At its simplest, biotechnology is technology based on biology – biotechnology harnesses cellular and biomolecular processes to develop technologies and products that help improve our lives and the health of our planet. We have used the biological processes of microorganisms for more than 6,000 years to make useful food products, such as bread and cheese, and to preserve dairy products.
Modern biotechnology provides breakthrough products and technologies to combat debilitating and rare diseases, reduce our environmental footprint, feed the hungry, use less and cleaner energy, and have safer, cleaner and more efficient industrial manufacturing processes.
Currently, there are more than 250 biotechnology health care products and vaccines available to patients, many for previously untreatable diseases. More than 13.3 million farmers around the world use agricultural biotechnology to increase yields, prevent damage from insects and pests and reduce farming’s impact on the environment. And more than 50 biorefineries are being built across North America to test and refine technologies to produce biofuels and chemicals from renewable biomass, which can help reduce greenhouse gas emissions.
Cells and Molecular Biotechnology
Cell biology is a branch of biology that studies cells physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division, death and cell function. This is done both on a microscopic and molecular level. Cellular Biology is also referred to as Cytology. Cellular Biology mainly revolves around the basic and fundamental concept that cell is the fundamental unit of life. The most important concept of Cellular Biology is the cell theory which states mainly 3 points: a: All organisms are composed of one or more cells, b: The cell is the basic unit of life in all living things and c: All cells are produced by the division of preexisting cells.
Cellular and Molecular Biology, Biotechnology and Biomedical Science together drive the modern life sciences enterprise in the 21st century. Dedicated professionals working in these fields have improved the quality of life by researching the fundamental biological questions that have led, for example, to developing novel drug therapies and the advent of personalized medicine.
Molecular biotechnology results from the convergence of many areas of research, such as molecular biology, microbiology, biochemistry, immunology, genetics, and cell biology. It is an exciting field fueled by the ability to transfer genetic information between organisms with the goal of understanding important biological processes or creating a useful product. The completion of the human genome project has opened a myriad of opportunities to create new medicines and treatments, as well as approaches to improve existing medicines.
Molecular biotechnology is a rapidly changing and dynamic field. As the pace of advances accelerates, its influence will increase.
Important Techniques used in Biotechnology
Bioprocess Technology is the sub-discipline within Biotechnology that combines living matter, in the form of organisms or enzymes, with nutrients under specific optimal conditions to make a desired product. It is responsible for translating discoveries of life sciences into practical and industrial products, processes and techniques that can serve the needs of society. The stages involved in Bioprocess includes the preparation stage of raw materials, substrates and media, the conversion state, biocatalysts, downstream processing, volume production, purification and final product processing.
A bioprocess is any process that uses complete living cells or their components (e.g., bacteria, enzymes, chloroplast) to obtain desired products. This process is commonly referred to as Fermentation.
Fermentation involves the conversion of substrates to desired product with the help of biological agents such as microorganisms. It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale. They can be divided into surface (emersion) and submersion techniques. The latter may be run in batch, fed batch, continuous reactors. In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry
In the submersion processes, the microorganisms grow in a liquid medium.
Monoclonal Antibodies are cells derived by cell division from a single ancestral cell. Monoclonals are a class of antibodies with identical offspring of a hybridoma and are very specific for a particular location in the body derived from a single clone and can be grown indefinitely. Monoclonal Antibodies recognize and bind to antigens in order to discriminate between specific epitopes which provides protection against disease organisms.
Monoclonal antibodies target various proteins that influence cell activity such as receptors or other proteins present on the surface of normal and cancer cells.The specificity of Monoclonal Antibodies allows its binding to cancerous cells by coupling a cytotoxic agent such as a strong radioactive which then seek outs to destroy the cancer cells while not harming the healthy ones.
Monoclonal antibodies are artificially produced against a specific antigen in order to bind to their target antigens. Laboratory production of monoclonal antibodies is produced from clones of only 1 cell which means that every monoclonal antibody produced by the cell is the same.
Fusion of cell culture myeloma cells with mammalian spleen cells antibodies result in hybrid cells/hybridomas which produces large amounts of monoclonal antibody. The cell fusion resulted in two different types of cells, one with the ability to grow continually, and the other with ability to produce bulk amounts of purified antibody. Hybrid cells produce only 1 exact antibody that is more pure than polyclonal antibodies produced by conventional techniques. Monoclonal Antibodies are far more effective than conventional drugs since drugs attack the foreign substance & the body’s own cells that cause harsh side effects & the monoclonal antibody only targets the foreign antigen/target molecule, without or only minor side effects.
