DNA Sequencing- Sanger’s dideoxy method or chain termination method

The chain terminator method is more efficient and uses fewer toxic chemicals and lower amount of radioactivity than the method of Maxam and Gilbert. Sanger method uses dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.

Chain termination method of DNA sequencing, is also referred to as dideoxynucleotide sequencing because of the use of the special types of ddNTPs. The ddNTPs are different from normal dNTPs. it possesses the hydrogen group instead of a hydroxyl group in the dNTPs.

Phosphodiester bond can’t form between two adjacent nucleotides due to the absence of 3'-hydroxyl group in ddNTPs. The nucleotide chain can’t synthesis further and hence it is known as the chain termination method.

The chain termination method requires a single-stranded DNA template,  DNA primer, a DNA polymerase, nucleotides radioactively or fluorescently labelled modified nucleotides that terminate DNA strand elongation.

The process of Sanger sequencing is broadly divided into 3 steps:

1. DNA extraction: using any of the DNA extraction protocols
2. PCR amplification: using the flanking primers, dNTPs,  ddNTPs, Taq DNA polymerase and PCR buffer. (ddNTPs  are fluorescent labelled, each with a characteristic dye, and appears in that colour during detection)
3. Identification of the amplified fragments: using autoradiography, PAGE, or capillary gel electrophoresis. 

PCR amplification is performed by designing four different reactions

Reaction	PCR reaction	modification
Reaction “A”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddATPs
Reaction “G”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddGTPs
Reaction “T”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddTTPs
Reaction “C”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddCTPs

 

●      Each tube contains the same amount of PCR reagents but in each tube, extra ddNTPs are added as shown in the table.

●      The primer binds at the region of interest.

●      Now in the next step, the Taq DNA polymerase adds the dNTPs to the DNA strand.

●    Taq polymerase expands the growing DNA strand by the addition of the dNTPs. Once  it adds the ddNTP instead of dNTP, the chain expansion is stopped or terminated.

●      The termination process is complete in 4 different tubes for 4 different ddNTPs. For example, in the ddATP tube, terminates the chain at all the positions where the ddATPs are going to bind.

●      The amplified PCR products are loaded onto the polyacrylamide gel electrophoresis. The DNA fragments migrate into the gel based on the size of the fragments. The smaller fragments run faster towards the positive charge than the larger fragments.  

●      Depending upon the types of labeling the gel is then analyzed.

●      The DNA bands are visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image.

●    A dark band in a lane indicates a DNA fragment that is result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). If fluorescent labelled, the characteristic colour inidcates incorporation of that dieoxynucleotide.

 

Sangers chain termination method of DNA sequencing.

In electrophoresis, the smaller DNA fragments migrate faster than larger ones. So start reading the sequence from the smallest fragment on the positive side.

The first point in sequencing the DNA is to match the size of DNA. if the bands obtained in gel and the nucleotide sequence of DNA are similar, the reaction is completed properly.

For instance, if sequence length is 16, then 16 bands must be present in the gel. Arrange the sequences accordingly.

Sanger sequencing is the gold standard method for research as well as in the diagnosis because of its easy setup and high reproducibility. its automation had done easily.

 Traditionally, the results are interpreted on PAGE manually but now, the process is automated. A detector detects the fluorescence signals each time when the chain is terminated. The signals are recorded and analyzed computationally. The computational software generates various fluorescence peaks depending upon the amount of fluorescence emitted

Advantage

Chain termination methods have greatly simplified DNA sequencing.

Limitations

●       Non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence.

●       DNA secondary structures affecting the fidelity of the sequence.

 

Fimbriae, Pili

 Fimbriae

· Short, fine, hair like appendages thinner than flagella and not involved in motility - a cell may be covered with up to 1,000 fimbriae - Only visible in an electron microscope

· Slender tubes composed of helically arranged fimbrillin protein subunits - 3 to 10 nm in diameter & several μm long 

· Attach bacteria to solid surfaces such as rocks in streams and host tissues

Pili

· Sex pili - about 1 to 10 per cell- often are larger than fimbriae (around 9 to 10 nm in diameter) - made of pilin protein subunits

· Genetically determined by sex factors or conjugative plasmids; required for bacterial mating

· Some bacterial viruses attach specifically to receptors on sex pili at the start of their reproductive cycle.

