structural choices can be optimized to reduce embodied carbon and improve operational efficiency before moving forward in development stages. Structural materials, such as steel and concrete, contribute significantly to a building's embodied carbon footprint. Strategies to mitigate these impacts include material substitution, incorporating recycled and reused materials, and adopting low-carbon manufacturing processes.

Challenges in addressing embodied carbon include insufficient data, lack of standardization, cost considerations, and regulatory barriers. Reliable databases are often limited, region-specific, and inconsistent, making it difficult to apply universally. Existing standards are often voluntary and vary in scope, making comparisons and benchmarking difficult. Life cycle assessment standards for evaluating building embodied carbon include ISO 14040, ISO 14044, EN 15978, PAS 2050, and ReCiPe. These frameworks provide structured approaches to evaluate and quantify life cycle environmental impacts, such as embodied carbon.

Addressing embodied carbon is a growing aspect of building science, becoming critical for advancing building sustainability efforts and reducing the environmental impact of the built environment.

Post-Occupancy Evaluation (POE)

POE is a survey-based method to measure the building performance after the built environment was occupied. The occupant responses were collected through structured or open inquiries. Statistical methods and data visualization were often used to suggest which aspects(features) of the building were supportive or problematic to the occupants. The results may become design knowledge for architects to design new buildings or provide a data-basis to improve the current environment.

Certification

Although there are no direct or integrated professional architecture or engineering certifications for building science, there are independent professional credentials associated with the disciplines. Building science is typically a specialization within the broad areas of architecture or engineering practice. However, there are professional organizations offering individual professional credentials in specialized areas. Some of the most prominent green building rating systems are:

• BREEAM (Building Research Establishment Environmental Assessment Method), which is the world's longest established sustainable building assessment system, developed by the Building Research Establishment;
• LEED (Leadership in Energy and Environmental Design), developed by the U.S. Green Building Council;
• Green Star (Australia), which is the main green building rating system in Australia, developed by the Green Building Council of Australia;
• WELL which is delivered by the International WELL Building Institute and administered by the Green Business Certification Inc.;
• CASBEE (Comprehensive Assessment System for Built Environment Efficiency), which is the main green building rating system in Japan;
• Living Building Challenge, developed by the International Living Future Institute;
• Passivhaus (Passive House), developed by the Passive House Institute, which is an internationally recognized, performance-based energy standard in construction.
• EcoChef, a sustainability certification designed specifically for commercial kitchens, evaluating energy efficiency, waste reduction, and operational performance in the foodservice industry, developed by Forward Dining Solutions.

There are other building sustainability accreditation and certification institutions as well. Also in the US, contractors certified by the Building Performance Institute, an independent organization, advertise that they operate businesses as Building Scientists. This is questionable due to their lack of scientific background and credentials. On the other hand, more formal building science experience is true in Canada for most of the Certified Energy Advisors. Many of these trades and technologists require and receive some training in very specific areas of building science (e.g., air tightness, or thermal insulation).

List of principal building science journals

Building and Environment: This international journal publishes original research papers and review articles related to building science, urban physics, and human interaction with the indoor and outdoor built environment. The journal's most cited articles cover topics such as occupant behavior in buildings, green building certification systems, and tunnel ventilation systems. Publisher: Elsevier. Impact Factor (2019): 4.971

Energy and Buildings: This international journal publishes articles with explicit links to energy use in buildings. The aim is to present new research results, and new proven practice aimed at reducing the energy needs of a building and improving indoor air quality. The journal's most cited articles cover topics such as prediction models for building energy consumption, optimization models of HVAC systems, and life cycle assessment. Publisher: Elsevier. Impact Factor (2019): 4.867

Indoor Air: This international journal publishes papers reflecting the broad categories of interest in the field of indoor environment of non-industrial buildings, including health effects, thermal comfort, monitoring and modelling, source characterization, and ventilation (architecture) and other environmental control techniques. The journal's most cited articles cover topics such as the impact of indoor air pollutants and thermal conditions on occupant performance, the movement of droplets in indoor environments, and the effects of ventilation rates on occupant health. Publisher: John Wiley & Sons. Impact Factor (2019): 4.739

Architectural Science Review: Founded at the University of Sydney, Australia in 1958, this journal aims to promote the development, accumulation, and application of scientific knowledge on a wide range of environmental topics. According to the journal description, the topics may include but not limited to building science and technology, environmental sustainability, structures and materials, audio and acoustics, illumination, thermal systems, building physics, building services, building climatology, building economics, ergonomics, history and theory of architectural science, the social sciences of architecture. Publisher: Taylor & Francis Group

Building Research and Information: This journal focuses on buildings, building stocks and their supporting systems. Unique to BRI is a holistic and transdisciplinary approach to buildings, which acknowledges the complexity of the built environment and other systems over their life. Published articles utilize conceptual and evidence-based approaches which reflect the complexity and linkages between culture, environment, economy, society, organizations, quality of life, health, well-being, design and engineering of the built environment. The journal's most cited articles cover topics such as the gap between performance and actual energy consumption, barriers and drivers for sustainable building, and the politics of resilient cities. Publisher: Taylor & Francis Group. Impact Factor (2019): 3.887

Journal of Building Performance Simulation: This international, peer-reviewed journal publishes high quality research and state of the art “integrated” papers to promote scientifically thorough advancement of all the areas of non-structural performance of a building and particularly in heat transfer, air, moisture transfer. The journal's most cited articles cover topics such as co-simulation of building energy and control systems, the Buildings library, and the impact of occupant's behavior on building energy demand. Publisher: Taylor & Francis Group. Impact Factor (2019): 3.458