The presence of a large amount of a specific monoclonal antibody in the blood means that there is an abnormal protein. Typically this protein can be detected during a physical examination and is identified using a screening blood test called “protein electrophoresis. The source of abnormal production of monoclonal antibody is a small population of plasma cells in the bone marrow.
Plant Cell Culture
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation.
Insect cell culture
Insect cell culture is a common choice for heterologous protein expression. For large-scale production or basic research, insect cells are able to express large quantities of protein with complex post-translational modifications. Gibco insectmedia has been formulated for maximum growth and protein yields.
Mammalian cell culture
Cells isolated from animal tissues can be expanded in culture for use as a research tool, for the production of virus vaccines and various therapeutic proteins, and to generate functional cells or tissue analogues for regenerative medicine. Chemical engineers are actively involved in harnessing the full potential of mammalian cells, especially with regard to process design and optimization.
Mammalian cells can be made to produce vaccines through viral infection, and therapeutic proteins through genetic engineering. Many of these medicines are necessary for patients who either lack the normal form of a protein or cannot produce it in sufficient quantity. Other therapeutic proteins include antibodies and specific binding proteins that neutralize disease-causing molecules within the body. For example, the drug Etanercept (trade name Enbrel) binds to tumor necrosis factor (TNF), thus preventing it from causing an inflammatory reaction in rheumatoid arthritis patients.
Human cells, in particular, are poised to enable opportunities in cell-based therapy and regenerative medicine. We now have the capability to derive stem cells from many sources and guide them to become specific cell types for clinical applications.
A biosensor is an analytical device which converts a biological response into an electrical signal. The term ‘biosensor’ is often used to cover sensor devices used in order to determine the concentration of substances and other parameters of biological interest even where they do not utilise a biological system directly.
Biosensors represent a rapidly expanding field, at the present time, with an estimated 60% annual growth rate; the major impetus coming from the health-care industry (e.g. 6% of the western world are diabetic and would benefit from the availability of a rapid, accurate and simple biosensor for glucose) but with some pressure from other areas.
Research and development in this field is wide and multidisciplinary, spanning biochemistry, bioreactor science, physical chemistry, electrochemistry, electronics and software engineering. Most of this current endeavour concerns potentiometric and amperometric biosensors and colorimetric paper enzyme strips. However, all the main transducer types are likely to be thoroughly examined, for use in biosensors, over the next few years.
Nanobiotechnology is a discipline in which tools from nanotechnology are developed and applied to study biological phenomena. For example, nanoparticles can serve as probes, sensors or vehicles for biomolecule delivery in cellular systems.
Nano-biotechnology refers to the science of integration between biology and nanotechnology. This being a very emerging branch, nanobiology and bionanotechnology are its sister terms.
Various concepts that are being emerged from nanobiotechnology are nanodevices, nanocantilevers and nanoparticles. This usually uses biological systems to devise technical aspects for the development of novel products. Nanobiotechnology as defined in broad terms an interdisciplinary field, that includes fields such as diverse as molecular chemistry, molecular biology, material physics and quantum electronics. The areas related have a widespread scope in research that encompasses studies like device physics, molecular self assembly and atomics scale studies.
Nanobiotechnology basically relies on the idea of producing products that can integrate into the organism’s system whereby effectively carry out the function it was meant for. Ideally two main strategies are being employed in this study on nanobiotechnology: top-down and bottom-up. Whereas in one molecular components are being integrated into an assembly, other forms the basis of forming nano scale particles from larger molecules.
Areas such as nanoelectronics, nanophotonics, nanomechanics, and nanoionics etc. have played an elemental role in deciding the fate of this arena and have been immensely integrated to its core for the development of better bots. Molecular nanotechnology, also referred to as molecular manufacturing, studies the development of nanosystems that are devised, operating at molecular and sub-atomics levels.
Molecular nanotechnology is the study related to the nanoscale devices that operate maximally at molecular levels. This study devises an object by way of mechanosynthesis thereby adding atom one at a time to form the desired nanoscale device. However such nano scale devices manufacturing procedures should not be confused with conventional technologies that are ardent in making traditional carbon tubes, nanofibers, nanoscale particles etc.
An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots.
Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane). The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery.
An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene.
1. Microarray Expression Analysis: In this experimental setup, the cDNA derived from the mRNA of known genes is immobilized. The sample has genes from both the normal as well as the diseased tissues. Spots with more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease.
2. Microarray for Mutation Analysis: For this analysis, the researchers use gDNA. The genes might differ from each other by as less as a single nucleotide base.
A single base difference between two sequences is known as Single Nucleotide Polymorphism (SNP) and detecting them is known as SNP detection.