Fimbriae	Pili
Fimbriae are tiny bristle-like fibers arising from the surface of bacterial cells.	Pili are hair like microfibers that are thick tubular structure made up of pilin.
Shorter than pili	Longer than fimbriae.
Thin	Thicker than fimbriae.
200-400 per cell	less 1-10 per cell
Fimbrillin protein	Pilin protein
Less rigid	More rigid than fimbriae
Both gram positive and gram negative bacteria	Only gram negative bacteria
Is governed by bacterial genes in the nucleoid region	Is governed by plasmid genes.
Responsible for cell to surface attachment. Specialized for attachment i.e. enable the cell to adhere the surfaces of other bacteria.	Responsible for bacterial conjugation. Two basic function of pili. They are gene transfer and attachment.
Do not function in active motility	Type IV pili shows twitching type of motility.
No receptors of other	Serve as receptor for certain viruses
Salmonella typhimurium, Shigella dysenteriae.	Escherichia coli, Neisseria gonorrhoeae.
Shigella dysenteriae uses its fimbriae to attach to the intestine and then produces a toxin that causes diarrhea.	Neisseria gonorrhoeae, the cause of gonorrhea, uses pili to attach to the urogenital and cervical epithelium when it causes disease.

 

Inventorying and Monitoring of Biodiversity

    Biological diversity is a result of millions of years of evolution and thousands of years of cultivation of plants and domestication of animals. For a successful, long-term conservation of an ecosystem,  knowledge of its biological diversity and efforts to protect and manage that diversity is important. 

    Understanding biological diversity is critical for its sustainable use and safeguarding it for the benefit of future generations.  The biological diversity is dynamic, continually evolving and changing in response to biotic and abiotic fluctuations and other environmental pressures. It is necessary to record in time and space its status and, subsequently, monitor it in order to identify changes and assess their impacts. 

    Recent years saw accelerating destruction of life on Earth which led to the signing of international agreements, such as the Convention for Biological Diversity and Agenda  for increased efforts to inventory and monitor the world’s biodiversity.    

     Inventorying or the identification and monitoring the components of biological diversity is  important for its conservation and sustainable use. Identifying and monitoring biological diversity is a huge and infinite task. The accuracy, adequacy and interpretation of inventories are important when determining the relevance of species lists to the conservation and management of biological diversity.

    For example, it is crucial to identify species present in areas of natural habitat ahead of any changes in land use in order to assess what diversity may be lost from a locality. This is particularly important to tropical ecosystems, where endemic species are more and, hence the risks of species becoming globally extinct may be greater. 

 Inventorying is the surveying, sorting, cataloguing, quantifying and mapping of genes, individuals, populations, species, habitats, biotopes, ecosystems and landscapes or their components. The resulting information can be analysed for various processes. Inventories involve the extensive application of systematics, ecology, biogeography and management. Inventories provide a snapshot of the state of biodiversity and baseline information for the assessment of change. Recording these changes is monitoring.

To monitor a process or a dynamic system (origin: from Latin “monere” = “to remind”, “to warn”) means to observe or measure the relevant parameters which describe the change of a system adequately. Thus, monitoring is intermittent surveillance to ascertain the maintenance or changes from a predetermined standard. Monitoring of biological diversity is usually goal-oriented and provides a framework for predicting the behaviour of key variables for improving management, increasing management options and providing early warning of system change. 

These attempt to identify, record and monitor organisms and their distributions is an enormous task. The inventories can contribute to solving the biodiversity crisis or prioritizing areas for conservation. Human exploitation of biodiversity is currently so high that monitoring is essential to avoid a catastrophe.

The Convention for Biological Diversity requires signatory nations to ‘identify components of biodiversity for conservation and sustainable use and monitor, through sampling and other techniques, the components of biological diversity identified.’ It also calls for signatories to ‘identify processes and categories of activities which are likely to have significant adverse impacts on the conservation and sustainable use of biological diversity, and monitor their effects’ and to ‘maintain and organise data derived from identification and monitoring activities.’