LEUKOS: This journal publishes engineering developments, scientific discoveries, and experimental results related to light applications. Topics of interest include optical radiation, light generation, light control, light measurement, lighting design, daylighting, energy management, energy economics, and sustainability. The journal's most cited articles cover topics such as lighting design metrics, psychological processes influencing lighting quality, and the effects of lighting quality and energy-efficiency on task performance, mood, health, satisfaction, and comfort. Publisher: Taylor & Francis Group. Impact Factor (2019): 2.667

Building Simulation: This international journal publishes original, high quality, peer-reviewed research papers and review articles dealing with modeling and simulation of buildings including their systems. The goal is to promote the field of building science and technology to such a level that modeling will eventually be used in every aspect of building construction as a routine instead of an exception. Of particular interest are papers that reflect recent developments and applications of modeling tools and their impact on advances of building science and technology. Publisher: Springer Nature. Impact Factor (2019): 2.472

Applied Acoustics: This journal covers research findings related to practical applications of acoustics in engineering and science. The journal's most cited articles related to building science cover topics such as the prediction of the sound absorption of natural materials, the implementation of low-cost urban acoustic monitoring devices, and sound absorption of natural kenaf fibers. Publisher: Elsevier. Impact Factor (2019): 2.440

Lighting Research & Technology: This journal covers all aspects of light and lighting, including the human response to light, light generation, light control, light measurement, lighting design equipment, daylighting, energy efficiency of lighting design, and sustainability. The journal's most cited articles cover topics such as light as a circadian stimulus for architectural lighting, human perceptions of color rendition, and the influence of color gamut size and shape on color preference. Publisher: SAGE Publishing. Impact Factor (2019): 2.226

Microbiomes of the built environment

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/Microbiomes_of_the_built_environment

Microbiomes of the built environment is a field of inquiry into the communities of microorganisms that live in human constructed environments like houses, cars and water pipes. It is also sometimes referred to as microbiology of the built environment.

• The field has accelerated somewhat in recent years, with significant funding from the Alfred P. Sloan Foundation and with the increase attention being given to microbiomes and communities of microbes generally.
• The National Academies of Sciences, Engineering, and Medicine of the USA is conducting a study of this field with the study entitled "Microbiomes of the Built Environment: From Research to Application".
• The American Association for the Advancement of Science ran a symposium on the topic in 2014.
• The American Academy of Microbiology had a colloquium on this topic in September 2015 and published a report "Microbiology of Built Environments".

A 2016 paper by Brent Stephens highlights some of the key findings of studies of "microbiomes of the indoor environment". These key findings include those listed below:

• "Culture-independent methods reveal vastly greater microbial diversity compared to culture-based methods"
• "Indoor spaces often harbor unique microbial communities"
• "Indoor bacterial communities often originate from indoor sources."
• "Humans are also major sources of bacteria to indoor air"
• "Building design and operation can influence indoor microbial communities."

The microbiomes of the built environment are being studied for multiple reasons including how they may impact the health of humans and other organisms occupying the built environment but also some non health reasons such as diagnostics of building properties, for forensic application, impact on food production, impact on built environment function, and more.

Studied environments

Extensive research has been conducted on individual microbes found in the built environment. More recently there has been a significant expansion in the number of studies that are examining the communities of microbes found in the built environment. Such studies have covered a range of environments.

• Buildings. Examples include homes, dormitories, offices, hospitals, operating rooms, NICUs, classrooms, transportation facilities such as train and subway stations, food production facilities  (e.g. dairies, wineries, cheesemaking facilities, sake breweries and beer breweries, aquaria, libraries, cleanrooms, zoos, animal shelters, farms, and chicken coops and housing.
• Vehicles. Examples include airplanes, ships, trains, automobiles and space vehicles including the International Space Station, MIR, the Mars Odyssey, the Herschel Spacecraft.
• Water Systems. Examples include shower heads, children's paddling pools, municipal water systems, recirculating aquaculture systems, drinking water and premise plumbing systems and saunas.
• Other. Examples include art and cultural heritage items, clothing, kitchen sponges, and household appliances such as dishwashers  and washing machines.

Findings

General biogeography

Overall the many studies that have been conducted on the microbiomes of the built environment have started to identify some general patterns regarding the microbes are found in various places. Different areas and kinds of buildings are linked to different sorts of microbiota. Pakpour et al. in 2016 reviewed the patterns relating to the presence of archaea in indoor environments (based on analysis of rRNA gene sequence data).

Human health

Many studies have documented possible human health implications of the microbiomes of the built environment.

• Newborn colonization. The microbes that colonize newborns come in part from the built environment (e.g., hospital rooms). This appears to be especially true for babies born by C-section (see for example Shin et al. 2016) and also babies that spend time in a NICU.
• Risk of allergy and asthma. The risk of allergy and asthma is correlated to differences in the built environment microbiome. Some experimental tests (e.g., in mice) have suggested that these correlations may actually be causal (i.e., the differences in the microbiomes may actually lead to differences in risk of allergy or asthma). Review papers on this topic include Casas et al. 2016 and Fujimura and Lynch 2015. The microbiome of household dust is correlated to the childhood risk of allergy, asthma and phenotypes connected to these ailments. The impact of the microbiome of the built environment on the risk of allergy and asthma and other inflammatory or immune conditions is a possible mechanism underlying what is known as the hygiene hypothesis.
• Mental health. In a 2015 review Hoisington et al. discuss possible connections between the microbiology of the built environment and human health. The concept presented in this paper is that more and more evidence is accumulating that the human microbiome has some impact on the brain and thus if the built environment either directly or indirectly impacts the human microbiome, this in turn could have impacts on human mental health.
• Pathogen transmission. Many pathogens are transmitted in the built environment and may also reside in the built environment for some period of time. Good examples include influenza, norovirus, legionella, and MRSA. The study of the transmission and survival of these pathogens is a component of studies of microbiomes of the built environment.
• Indoor Air Quality. The study of indoor air quality and the health impact of such air quality is linked at least in part to microbes in the built environment since they can impact directly or indirectly indoor air quality.