Applications of Microarrays
1. Gene Discovery: DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels under different conditions.
2. Disease Diagnosis: DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.
3. Drug Discovery: Microarray technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.
4. Toxicological Research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants.
DNA microarray analysis is one of the fastest-growing new technologies in the field of genetic research. Scientists are using DNA microarrays to investigate everything from cancer to pest control.
A DNA micorarray allows scientists to perform an experiment on thousands of genes at the same time. Each spot on a microarray contains multiple identical strands of DNA. The DNA sequence on each spot is unique. Each spot represents one gene. Microarrays can be the size of a microscope slide, or even smaller.
Protein microarrays, an emerging class of proteomic technologies, are fast becoming critical tools in biochemistry and molecular biology. Two classes of protein microarrays are currently available: analytical and functional protein microarrays.
Analytical protein microarrays, mostly antibody microarrays, have become one of the most powerful multiplexed detection technologies. Functional protein microarrays are being increasingly applied to many areas of biological discovery, including studies of protein interaction, biochemical activity, and immune responses. Great progress has been achieved in both classes of protein microarrays in terms of sensitivity, specificity, and expanded application.
Protein microarrays, also known as protein chips, are miniaturized and parallel assay systems that contain small amounts of purified proteins in a high-density format. They allow simultaneous determination of a great variety of analytes from small amounts of samples within a single experiment. Protein microarrays are typically prepared by immobilizing proteins onto a microscope slide using a standard contact spotter (1,2) or noncontact microarrayer (3,4,5).
A variety of slide surfaces can be used. Popular types include aldehyde-and epoxy-derivatized glass surfaces for random attachment through amines (2,6), nitrocellulose (7,8), or gel-coated slides (9,10) and nickel-coated slides for affinity attachment of His6-tagged proteins. The last type was reported to provide 10-fold better signals than those obtained with other random attachment methods. After proteins are immobilized on the slides, they can be probed for a variety of functions/ activities. Finally, the resulting signals are usually measured by detecting fluorescent or radio-isotope labels.
Tissue microarray is a recent innovation in the field of pathology. A microarray contains many small representative tissue samples from hundreds of different cases assembled on a single histologic slide, and therefore allows high throughput analysis of multiple specimens at the same time.
Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates. Using this technique, up to 1000 or more tissue samples can be arrayed into a single paraffin block. It can permit simultaneous analysis of molecular targets at the DNA, mRNA, and protein levels under identical, standardized conditions on a single glass slide, and also provide maximal preservation and use of limited and irreplaceable archival tissue samples.
This versatile technique, in which data analysis is automated facilitates retrospective and prospective human tissue studies. It is a practical and effective tool for high-throughput molecular analysis of tissues that is helping to identify new diagnostic and prognostic markers and targets in human cancers, and has a range of potential applications in basic research, prognostic oncology and drug discovery. This article summarizes the technical aspects of tissue microarray construction and sectioning, advantages, application, and limitations.
Genetic engineering, sometimes called genetic modification, is the process of altering the DNA in an organism’s genome, This may mean changing one base pair (A-T or C-G), deleting a whole region of DNA, or introducing an additional copy of a gene. It may also mean extracting DNA from another organism’s genome and combining it with the DNA of that individual.
Genetic engineering is used by scientists to enhance or modify the characteristics of an individual organism. Genetic engineering can be applied to any organism, from a virus to a sheep.
A small piece of circular DNA called a plasmid is extracted from the bacteria or yeast cell. A small section is then cut out of the circular plasmid by restriction enzymes, ‘molecular scissors’. The gene for human insulin is inserted into the gap in the plasmid. This plasmid is now genetically modified. The genetically modified plasmid is introduced into a new bacteria or yeast cell. This cell then divides rapidly.
To create large amounts of the cells, the genetically modified bacteria or yeast are grown in large fermentation vessels that contain all the nutrients they need. When fermentation is complete, the mixture is filtered.
Genetic engineering has a number of useful applications, including scientific research, agriculture and technology. In plants, genetic engineering has been applied to improve the resilience, nutritional value and growth rate of crops such as potatoes, tomatoes and rice. In animals it has been used to develop sheep that produce a therapeutic protein in their milk that can be used to treat cystic fibrosis, or worms that glow in the dark to allow scientists to learn more about diseases such as Alzheimer’s.
Recombinant DNA technology
All organisms on Earth evolved from a common ancestor, so all organisms use DNA as their molecule of heredity. At the chemical level, DNA is the same whether it is taken from a microscopic bacterium or a blue whale. As a result, DNA from different organisms can be “cut and pasted” together, resulting in “recombinant DNA”.