Biodiversity inventories and monitoring provide the essential biological information for many biological sciences, including systematics, population biology and ecology, as well as for many applied sciences, such as biotechnology, soil science, agriculture, forestry and fisheries science, conservation and environmental sciences. 

We also see inventorying and monitoring as vital for identifying key issues for policy and management goals, for example, in assessing priorities for conservation, land use and sustainable management, pollution control, environmental impact assessments, and for informing policy makers and the public on the state of biodiversity. 

Historical collections of organisms provide extremely important baseline information on how the range, abundance and form of species may change over time. These are essential for the identification and verification of field data and provide a permanent record. 

The need for inventorying and monitoring of biodiversity.

 Over the past millennia and centuries, human activities on our planet causes rapid changes of biodiversity, including extinction of phylogenetic lineages, invasion of exotic taxa and even the spread of artificially constructed genetically manipulated organisms (GMO´s) or part of their genome.

    Many of these changes are beneficial, others have negative impact on ecosystem functions, on other organisms and on goods and services, consumed by humans. Conservation, management and, monitoring of biodiversity is of high relevance for biodiversity and for sustainable development. With rising awareness of global environmental changes, monitoring data are needed for a variety of goals, including

(a) to understand the role and impact of drivers and causes of change

(b) to be able to analyze processes and mechanisms of change

(c) to lay the foundation for modeling and prediction of future changes etc.

Methods of inventorying and monitoring of biodiversity and their limitations

Methods for carrying out inventories and monitoring at the population, species, ecosystem and landscape level are important for understanding how that population is changing in the face of anthropogenic disturbance.

Methods of inventorying

Inventorying or the identification and monitoring the components of biological diversity is important for its conservation and sustainable use.  The accuracy, adequacy and interpretation of inventories are important when determining the relevance of species with reference to the conservation and management of biological diversity.

Benefits

Biological inventories provide a finer view of biological diversity and can be used to establish national conservation programs and policies

1)     These efforts tell us the status of biodiversity, and also identify valuable biological resources, some of which are unknown, while others are locally known but have potential for much wider use.

2)    Inventories and surveys also provide baseline data against which to monitor changes in biological diversity and to trace the environmental impacts of development projects.

Assessment Methods

Status and state of a resource at a point in time is analysed and recorded in the processes biodiversity inventory.

• Distribution and composition of species, habitats and populations is measured in this process.
• The analysis can be  qualitative or quantitative.
• This helps to assess the presence or infer the absence of species within an area.

A range of methods may be more or less applicable and more or less expensive.

• Censusing and related techniques are important for estimating population size and hence rarity/ threatened status of species.
• Species inventories, will  remain the mainstay of inventorying and in the selection of protected, ecologically important and economically sensitive areas.
• Remote sensing, coupled with ground truthing, can monitor vegetation cover, land use, forest loss and other aspects of biodiversity change at the biotope and eco system level.

 Traditional forest inventory and vegetation analysis.

Foresters have historically developed the science of forest inventory, principally for estimates of standing volumes of wood in forests and for recurrent measurements to indicate changes with time or management; the numbers and densities of non-wood plant species are occasionally recorded. Systems of permanent and temporary sample plots in forests have sometimes been established for these forestry purposes. 

Substantial work in Australia by the Commonwealth Scientific and Industrial Research Organization (CSIRO) has expanded such traditional forest inventories into multi-taxa surveys. There has also been considerable international activity recently to establish biodiversity monitoring plots e.g. United Nations Educational, Scientific and Cultural Organization (UNESCO) Man and the Biosphere Programme; Smithsonian Institution, Washington; and the Food and Agriculture Organization of the United Nations (FAO).

Molecular methods.

The last decade has seen an increasing use of high profile molecular genetic techniques to study genetic diversity, systematics and population genetics at the DNA and protein levels. These technologies have included isozyme, restriction fragment length polymorphism (RFLPs), randomly amplified polymorphic DNA (RAPD), DNA fingerprinting and, more recently micro-satellites.

 

Remote sensing.