Components of the Built Environment that Likely Impact Microbiomes

A major component of studies of microbiomes of the built environment involves determining how components of the built environment impact these microbes and microbial communities. Factors that are thought to be important include humidity, pH, chemical exposures, temperature, filtration, surface materials, and air flow. There has been an effort to develop standards for what built environment "metadata" to collect associated with studies of the microbial communities in the built environment. A 2014 paper reviews the tools that are available to improve the built environment data that is collected associated with such studies. Data covered in this review include building characteristics and environmental conditions, HVAC system characteristics and ventilation rates, human occupancy and activity measurements, surface characterizations and air sampling and aerosol dynamics.

Impact of Microbiomes on the Built Environment

Just as the built environment has an impact on the microbiomes found therein, the microbial communities of the built environment can impact the built environment itself. Examples include degradation of building materials, altering fluid and airflow, generating volatiles, and more.

Possible Uses in Forensics

The microbiome of the built environment has some potential for being used as a feature for forensic studies. Most of these applications are still in the early research phase. For example, it has been shown that people leave behind a somewhat diagnostic microbial signature when they type on computer keyboards, use phones or occupy a room.

Odor

There has been a significant amount of research on the role that microbes play in various odors in the built environment. For example, Diekmann et al. examined the connection between volatile organic emissions in automobile air conditioning units. They reported that the types of microbes found were correlated to the bad odors found. Park and Kim examined which microbes found in an automobile air conditioner could produce bad smelling volatile compounds and identified candidate taxa producing some such compounds.

Methods

Many methods are used to study microbes in built environment. A review of such methods are some of the challenges in using them was published by NIST. Hoisington et al. in 2014 reviewed methods that could be used by building professionals to study the microbiology of the built environment. Methods used in the study of microbes in the built environment include culturing (with subsequent studies of the cultured microbes), microscopy, air, water and surface sampling, chemical analyses, and culture independent DNA studies such as ribosomal RNA gene PCR and metagenomics.

Microbial ecology

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Microbial_ecology

The great plate count anomaly. Counts of cells obtained via cultivation are orders of magnitude lower than those directly observed under the microscope. This is because microbiologists are able to cultivate only a minority of naturally occurring microbes using current laboratory techniques, depending on the environment.

Microbial ecology (or environmental microbiology) is a discipline where the interaction of microorganisms and their environment are studied. Microorganisms are known to have important and harmful ecological relationships within their species and other species. Many scientists have studied the relationship between nature and microorganisms: Martinus Beijerinck, Sergei Winogradsky, Louis Pasteur, Robert Koch, Lorenz Hiltner, Dionicia Gamboa and many more; to understand the specific roles that these microorganisms have in biological and chemical pathways and how microorganisms have evolved. Currently, there are several types of biotechnologies that have allowed scientists to analyze the biological/chemical properties of these microorganisms also.

Many of these microorganisms have been known to form different symbiotic relationships with other organisms in their environment. Some symbiotic relationships include mutualism, commensalism, amenalism, and parasitism.

In addition, it has been discovered that certain substances in the environment can kill microorganisms, thus preventing them from interacting with their environment. These substances are called antimicrobial substances. These can be antibiotic, antifungal, or antiviral.

Influential Scientists

Louis Pasteur

Martinus Beijerinck invented the enrichment culture, a fundamental method of studying microbes from the environment. Sergei Winogradsky was one of the first researchers to attempt to understand microorganisms outside of the medical context—making him among the first students of microbial ecology and environmental microbiology—discovering chemosynthesis and developing the Winogradsky column in the process.

Louis Pasteur was a French chemist who derived key microbial principles that we use today: microbial fermentation, pasteurization, germ theory, and vaccines. These principles have served as a foundation for scientists in viewing the relationship between microbes and their environment. For example, Pasteur disproved the theory of spontaneous generation, the belief of life arising from nonliving materials. Pasteur stated that life can only come from life and not nonliving materials. This led to the idea that microorganisms were responsible for the microbial growth in any environment.

Robert Koch was a physician-scientist who implemented oil-immersion lens and a condenser while using microscopes, to increase the imagery of viewing bacteria. This led Koch to be the first publisher of bacteria photographs. As a result, Koch was able to study wound infections in animals at the microscopic level. He was able to distinguish distinct bacteria species, which led him to believe that the best way to study a certain disease is to focus on a specific pathogen. In 1879, Koch started to develop "pure" cultures to grow bacteria colonies. These advancements led Koch to solve the Cholera endemic in India during the year 1883. Koch's laboratory techniques and materials led him to conclude that the use of unfiltered water was causing the Cholera endemic, since it contained bacteria causing intestinal harm in humans.

Lorenz Hiltner is known as one of the pioneers in "microbial ecology." His research focused on how microbials in the rhizosphere provided nutrients to plants. Hiltner stated that the quality of plant products was a result of the plant's roots microflora. One of Hiltner contributions to the study of plant nutrition and soil bacteriology was creating antimicrobial seeds covered with mercury chloride. The sole purpose of creating the antimicrobial seeds were to protect the seeds from the harmful effects of pathogenic fungi. In addition, he recognized the known bacteria that were responsible for the nitrogen cycle: denitrification, nitrification, and nitrogen fixation.

Dionicia Gamboa is a prime example of how scientists are still trying to understand the relationship between microorganisms and nature. Gamboa is a Peruvian biologist who has dedicated her career towards treating malaria and leishmaniasis microorganisms. In 2009, Gamboa and her colleagues published a paper on treating different strains of malaria and leishmaniasis microorganisms, using plant extracts from the amazon. To add on, Gamboa has studied different ways to accurately detect malaria and leishmaniasis microorganisms in humans, using PCR and serology. Her studies have helped understand the epidemiology of these microorganisms, to reduce the interaction with them in nature and their harmful effects.