If the DNA from the different sources is cut with the same restriction enzyme, the cut ends can be joined together and then sealed into a continuous DNA strand by the enzyme ligase. In 1973, the first organism to contain recombinant DNA was engineered by Herb Boyer (UCSF) and Stanley Cohen (Stanford University). Together they introduced an antibiotic resistance gene into E.coli bacteria. Notably, they also produced bacteria that contained genes from the toad Xenopus laevis , which showed DNA from very different species could be spliced together. Paul Berg was awarded the 1980 Nobel Prize in Chemistry “for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA”.
The ability to cut, paste, and copy molecules of DNA was not only a watershed moment for scientific research but spawned an entire industry built on genetic engineering.
Today recombinant DNA technology is used extensively in research laboratories worldwide to explore myriad questions about gene structure, function, expression pattern, regulation, and much more. One widely used application involves genetically engineering “knock-out” animals (typically mice) to contain a non-functional form of a particular gene of interest. The goal of such experiments is to determine gene function by analyzing the consequences of the missing gene. While knockout mice are generated to answer questions in many different fields, they are particularly useful in developmental biology and have led to an understanding of some of the essential genes involved in the development of an organism from a single fertilized egg.
Recombinant DNA techniques are also a cornerstone of the biotechnology industry. One example is the generation of genetically engineered plants to produce an insect toxin called Bt toxin. The Bt gene is derived from a bacterium called Bacillus thuringiensis and produces a toxin that disrupts gut function in the larvae (caterpillars) of certain insects that are crop pests. The gene that produces Bt toxin is introduced into such plants by recombinant DNA technology, and results in the selective killing of crop-feeding insects. This development has had a major economic impact and reduced the expenses of pesticides used per year and has increased the longevity and success of several crops.
A widely debated application of recombinant DNA technology is in the production of genetically modified foods. Genes can be derived from plants or even other organisms to give plants characteristics that are beneficial to both producers and consumers of agricultural products:
1. Delayed fruit ripening for longer shelf life during transportation
2. Resistance to insects and plant viruses
3. Enhanced flavor and nutritional content
4. Edible vaccines to prevent widespread diseases in developing countries
The technology behind genetic modification of foods is similar to the one used to produce human insulin, with an additional step. After the bacteria multiply the gene of interest, the gene is then introduced into plant cells so that the plant will manufacture the gene product: whether it is an insecticide, vaccine, or other plant substance.
Because agriculture is conducted on such a large scale, the use of genetically modified plants poses ecological implications that must be considered carefully.
Protein engineering is the conception and production of unnatural polypeptides, often through modification of amino acid sequences that are found in nature. Synthetic protein structures and functions can now be designed entirely on a computer or produced through directed evolution in the laboratory.
It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It is also a product and services market, with an estimated value of $168 billion by 2017.
There are two general strategies for protein engineering: rational protein design and directed evolution. These methods are not mutually exclusive; researchers will often apply both. In the future, more detailed knowledge of protein structure and function, and advances in high-throughput screening, may greatly expand the abilities of protein engineering. Eventually, even unnatural amino acids may be included, via newer methods, such as expanded genetic code, that allow encoding novel amino acids in genetic code.
Computing methods have been used to design a protein with a novel fold, named Top7, and sensors for unnatural molecules. The engineering of fusion proteins has yielded rilonacept, a pharmaceutical that has secured Food and Drug Administration (FDA) approval for treating cryopyrin-associated periodic syndrome.
Another computing method, IPRO, successfully engineered the switching of cofactor specificity of Candida boidinii xylose reductase. Iterative Protein Redesign and Optimization (IPRO) redesigns proteins to increase or give specificity to native or novel substrates and cofactors. This is done by repeatedly randomly perturbing the structure of the proteins around specified design positions, identifying the lowest energy combination of rotamers, and determining whether the new design has a lower binding energy than prior ones.
Computation-aided design has also been used to engineer complex properties of a highly ordered nano-protein assembly.
Antisense and RNA Interference
MicroRNA (miRNA) are gene-regulatory RNAs that are loaded onto the RNA-induced silencing complex (RISC) and interact with partially-complementary targets on mRNA to suppress protein expression.
The miRNA is originally double-stranded and composed of strands about 21 nucleotides long; on loading onto RISC, one strand is degraded and the other, the “guide” strand, is held on the surface of RISC where it can interact with mRNA. The targets recognized by the guide strand are most commonly on the 3′-untranslated region (UTR) of an RNA.