 A large array of technologies now exists for examination of terrestrial resources including aerial photography and satellite imagery in various electromagnetic wavebands. Their scales and precision differ but the locations and changes in forest or ecosystem boundaries can be identified easily and the standing volume of wood in some forest types can be estimated reasonably precisely. If coupled to appropriately detailed ground truthing, the techniques are applicable to identifying rare communities and vulnerable remnants, for mapping vegetation and for zonation and land use planning. 

However, none of these techniques are yet refined sufficiently to identify individual plants unequivocally at a scale and precision that would permit biodiversity monitoring within ecosystems.

Databases and geographic information systems.

All of the historical and current data collected by any of these technologies may now be combined into electronic databases and portrayed by a large number of geographic information systems that are commercially available. One of the most intensive and extensive database systems now available appears to be the Biological and Conservation Database (BCD), of the United States Nature Conservancy - this allows rare taxa to be identified and critical sites to be recognized. 

The success of such research is dependent on the careful, repetitive and tedious recording and management of primary data.

  

Sample	inventory and monitoring tools and techniques
Population Species	Censuses (observations, counts, captures, signs, radio-tracking); remote sensing; habitat suitability index (HSI); species-habitat modeling; population viability analysis
Genetic	Electrophoresis; karyotypic analysis; DNA sequencing; offspring-parent regression; sib analysis; morphological analysis
Regional Landscape	Aerial photographs (satellite and conventional aircraft) and other remote sensing data; Geographic Information System (GIS) technology; time series analysis; spatial statistics; mathematical indices (of pattern, heterogeneity, connectivity, layering, diversity, edge, morphology, autocorrelation, fractal dimension)
Community Ecosystem	Aerial photographs and other remote sensing data; ground-level photo stations; time series analysis; physical habitat measures and resource inventories; habitat suitability indices (HSI, multispecies ); observations, censuses and inventories, captures, and other sampling methodologies; mathematical indices (e.g., of diversity, heterogeneity, layering dispersion, biotic integrity)

 

Monitoring, i.e. the measurement of recent changes of biodiversity, is providing important information for an understanding of our activities. Monitoring the population sizes of protected species in their conservation areas gives feedback on the success of conservation measures. Monitoring the spread of a toxic invading species or of an infectious organism can feed into an early warning system for farmers or for medical services. 

 Methods of Monitoring

There are different approaches to monitoring such as:

1. Neutral observation (“What happens?”)

This approach of pure observation can document the consequences of change, particularly of properties of the ecosystem such as soil quality, microclimate, land use. New observations such as the impact of rare events can also be considered in this pure observation approach

2. Early warning system (“When must we take action?”)

Change of biodiversity can have important consequences for ecosystem function and use of resources. Single events can cause cascades of secondary effects, and transitions from one phase of a system into another new phase may take place suddenly once thresholds have been passed (e.g. state of an ecosystem, size of a population or area of distribution). Therefore, an observation system should serve as an early warning system, which allows action to be taken well before irreversible damage has taken place.

3. Indicators of biodiversity change (“What is important?”)

The observation or measurement of a limited set of specific qualities and/or quantities that can act as indicators is a more practical approach because observation of a large number of parameters and processes requires large investments of time and manpower.

4. Causality approach (“Why does change happen?”)

Observation of the type and intensity of change within a given ecosystem can allow identification of the drivers/causes of change such as specific climatic changes, specific land use practices.

5. Process analysis (“How does change happen?”)

If observation is designed to provide additional scientific understanding of the mechanisms and processes of change, more detailed studies are needed. The resulting knowledge can also form the basis for the prediction of future developments:

6. Model-based approach (“Do we understand the full picture?”)

Observations can be used to test (verify or falsify) and validate results based on models.

7. Experimental approach (“How can we intervene?”)

One of the ultimate goals of biodiversity research is the implementation of sustainable use and conservation of biodiversity. For this purpose, observation can integrate a variety of experimental approaches for testing and measuring vulnerability, resilience, restorability and other system properties. If successful, this approach help in deriving management recommendations.

 

Examples of monitoring at each Biodiversity level

1.     Landscape Monitoring

Landscape diversity is the number of ecosystems, or combinations of ecosystems, and types of interactions and disturbances present within a given landscape. Two approaches to assessing biological diversity at a landscape scale include measuring landscape patterns and comparing current conditions to historic reference conditions. Each of these approaches relies on the use of geographic information systems (GISs) and requires mapped vegetation and other layers that can be analyzed with GIS technologies.