Important Microbial Roles in The Environment

Microorganisms are the backbone of all ecosystems, even in areas where photosynthesis cannot take place. For example, chemosynthetic microorganisms are the primary producers in extreme environments, such as high temperature geothermal environments. In these extreme conditions, the chemosynthetic microbes provide energy and carbon to other organisms. Chemosynthetic microorganisms gain energy by oxidizing inorganic compounds such as hydrogen, nitrite, ammonia, sulfur and iron (II). These organisms can be found in both aerobic and anaerobic environment.

The nitrogen cycle, phosphorus cycle, sulphur cycle, and carbon cycle depend on microorganisms also. Each cycle involves microorganisms in certain processes. For example, nitrogen gas makes up 78% of the Earth's atmosphere, but it is almost chemically inert; as a result, it is unavailable to most organisms. It has to be converted biologically to an available form by microorganism, through nitrogen fixation. Through these biogeochemical cycles, microorganisms are able to make nutrients such as nitrogen, phosphorus and potassium available in the soil. Microorganisms play a role in solubilizing phosphate, improving soil health, and plant growth.

Microbial Applications in Biotechnology

Microbial interactions are found in bioremediation. Bioremediation is a technology that removes contaminants from soil and wastewater using microorganisms. Examples of some microorganisms that play a role in bioremediation are the following: Pseudomonas, Bacillus, Arthrobacter, Corynebacterium, Methosinus, Rhodococcus, Stereum hirsutum, methanogens, Aspergilus niger, Pleurotus ostreatus, Rhizopus arrhizus, Azotobacter, Alcaligenes, Phormidium valderium, and Ganoderma applantus.

Microbial Evolution

Due to high levels of horizontal gene transfer among microbial communities, microbial ecology is also important to the studies of evolution.

Microbial Symbiotic Relationships

Mutualism

Mutualism is a close relationship between two different species in which each has a positive effect on the other . In mutualism, one partner provides service to the other partner and also receives service from the other partner as well. Mutualism in microbial ecology is a relationship between microbial species and other species (example humans) that allows for both sides to benefit. Microorganisms form mutualistic relationship with other microorganism, plants or animals. One example of microbe-microbe interaction would be syntrophy, also known as cross-feeding, of which Methanobacterium omelianskii is a classical example. This consortium is formed by an ethanol fermenting organism and a methanogen. The ethanol-fermenting organism provides the archaeal partner with the H₂, which this methanogen needs in order to grow and produce methane. Syntrophy has been hypothesized to play a significant role in energy and nutrient-limited environments, such as deep subsurface, where it can help the microbial community with diverse functional properties to survive, grow and produce maximum amount of energy. Anaerobic oxidation of methane (AOM) is carried out by mutualistic consortium of a sulfate-reducing bacterium and an anaerobic methane-oxidizing archaeon. The reaction used by the bacterial partner for the production of H₂ is endergonic (and so thermodynamically unfavored) however, when coupled to the reaction used by archaeal partner, the overall reaction becomes exergonic. Thus the two organisms are in a mutualistic relationship which allows them to grow and thrive in an environment, deadly for either species alone. Lichen is an example of a symbiotic organism.

Microorganisms also engage in mutualistic relationship with plants and a typical example of such relationship is arbuscular mycorrhizal (AM) relationship, a symbiotic relationship between plants and fungi. This relationship begins when chemical signals are exchange between the plant and the fungi leading to the metabolic stimulation of the fungus. The fungus then attacks the epidermis of the plant’s root and penetrates its highly branched hyphae into the cortical cells of the plant. In this relationship, the fungi gives the plant phosphate and nitrogen obtained from the soil with the plant in return providing the fungi with carbohydrate and lipids obtained from photosynthesis. Also, microorganisms are involve in mutualistic relationship with mammals such as humans. As the host provides shelter and nutrient to the microorganisms, the microorganisms also provide benefits such as helping in the growth of the gastrointestinal tract of the host and protecting host from other detrimental microorganisms.

Commensalism

Commensalism is very common in microbial world, literally meaning "eating from the same table". It is a relationship between two species where one species benefits with no harm or benefit for the other species. Metabolic products of one microbial population are used by another microbial population without either gain or harm for the first population. There are many "pairs "of microbial species that perform either oxidation or reduction reaction to the same chemical equation. For example, methanogens produce methane by reducing CO₂ to CH₄, while methanotrophs oxidise methane back to CO₂.

Amensalism

Amensalism (also commonly known as antagonism) is a type of symbiotic relationship where one species/organism is harmed while the other remains unaffected. One example of such a relationship that takes place in microbial ecology is between the microbial species Lactobacillus casei and Pseudomonas taetrolens. When co-existing in an environment, Pseudomonas taetrolens shows inhibited growth and decreased production of lactobionic acid (its main product) most likely due to the byproducts created by Lactobacillus casei during its production of lactic acid.

Parasitism

Certain microorganisms are known to have a host-parasite interaction with other organisms. For example, phytopathogenic fungi are known to infect and damage plants. The phytopathogenic fungi is a major issue in agriculture, because it has the capacity to infect its host by their root system. This is a major issue because the symptoms of the infection are not easily detected. Another example of a parasitic microorganism is the nematode. These organisms are known to cause river blindness and lymphatic filariasis in humans. These organisms are transmitted to hosts through different mosquito species from the following groups: Aedes, Anopheles, and Culex.