Antisense is a nucleic acid strand (or nucleic acid analog) that is complementary to an mRNA sequence. Antisense occurs naturally and can trigger RNA degradation by the action of the enzyme RNase H. Originally natural antisense RNA was tried as a method for silencing a gene, but the poor stability of RNA in cells led to development of nucleic acid analogs that are more nuclease resistant and still activate RNase H (such as phosphorothioate RNA) and other nucleic acid analogs that bind to RNA and sterically inhibit processes without activating RNase H (such as 2′-O-methyl phosphorothioate RNA, Morpholino oligos, locked nucleic acids, or peptide nucleic acids).
These latter RNase-H independent oligos do not trigger degradation of mRNA but they can be used like molecular masking tape to block translation, alter splicing of pre-mRNA, inhibit activity of miRNA, block ribozyme activity, and interfere with various other processes that require some other factor to bind to a particular sequence on an RNA molecule.
DNA fingerprinting is a laboratory technique used to establish a link between biological evidence and a suspect in a criminal investigation. A DNA sample taken from a crime scene is compared with a DNA sample from a suspect. If the two DNA profiles are a match, then the evidence came from that suspect. Conversely, if the two DNA profiles do not match, then the evidence cannot have come from the suspect. DNA fingerprinting is also used to establish paternity
DNA Typing Techniques
DNA typing is a procedure wherein DNA extracted from a biological sample obtained from an individual is analyzed. The DNA is processed to generate a pattern for each person that is generally termed as a ‘ DNA profile’. This profile is unique for each person excepting that derived from identical twins.
DNA typing is founded on a number of genetic and molecular principles. Basic to the understanding of the complexity of DNA typing is the concept of the cell. A cell is the building unit of an organism made up of its component parts, one of which is the nucleus that functions as its command center. As the command center of the cell, the nucleus houses the DNA or deoxyribonucleic acid that codes for genetic information responsible for all cellular processes.
Several DNA molecules comprise genes which in turn are located in minute bodies called chromosomes. In humans, there are 23 pairs of chromosomes within a cell thus making up a total of 46 chromosomes. Some chromosomal regions contain repeating units of the same type of DNA molecule which may or may not code for a specific protein. The number of repeating units in individuals may vary hence chromosomal regions with short tandem repeating DNA units (known as Short Tandem Repeat or STR markers) have been used as markers for human identification in forensic casework.
DNA can also be found in another part of the cell called the mitochondrion. Unlike nuclear DNA with two copies per cell, multiple copies of mitochondrial DNA (up to 100,000 copies) are present per human cell. Because of this, mitochondrial DNA analysis is the method of choice when dealing with environmentally challenged samples, e.g. identification of mass disaster victims, exhumed human remains.
Steps in DNA Fingerprinting
DNA fingerprinting involves a number of intensive and important steps in order to fully complete and develop and DNA fingerprint of a father, a suspect or a person involved in an immigration problem. The process of DNA fingerprinting starts with isolating DNA from any part of the body such as blood, semen, vaginal fluids, hair roots, teeth, bones, etc.
Polymerase chain reaction (PCR) is the next step in the process. In many situations, there is only a small amount of DNA available for DNA fingerprinting. Because of this, in a test tube, DNA replication is must occur to make more DNA. The DNA and the cells will undergo DNA replication in order to make more DNA to be tested.
After the DNA is isolated and more copies of the DNA have been made, the DNA will be tested. The scientist will treat DNA with restriction enzymes. This will produce different sized fragments which are known as restriction fragment length polymorphisms (RFLPs). These fragments can then be observed doing an experiment called gel electrophoresis which separates DNA based on fragment sizes.
Gel electrophoresis is the next step in this process of DNA fingerprinting. During gel electrophoresis, an electrical current is applied to a gel mixture, which includes the samples of the DNA.
The electric current causes the DNA strands to move through the gel. This separates the molecules of different sizes.
The fragments of separated DNA are sieved out of the gel using a nylon membrane (treated with chemicals that allow for it to break the hydrogen bonds of DNA so there are sing strands). The DNA (single stranded) is cross-linked against the nylon using heat or a UV light.
The probe shows up on photographic film because the strands of DNA decay and give off light. In the end it leaves dark spots on the film which are also known as the DNA bands of a person. What make up the fingerprint are the unique patterns of bands. The pattern of bands are different because we are all different and unique (other than identical twins).
Once the filter is exposed to the x-ray film, the radioactive DNA sequences are shown and can be seen with the naked eye. This creates a banding pattern or what we know as DNA fingerprints. This technique is called southern blotting. These fingerprints can be used to determine which hair strand belongs to which person for example.
DNA fingerprints of children should be similar to the their parents’ fingerprints, although they may not be the same. Some bands will match one parent and other bands can match the other parent. With the bands of both of those parents, they make the bands and the identity of the child.