2.     Community-Ecosystem Monitoring

    A community comprises the populations of some or all species coexisting at a site. An ecosystem includes the abiotic aspects of the environment and the biotic community. Monitoring at this level is important to the maintenance of ecosystem functions and integrity that have been identified as a main theme of ecosystem management. A common way of assessing biodiversity is by measuring the number and relative abundance of species in a community or ecosystem, often referred to as species diversity. Species diversity is a function of the number of species present (richness) and the evenness or equitability (relative abundance) of each.

      3.     Diversity Indices

In general, three main categories of measures are used to assess species diversity: (1) species richness indices, which measure the number of species in a sampling unit; (2) species abundance models, which have been developed to describe the distribution of species abundances; and (3) indices that are based on the proportional abundances of species such as the Shannon and Simpson indices.

To quantify community-level biodiversity, an inventory or sample of the species present and their relative abundance must be completed. Various methods have been used to Inventory plant, wildlife, and fish species.

 4.     Functional Groups or Guilds

Some investigators have taken a different viewpoint by lumping species into functional groups or guilds. Many approaches for grouping species based on habitat or behavioral similarities and their potential problems. Species are grouped into guilds based on their function in the ecosystem, and then the relative importance of each guild is considered based on how a change in their abundance affects ecosystem and community process.

 5.     Rapid Assessment Techniques

They estimated species richness of spiders, ants, polychaetes, and mosses, and divided them into recognizable taxonomic units (RTUs). These RTUs are taxa that are readily separated by morphological differences that are obvious to individuals with less training than professional taxonomists. They found that by using RTUs, there is little difference between classifications made by a biodiversity technician and those made by a taxonomy specialist. This could result in a considerable savings of time and money to complete the inventories needed to assess this one facet of biological diversity. This technique could be applied at different time periods to monitor the trends in the numbers of individuals within RTUs.

 6.     GAP ANALYSIS

Gap analysis, provides a framework in which to obtain an overview of the distribution and conservation status of several components of biodiversity. The approach involves mapping, digitizing, and ground-truthing vegetation and species distribution data; digitizing biodiversity management areas and landownership maps; adding location data on all species and high-interest habitats such as wetlands and streams; and mapping, delineating, and ranking areas of high community diversity and species richness. These data are then used to identify "gaps" in protection of vegetation types and species-rich areas and provide land managers with information needed to make informed decisions about reserve selection and design, land management policy, and other conservation actions.

7.  Abundance Indices

 An index is usually a count statistic that is obtained in the field and carries information about a population. Abundance indices are divided into direct indices and indirect indices. Direct indices are based on direct observation of animals, either visually or through capture or harvest. ndirect indices are based on evidence of an animal’s presence.

 8. Population Viability Analysis

 Population viability analysis (PYA) estimates the conditions necessary for a population to persist for a given period of time in a given place

 9.     Genetic Monitoring

 Genetic diversity refers to the breadth of genetic variation within and among individual populations and species

10.Variation in DNA

The development of recombinant DNA technologies allows the direct measurement of genetic variation, which differs from indirect methods such as estimation of variation from a phenotype. analysis of mitochondrial DNA has been used to deduce population structure.

Intensive and extensive biodiversity monitoring are two different methods for observing and measuring changes in biodiversity over time: 

Intensive monitoring

Provides the best evaluation of species presence and abundance, but is limited in geographic coverage. 

Extensive monitoring

Covers a large geographic area with relatively little effort per site, but provides long-term data on population abundance.

   The constantly moving and three-dimensional nature of the aquatic and marine environments presents challenges in the process of inventory and monitoring. Biological organisms are not restricted by political boundaries. Long-term research sites and programmes provide essential information on how biodiversity changes, and are important in distinguishing anthropogenic from natural change. 

If institutions concerned with inventorying and monitoring are to become more effective, they need to be better resourced, their collections better maintained and a new cadre of professional researchers and technicians trained and funded. There should be a sharing of scientific expertise, facilities and information by collaboration between nations and agencies, if  we are to follow international agreements on inventorying and monitoring biodiversity.