Antimicrobials

Antimicrobials are substances that are capable of killing microorganism. Antimicrobial can be antibacterial or antibiotic, antifungal or antiviral substance and most of these substance are natural products or may have been obtain from natural products. Natural products are therefore vital in the discovery of pharmaceutical agents. Most of the naturally obtained antibiotics are produced by organism under the phylum Actinobacteria. The genus Streptomyces are responsible for most of the antibiotic substances produced by Actinobacteria. These natural products with antimicrobial properties belong to the terpenoids, spirotetronate, tetracenedione, lactam, and other groups of compounds. Examples include napyradiomycin, nomimicin, formicamycin, and isoikarugamycin, Some metals, particularly copper, silver, and gold also have antimicrobial properties. Using antimicrobial copper-alloy touch surfaces is a technique that has begun to be used in the 21st century to prevent the transmission of bacteria. Silver nanoparticles have also begun to be incorporated into building surfaces and fabrics, although concerns have been raised about the potential side-effects of the tiny particles on human health. Due to the antimicrobial properties certain metals possess, products such as medical devices are made using those metals.

SNP genotyping

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/SNP_genotyping 

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles (where the rare allele frequency is > 1%). SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

Hybridization-based methods

Several applications have been developed that interrogate SNPs by hybridizing complementary DNA probes to the SNP site. The challenge of this approach is reducing cross-hybridization between the allele-specific probes. This challenge is generally overcome by manipulating the hybridization stringency conditions.

Dynamic allele-specific hybridization

Dynamic allele-specific hybridization (DASH) genotyping takes advantage of the differences in the melting temperature in DNA that results from the instability of mismatched base pairs. The process can be vastly automated and encompasses a few simple principles.

In the first step, a genomic segment is amplified and attached to a bead through a PCR reaction with a biotinylated primer. In the second step, the amplified product is attached to a streptavidin column and washed with NaOH to remove the unbiotinylated strand. An allele-specific oligonucleotide is then added in the presence of a molecule that fluoresces when bound to double-stranded DNA. The intensity is then measured as temperature is increased until the melting temperature (Tm) can be determined. A SNP will result in a lower than expected Tm.

Because DASH genotyping is measuring a quantifiable change in Tm, it is capable of measuring all types of mutations, not just SNPs. Other benefits of DASH include its ability to work with label free probes and its simple design and performance conditions.

Molecular beacons

SNP detection through molecular beacons makes use of a specifically engineered single-stranded oligonucleotide probe. The oligonucleotide is designed such that there are complementary regions at each end and a probe sequence located in between. This design allows the probe to take on a hairpin, or stem-loop, structure in its natural, isolated state. Attached to one end of the probe is a fluorophore and to the other end a fluorescence quencher. Because of the stem-loop structure of the probe, the fluorophore is close to the quencher, thus preventing the molecule from emitting any fluorescence. The molecule is also engineered such that only the probe sequence is complementary to the genomic DNA that will be used in the assay (Abravaya et al. 2003).

If the probe sequence of the molecular beacon encounters its target genomic DNA during the assay, it will anneal and hybridize. Because of the length of the probe sequence, the hairpin segment of the probe will be denatured in favour of forming a longer, more stable probe-target hybrid. This conformational change permits the fluorophore and quencher to be free of their tight proximity due to the hairpin association, allowing the molecule to fluoresce.

If on the other hand, the probe sequence encounters a target sequence with as little as one non-complementary nucleotide, the molecular beacon will preferentially stay in its natural hairpin state and no fluorescence will be observed, as the fluorophore remains quenched.

The unique design of these molecular beacons allows for a simple diagnostic assay to identify SNPs at a given location. If a molecular beacon is designed to match a wild-type allele and another to match a mutant of the allele, the two can be used to identify the genotype of an individual. If only the first probe's fluorophore wavelength is detected during the assay then the individual is homozygous to the wild type. If only the second probe's wavelength is detected then the individual is homozygous to the mutant allele. Finally, if both wavelengths are detected, then both molecular beacons must be hybridizing to their complements and thus the individual must contain both alleles and be heterozygous.

SNP microarrays

In high-density oligonucleotide SNP arrays, hundreds of thousands of probes are arrayed on a small chip, allowing for many SNPs to be interrogated simultaneously. Because SNP alleles only differ in one nucleotide and because it is difficult to achieve optimal hybridization conditions for all probes on the array, the target DNA has the potential to hybridize to mismatched probes. This is addressed somewhat by using several redundant probes to interrogate each SNP. Probes are designed to have the SNP site in several different locations as well as containing mismatches to the SNP allele. By comparing the differential amount of hybridization of the target DNA to each of these redundant probes, it is possible to determine specific homozygous and heterozygous alleles. Although oligonucleotide microarrays have a comparatively lower specificity and sensitivity, the scale of SNPs that can be interrogated is a major benefit. The Affymetrix Human SNP 5.0 GeneChip performs a genome-wide assay that can genotype over 500,000 human SNPs (Affymetrix 2007)..

Enzyme-based methods

A broad range of enzymes including DNA ligase, DNA polymerase and nucleases have been employed to generate high-fidelity SNP genotyping methods.

Restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) is considered to be the simplest and earliest method to detect SNPs. SNP-RFLP makes use of the many different restriction endonucleases and their high affinity to unique and specific restriction sites. By performing a digestion on a genomic sample and determining fragment lengths through a gel assay it is possible to ascertain whether or not the enzymes cut the expected restriction sites. A failure to cut the genomic sample results in an identifiably larger than expected fragment implying that there is a mutation at the point of the restriction site which is rendering it protection from nuclease activity.

The combined factors of the high complexity of most eukaryotic genomes, the requirement for specific endonucleases, the fact that the exact mutation cannot necessarily be resolved in a single experiment, and the slow nature of gel assays make RFLP a poor choice for high throughput analysis.