Applications of DNA Fingerprinting
In 1984, Alec Jeffreys, a British geneticist, identified the presence of minisatellites within the boundaries of genes. These minisatellites do not contribute to the functioning of genes and are distributed throughout the cellular DNA of an organism in a unique and inheritable pattern. The DNA fingerprint can be revealed by processing cells collected from individuals through one of several different techniques. These different techniques for genetic fingerprinting have been applied to identify and isolate disease genes, develop cures for diseased genes, and diagnose genetic diseases.
Testing paternity samples requires the collection of cells and comparison of DNA fingerprints from and between children and potential parents. Children will have a mix of DNA fingerprints inherited from each parent. When a child is conceived, each parent provides half of the genetic information. Most often the test is performed when the mother of the child is known but the father is in question.
Since it is highly unlikely that any two people will have the same genetic fingerprint, paternity testing using DNA fingerprints is a reliable way to determine the parentage of a child.
A crime scene can contain biological samples, including blood, semen, saliva, skin, urine and hair, from perpetrators, victims and bystanders that can be processed to provide DNA fingerprints. The DNA fingerprints obtained are used to search existing databases for matches and to identify victims or suspects.
The biological evidence and the DNA fingerprints can be used in trials to help prove guilt or innocence. The United States military has been storing DNA fingerprints of all military personnel for identification of casualties and those missing in action. The military has found the technology to be superior to identification methods used previously.
Plants and Animals
DNA fingerprinting of plants and animals is performed for food security, food safety, identification and parentage. In food animals, DNA fingerprinting can be used to trace meat to the source animal. The technique can be used to identify endangered and non-endangered fish species, while the sources of plants can be verified to prevent counterfeiting of seeds and stock. Pathogenic food organisms can be quickly identified by their DNA fingerprints, allowing doctors to provide timely, targeted treatment
Clones are organisms that are exact genetic copies. Every single bit of their DNA is identical. Clones can happen naturally—identical twins are just one of many examples. Or they can be made in the lab.
There are two ways to make an exact genetic copy of an organism in a lab: artificial embryo twinning and somatic cell nuclear transfer.
1. Artificial Embryo Twinning
Artificial embryo twinning is a relatively low-tech way to make clones. As the name suggests, this technique mimics the natural process that creates identical twins.
In nature, twins form very early in development when the embryo splits in two. Twinning happens in the first days after egg and sperm join, while the embryo is made of just a small number of unspecialized cells. Each half of the embryo continues dividing on its own, ultimately developing into separate, complete individuals. Since they developed from the same fertilized egg, the resulting individuals are genetically identical.
Artificial embryo twinning uses the same approach, but it is carried out in a Petri dish instead of inside the mother. A very early embryo is separated into individual cells, which are allowed to divide and develop for a short time in the Petri dish. The embryos are then placed into a surrogate mother, where they finish developing. Again, since all the embryos came from the same fertilized egg, they are genetically identical.
2. Somatic Cell Nuclear Transfer
Somatic cell nuclear transfer (SCNT), also called nuclear transfer, uses a different approach than artificial embryo twinning, but it produces the same result: an exact genetic copy, or clone, of an individual. This was the method used to create Dolly the Sheep.
A somatic cell is any cell in the body other than sperm and egg, the two types of reproductive cells. Reproductive cells are also called germ cells. In mammals, every somatic cell has two complete sets of chromosomes, whereas the germ cells have only one complete set. The nucleus is a compartment that holds the cell’s DNA. The DNA is divided into packages called chromosomes, and it contains all the information needed to form an organism. It is small differences in our DNA that make each of us unique.
The lamb, Dolly, was an exact genetic replica of the adult female sheep that donated the somatic cell. She was the first-ever mammal to be cloned from an adult somatic cell.
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMO).
This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as “clones”. Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them.
The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes.
Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. Another characteristic that limits chances of success is large size of DNA sequence. Inserts larger than 10kbp have very limited success, but bacteriophages such as bacteriophage λ can be modified to successfully insert a sequence up to 40 kbp.
In reproductive cloning, researchers remove a mature somatic cell, such as a skin cell, from an animal that they wish to copy. They then transfer the DNA of the donor animal’s somatic cell into an egg cell, or oocyte, that has had its own DNA-containing nucleus removed.
Researchers can add the DNA from the somatic cell to the empty egg in two different ways. In the first method, they remove the DNA-containing nucleus of the somatic cell with a needle and inject it into the empty egg. In the second approach, they use an electrical current to fuse the entire somatic cell with the empty egg. In both processes, the egg is allowed to develop into an early-stage embryo in the test-tube and then is implanted into the womb of an adult female animal.