PCR-based methods

Tetra-primer amplification refractory mutation system PCR, or ARMS-PCR, employs two pairs of primers to amplify two alleles in one PCR reaction. The primers are designed such that the two primer pairs overlap at a SNP location but each match perfectly to only one of the possible SNPs. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. As a result, if a given allele is present in the PCR reaction, the primer pair specific to that allele will produce product but not to the alternative allele with a different SNP. The two primer pairs are also designed such that their PCR products are of a significantly different length allowing for easily distinguishable bands by gel electrophoresis or melt temperature analysis. In examining the results, if a genomic sample is homozygous, then the PCR products that result will be from the primer that matches the SNP location and the outer opposite-strand primer, as well from the two outer primers. If the genomic sample is heterozygous, then products will result from the primer of each allele and their respective outer primer counterparts as well as the outer primers.

An alternative strategy is to run multiple qPCR reactions with different primer sets that target each allele separately. Well-designed primers will amplify their target SNP at a much earlier cycle than the other SNPs. This allows more than two alleles to be distinguished, although an individual qPCR reaction is required for each SNP. To achieve high enough specificity, the primer sequence may require placement of an artificial mismatch near its 3'-end, which is an approach generally known as Taq-MAMA.

Flap endonuclease

Flap endonuclease (FEN) is an endonuclease that catalyzes structure-specific cleavage. This cleavage is highly sensitive to mismatches and can be used to interrogate SNPs with a high degree of specificity

In the basic Invader assay, a FEN called cleavase is combined with two specific oligonucleotide probes, that together with the target DNA, can form a tripartite structure recognized by cleavase.^[7] The first probe, called the Invader oligonucleotide is complementary to the 3’ end of the target DNA. The last base of the Invader oligonucleotide is a non-matching base that overlaps the SNP nucleotide in the target DNA. The second probe is an allele-specific probe which is complementary to the 5’ end of the target DNA, but also extends past the 3’ side of the SNP nucleotide. The allele-specific probe will contain a base complementary to the SNP nucleotide. If the target DNA contains the desired allele, the Invader and allele-specific probes will bind to the target DNA forming the tripartite structure. This structure is recognized by cleavase, which will cleave and release the 3’ end of the allele-specific probe. If the SNP nucleotide in the target DNA is not complementary to the allele-specific probe, the correct tripartite structure is not formed and no cleavage occurs. The Invader assay is usually coupled with fluorescence resonance energy transfer (FRET) system to detect the cleavage event. In this setup, a quencher molecule is attached to the 3’ end and a fluorophore is attached to the 5’ end of the allele-specific probe. If cleavage occurs, the fluorophore will be separated from the quencher molecule generating a detectable signal.

Only minimal cleavage occurs with mismatched probes making the Invader assay highly specific. However, in its original format, only one SNP allele could be interrogated per reaction sample and it required a large amount of target DNA to generate a detectable signal in a reasonable time frame. Several developments have extended the original Invader assay. By carrying out secondary FEN cleavage reactions, the Serial Invasive Signal Amplification Reaction (SISAR) allows both SNP alleles to be interrogated in a single reaction. SISAR Invader assay also requires less target DNA, improving the sensitivity of the original Invader assay. The assay has also been adapted in several ways for use in a high-throughput format. In one platform, the allele-specific probes are anchored to microspheres. When cleavage by FEN generates a detectable fluorescent signal, the signal is measured using flow-cytometry. The sensitivity of flow-cytometry, eliminates the need for PCR amplification of the target DNA (Rao et al. 2003). These high-throughput platforms have not progressed beyond the proof-of-principle stage and so far the Invader system has not been used in any large scale SNP genotyping projects.

Primer extension

Primer extension is a two step process that first involves the hybridization of a probe to the bases immediately upstream of the SNP nucleotide followed by a ‘mini-sequencing’ reaction, in which DNA polymerase extends the hybridized primer by adding a base that is complementary to the SNP nucleotide. This incorporated base is detected and determines the SNP allele (Goelet et al. 1999; Syvanen 2001). Because primer extension is based on the highly accurate DNA polymerase enzyme, the method is generally very reliable. Primer extension is able to genotype most SNPs under very similar reaction conditions making it also highly flexible. The primer extension method is used in a number of assay formats. These formats use a wide range of detection techniques that include MALDI-TOF Mass spectrometry (see Sequenom) and ELISA-like methods.

Generally, there are two main approaches which use the incorporation of either fluorescently labeled dideoxynucleotides (ddNTP) or fluorescently labeled deoxynucleotides (dNTP). With ddNTPs, probes hybridize to the target DNA immediately upstream of SNP nucleotide, and a single, ddNTP complementary to the SNP allele is added to the 3’ end of the probe (the missing 3'-hydroxyl in didioxynucleotide prevents further nucleotides from being added). Each ddNTP is labeled with a different fluorescent signal allowing for the detection of all four alleles in the same reaction. With dNTPs, allele-specific probes have 3’ bases which are complementary to each of the SNP alleles being interrogated. If the target DNA contains an allele complementary to the probe's 3’ base, the target DNA will completely hybridize to the probe, allowing DNA polymerase to extend from the 3’ end of the probe. This is detected by the incorporation of the fluorescently labeled dNTPs onto the end of the probe. If the target DNA does not contain an allele complementary to the probe's 3’ base, the target DNA will produce a mismatch at the 3’ end of the probe and DNA polymerase will not be able to extend from the 3' end of the probe. The benefit of the second approach is that several labeled dNTPs may get incorporated into the growing strand, allowing for increased signal. However, DNA polymerase in some rare cases, can extend from mismatched 3’ probes giving a false positive result.