Ultimately, the adult female gives birth to an animal that has the same genetic makeup as the animal that donated the somatic cell. This young animal is referred to as a clone. Reproductive cloning may require the use of a surrogate mother to allow development of the cloned embryo, as was the case for the most famous cloned organism, Dolly the sheep.
Nucleus and Cytoplasm
The nucleus and the cytoplasm are two very different parts of cells, but they also work together in a number of key ways, particularly where protein production and cell division are concerned. Nearly all cells, whether human, plant, or animal, have both of these elements.
The nucleus is basically the “brain” of the cell, and is where all of the important data and cellular materials are housed. It looks sort of like the yolk in the center of an egg. The cytoplasm, by contrast, is more analogous to an egg white. It is the fluid that keeps the cell’s internal environment suspended, and the nucleus floats within it. Materials can and do pass from the cytoplasm into the nucleus and then from the nucleus back into the cytoplasm, but the process is highly regulated.
Protein production is one of the most important things to require this sort of free passage, and is what allows cells to reproduce and grow. The contents of the nucleus don’t usually come into contact with the cytoplasm, but two scenarios — cell division and cell death — are exceptions.
Controversies over Animal Cloning
The public does not support animal cloning, and the technology is riddled with problems that cause animal suffering. Our government must act to address the public’s concerns and keep these products off of grocery store shelves.
Despite years of research, over 95% of cloning attempts fail, even with extensive veterinary intervention. Birth defects, physiological impairments, illness, and premature death continue to be the norm, not the exception, with cloning. Seemingly healthy clones have unexpectedly developed problems. Problems occur with cloning far more often than with any other method of reproduction.
Large Offspring Syndrome, a typically fatal condition associated with a host of abnormalities, occurs in over 50% of cow clones, but in fewer than 6% of conventionally bred animals. Hydrops, another typically fatal condition in which the animal swells with fluid, occurs in 28% of cow clones, but very rarely otherwise. A high rate of late-term pregnancy loss, pregnancy complications, painful labor, and surgical intervention is unique to clone pregnancies.
The public is worried about the moral and ethical implications of animal cloning Cloning affects animal welfare and promotes intensive farming practices and the commodification of animals. Many feel that cloning is “not natural,” or is “playing God.”
Governments around the world are debating the ethics of cloning animals for food. The expert European Group on Ethics concluded that there is no ethical justification to clone animals. The debate over cloning illustrates how complex innovations in biotechnology often outpace society’s ability to make sense of them.
Advocates of the process contend that animal cloning has been studied rigorously for decades and has been shown to be safe. Cloning speeds up reproduction of the healthiest and most productive livestock. Most consumers will likely never eat a cloned animal because clones are expensive; it is their progeny that will enter the food chain. Additionally, cloning could lead to creating lines of animals resistant to diseases harmful to humans, such as bovine spongiform encephalopathy.
Cloning food animals could have unintended consequences, such as accelerating monoculture in animal agriculture. A new or emerging pathogen could wipe out entire herds because of the lack of genetic diversity among the animals.
Classification of Stem Cells based on Differentiation
Currently, there is not an agreed upon number of stem cell types in existence, because one can classify stem cells either by differentiation potential (what they can turn into) or by origin (from where they are sourced). While classifying stem cells by the source tissue from which they are derived is descriptive, it is not valuable for classification purposes, because there is the potential to label stem cells into dozens (if not hundreds) of different categories.
There are also a number of cell types that have varying capacities for differentiation. For these cells, some researchers label them as “stem cells,” while others consider them to be either “progenitor cells” or simply cells with diverse functional capabilities. All stem cells that exist can be classified into one of five groups based on their differentiation potential. These groups are:
1. Totipotent (or Omnipotent) Stem Cells
These stem cells are the most powerful that exist. They can differentiate into embryonic, as well as extra-embryonic tissues, such as chorion, yolk sac, amnion, and the allantois. In humans and other placental animals, these tissues form the placenta. The most important characteristic of a totipotent cell is that it can generate a fully-functional, living organism.
The best known example of a totipotent cell is a fertilized egg (formed when a sperm and egg unite to form a zygote).
2. Pluripotent Stem Cells
The next most powerful type of stem cell is the pluripotent stem cell. The importance of this cell type is that it can self-renew and differentiate into any of the three germ layers, which are: ectoderm, endoderm and mesoderm. These three germ layers further differentiate to form all tissues and organs within a human being. Among the natural pluripotent stem cells, embryonic stem cells are the best example. However, a type of “human-made” pluripotent stem cell also exists, which is the induced pluripotent stem cell (iPS cell).