A different approach is used by Sequenom's iPLEX SNP genotyping method, which uses a MassARRAY mass spectrometer. Extension probes are designed in such a way that 40 different SNP assays can be amplified and analyzed in a PCR cocktail. The extension reaction uses ddNTPs as above, but the detection of the SNP allele is dependent on the actual mass of the extension product and not on a fluorescent molecule. This method is for low to medium high throughput, and is not intended for whole genome scanning.

The flexibility and specificity of primer extension make it amenable to high throughput analysis. Primer extension probes can be arrayed on slides allowing for many SNPs to be genotyped at once. Broadly referred to as arrayed primer extension (APEX), this technology has several benefits over methods based on differential hybridization of probes. Comparatively, APEX methods have greater discriminating power than methods using this differential hybridization, as it is often impossible to obtain the optimal hybridization conditions for the thousands of probes on DNA microarrays (usually this is addressed by having highly redundant probes). However, the same density of probes cannot be achieved in APEX methods, which translates into lower output per run.

Illumina Incorporated's Infinium assay is an example of a whole-genome genotyping pipeline that is based on primer extension method. In the Infinium assay, over 100,000 SNPs can be genotyped. The assay uses hapten-labelled nucleotides in a primer extension reaction. The hapten label is recognized by anti-bodies, which in turn are coupled to a detectable signal (Gunderson et al. 2006).

APEX-2 is an arrayed primer extension genotyping method which is able to identify hundreds of SNPs or mutations in parallel using efficient homogeneous multiplex PCR (up to 640-plex) and four-color single-base extension on a microarray. The multiplex PCR requires two oligonucleotides per SNP/mutation generating amplicons that contain the tested base pair. The same oligonucleotides are used in the following step as immobilized single-base extension primers on a microarray (Krjutskov et al. 2008).

5’- nuclease

Taq DNA polymerase's 5’-nuclease activity is used in the TaqMan assay for SNP genotyping. The TaqMan assay is performed concurrently with a PCR reaction and the results can be read in real-time as the PCR reaction proceeds (McGuigan & Ralston 2002). The assay requires forward and reverse PCR primers that will amplify a region that includes the SNP polymorphic site. Allele discrimination is achieved using FRET combined with one or two allele-specific probes that hybridize to the SNP polymorphic site. The probes will have a fluorophore linked to their 5’ end and a quencher molecule linked to their 3’ end. While the probe is intact, the quencher will remain in close proximity to the fluorophore, eliminating the fluorophore's signal. During the PCR amplification step, if the allele-specific probe is perfectly complementary to the SNP allele, it will bind to the target DNA strand and then get degraded by 5’-nuclease activity of the Taq polymerase as it extends the DNA from the PCR primers. The degradation of the probe results in the separation of the fluorophore from the quencher molecule, generating a detectable signal. If the allele-specific probe is not perfectly complementary, it will have lower melting temperature and not bind as efficiently. This prevents the nuclease from acting on the probe (McGuigan & Ralston 2002).

Since the TaqMan assay is based on PCR, it is relatively simple to implement. The TaqMan assay can be multiplexed by combining the detection of up to seven SNPs in one reaction. However, since each SNP requires a distinct probe, the TaqMan assay is limited by the how close the SNPs can be situated. The scale of the assay can be drastically increased by performing many simultaneous reactions in microtitre plates. Generally, TaqMan is limited to applications that involve interrogating a small number of SNPs since optimal probes and reaction conditions must be designed for each SNP (Syvanen 2001).

Oligonucleotide Ligation Assay

DNA ligase catalyzes the ligation of the 3' end of a DNA fragment to the 5' end of a directly adjacent DNA fragment. This mechanism can be used to interrogate a SNP by hybridizing two probes directly over the SNP polymorphic site, whereby ligation can occur if the probes are identical to the target DNA. In the oligonucleotide ligase assay, two probes are designed; an allele-specific probe which hybridizes to the target DNA so that its 3' base is situated directly over the SNP nucleotide and a second probe that hybridizes the template upstream (downstream in the complementary strand) of the SNP polymorphic site providing a 5' end for the ligation reaction. If the allele-specific probe matches the target DNA, it will fully hybridize to the target DNA and ligation can occur. Ligation does not generally occur in the presence of a mismatched 3' base. Ligated or unligated products can be detected by gel electrophoresis, MALDI-TOF mass spectrometry or by capillary electrophoresis for large-scale applications. With appropriate sequences and tags on the oligonucleotides, high-throughput sequence data can be generated from the ligated products and genotypes determined (Curry et al., 2012). The use of large numbers of sample indexes allows high-throughput sequence data on hundreds of SNPs in thousands of samples to be generated in a small portion of a high-throughput sequencing run. This is a massive genotyping by sequencing technology (MGST).

Other post-amplification methods based on physical properties of DNA

The characteristic DNA properties of melting temperature and single stranded conformation have been used in several applications to distinguish SNP alleles. These methods very often achieve high specificity but require highly optimized conditions to obtain the best possible results.

Single strand conformation polymorphism

Main article: Single_strand_conformation_polymorphism

Single-stranded DNA (ssDNA) folds into a tertiary structure. The conformation is sequence dependent and most single base pair mutations will alter the shape of the structure. When applied to a gel, the tertiary shape will determine the mobility of the ssDNA, providing a mechanism to differentiate between SNP alleles. This method first involves PCR amplification of the target DNA. The double-stranded PCR products are denatured using heat and formaldehyde to produce ssDNA. The ssDNA is applied to a non-denaturing electrophoresis gel and allowed to fold into a tertiary structure. Differences in DNA sequence will alter the tertiary conformation and be detected as a difference in the ssDNA strand mobility (Costabile et al. 2006). This method is widely used because it is technically simple, relatively inexpensive and uses commonly available equipment. However compared to other SNP genotyping methods, the sensitivity of this assay is lower. It has been found that the ssDNA conformation is highly dependent on temperature and it is not generally apparent what the ideal temperature is. Very often the assay will be carried out using several different temperatures. There is also a restriction on the length of fragment because the sensitivity drops when sequences longer than 400 bp are used (Costabile et al. 2006).

Temperature gradient gel electrophoresis

The temperature gradient gel electrophoresis (TGGE) or temperature gradient capillary electrophoresis (TGCE) method is based on the principle that partially denatured DNA is more restricted and travels slower in a porous material such as a gel. This property allows for the separation of DNA by melting temperature. To adapt these methods for SNP detection, two fragments are used; the target DNA which contain the SNP polymorphic site being interrogated and an allele-specific DNA sequence, referred to as the normal DNA fragment. The normal fragment is identical to the target DNA except potentially at the SNP polymorphic site, which is unknown in the target DNA. The fragments are denatured and then reannealed. If the target DNA has the same allele as the normal fragment, homoduplexes will form that will have the same melting temperature. When run on the gel with a temperature gradient, only one band will appear. If the target DNA has a distinct allele, four products will form following the reannealing step; homoduplexes consisting of target DNA, homoduplexes consisting of normal DNA and two heterduplexes of each strand of target DNA hybridized with the normal DNA strand. These four products will have distinct melting temperatures and will appear as four bands in the denaturing gel.

Denaturing high performance liquid chromatography

Denaturing high performance liquid chromatography (DHPLC) uses reversed-phase HPLC to interrogate SNPs. The key to DHPLC is the solid phase which has differential affinity for single and double-stranded DNA. In DHPLC, DNA fragments are denatured by heating and then allowed to reanneal. The melting temperature of the reannealed DNA fragments determines the length of time they are retained in the column. Using PCR, two fragments are generated; target DNA containing the SNP polymorphic site and an allele-specific DNA sequence, referred to as the normal DNA fragment. This normal fragment is identical to the target DNA except potentially at the SNP polymorphic site, which is unknown in the target DNA. The fragments are denatured and then allowed to gradually reanneal. The reannaled products are added to the DHPLC column. If the SNP allele in the target DNA matches the normal DNA fragment, only identical homoduplexes will form during the reannealing step. If the target DNA contains a different SNP allele than the normal DNA fragment, heteroduplexes of the target DNA and normal DNA containing a mismatched polymorphic site will form in addition to homoduplexes. The mismatched heteroduplexes will have a different melting temperature than the homoduplexes and will not be retained in the column as long. This generates a chromatograph pattern that is distinctive from the pattern that would be generated if the target DNA fragment and normal DNA fragments were identical. The eluted DNA is detected by UV absorption.

DHPLC is easily automated as no labeling or purification of the DNA fragments is needed. The method is also relatively fast and has a high specificity. One major drawback of DHPLC is that the column temperature must be optimized for each target in order to achieve the right degree of denaturation.

High-resolution melting of the entire amplicon

High Resolution Melting analysis is the simplest PCR-based method to understand. Basically, the same thermodynamic properties that allowed for the gel techniques to work apply here, and in real-time. A fluorimeter monitors the post-PCR denaturation of the entire dsDNA amplicon. You make primers specific to the site you want to amplify. You "paint" the amplicon with a double-strand specific dye, included in the PCR mix. The ds-specific dye integrates itself into the PCR product. In essence, the entire amplicon becomes a probe. This opens up new possibilities for discovery. Either you position the primers very close to either side of the SNP in question (small amplicon genotyping, Liew, 2004) or amplify a larger region (100-400bp in length) for scanning purposes. For simple genotyping of an SNP, it is easier to just make the amplicon small to minimize the chances you mistake one SNP for another. The melting temperature (Tm) of the entire amplicon is determined and most homozygotes are sufficiently different (in the better instruments) in Tm to genotype. Heterozygotes are even easier to differentiate because they have heteroduplexes generated (refer to the gel-based explanations) which broadens the melt transition and usually gives two discernible peaks. Amplicon melting using a fluorescently-labeled primer has been described (Gundry et al., 2003) but is less practical than using ds-specific dyes due to the cost of the fluorogenic primer.

Scanning of larger amplicons is based on the same principles as outlined above. However, melting temperature and the overall shape of the melting curve become informative. For amplicons >c.150bp there are often >2 melting peaks, each of which can vary, depending on the DNA template composition. Numerous investigators have been able to successfully eliminate the majority of their sequencing through melt-based scanning, allowing accurate locus-based genotyping of large numbers of individuals. Many investigators have found scanning for mutations using high resolution melting as a viable and practical way to study entire genes.

Use of DNA mismatch-binding proteins

DNA mismatch-binding proteins can distinguish single nucleotide mismatches and thus facilitate differential analysis of SNPs. For example, MutS protein from Thermus aquaticus binds different single nucleotide mismatches with different affinities and can be used in capillary electrophoresis to differentiate all six sets of mismatches (Drabovich & Krylov 2006).

SNPlex

Main article: SNPlex

SNPlex is a proprietary genotyping platform sold by Applied Biosystems.

Surveyor nuclease assay

Main article: Surveyor_nuclease_assay

Surveyor nuclease is a mismatch endonuclease enzyme that recognizes all base substitutions and small insertions/deletions (indels), and cleaves the 3′ side of mismatched sites in both DNA strands.

Sequencing

Further information: SNV calling from NGS data

Next-generation sequencing technologies such as pyrosequencing sequence less than 250 bases in a read which limits their ability to sequence whole genomes. However, their ability to generate results in real-time and their potential to be massively scaled up makes them a viable option for sequencing small regions to perform SNP genotyping. Compared to other SNP genotyping methods, sequencing is in particular, suited to identifying multiple SNPs in a small region, such as the highly polymorphic Major Histocompatibility Complex region of the genome.