3. Multipotent Stem Cells
Multipotent stem cells are a middle-range type of stem cell, in that they can self-renew and differentiate into a specific range of cell types. An excellent example of this cell type is the mesenchymal stem cell (MSC). Mesenchymal stem cells can differentiate into osteoblasts (a type of bone cell), myocytes (muscle cells), adipocytes (fat cells), and chondrocytes (cartilage cells).
These cells types are fairly diverse in their characteristics, which is why mesenchymal stem cells are classified as multipotent stem cells.
4. Oligopotent stem cells
The next type of stem cells, oligopotent stem cells are similar to the prior category (multipotent stem cells), but they become further restricted in their capacity to differentiate. While these cells can self-renew and differentiate, they can only do so to a limited, they can only do so into closely related cell types. An excellent example of this cell type is the hematopoietic stem cell (HSC).
HSCs are cells derived from mesoderm that can differentiate into other blood cells. Specifically, HSCs are oligopotent stem cells that can differentiate into both myeloid and lymphoid cells.
5. Unipotent Stem Cells
The unipotent stem cells, are the least potent and most limited type of stem cell. An example of this stem cell type would be muscle stem cells. While muscle stem cells can self-renew and differentiate, they can only do so into a single cell type. They are uni-directional in their differentiation capacity.
Stem cell technology
Stem cell technology is a rapidly developing field that combines the efforts of cell biologists, geneticists, and clinicians and offers hope of effective treatment for a variety of malignant and non-malignant diseases. Stem cells are defined as totipotent progenitor cells capable of self renewal and multilineage differentiation.
Stem cells survive well and show stable division in culture, making them ideal targets for in vitro manipulation. Although early research has focused on haematopoietic stem cells, stem cells have also been recognised in other sites. Research into solid tissue stem cells has not made the same progress as that on haematopoietic stem cells. This is due to the difficulty of reproducing the necessary and precise three dimensional arrangements and tight cell-cell and cell-extracellular matrix interactions that exist in solid organs. However, the ability of tissue stem cells to integrate into the tissue cytoarchitecture under the control of the host microenvironment and developmental cues, makes them ideal for cell replacement therapy.
Embryonic Stem Cells
Embryonic stem cells are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro—in an in vitro fertilization clinic—and then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman’s body.
Human embryonic stem cells (hESCs) are generated by transferring cells from a preimplantation-stage embryo into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. In the original protocol, the inner surface of the culture dish was coated with mouse embryonic skin cells specially treated so they will not divide. This coating layer of cells is called a feeder layer. The mouse cells in the bottom of the culture dish provide the cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Researchers have now devised ways to grow embryonic stem cells without mouse feeder cells. This is a significant scientific advance because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
The process of generating an embryonic stem cell line is somewhat inefficient, so lines are not produced each time cells from the preimplantation-stage embryo are placed into a culture dish. However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed gently and plated into several fresh culture dishes. The process of re-plating or subculturing the cells is repeated many times and for many months. Each cycle of subculturing the cells is referred to as a passage. Once the cell line is established, the original cells yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line. At any stage in the process, batches of cells can be frozen and shipped to other laboratories for further culture and experimentation.
At various points during the process of generating embryonic stem cell lines, scientists test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells. This process is called characterization.
Scientists who study human embryonic stem cells have not yet agreed on a standard battery of tests that measure the cells’ fundamental properties. However, laboratories that grow human embryonic stem cell lines use several kinds of tests, including:
1. Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term growth and self-renewal. Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated.
2. Using specific techniques to determine the presence of transcription factors that are typically produced by undifferentiated cells. Two of the most important transcription factors are Nanog and Oct4. Transcription factors help turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. In this case, both Oct 4 and Nanog are associated with maintaining the stem cells in an undifferentiated state, capable of self-renewal.
3. Using specific techniques to determine the presence of particular cell surface markers that are typically produced by undifferentiated cells.
4. Examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed. It does not detect genetic mutations in the cells.
5. Determining whether the cells can be re-grown, or subcultured, after freezing, thawing, and re-plating.
6. Testing whether the human embryonic stem cells are pluripotent by 1) allowing the cells to differentiate spontaneously in cell culture; 2) manipulating the cells so they will differentiate to form cells characteristic of the three germ layers; or 3) injecting the cells into a mouse with a suppressed immune system to test for the formation of a benign tumor called a teratoma. Since the mouse’s immune system is suppressed, the injected human stem cells are not rejected by the mouse immune system and scientists can observe growth and differentiation of the human stem cells. Teratomas typically contain a mixture of many differentiated or partly differentiated cell types—an indication that the embryonic stem cells are capable of differentiating into multiple cell types.
Adult Stem Cells
An adult stem cell is thought to be an undifferentiated cell, found among differentiated cells in a tissue or organ. The adult stem cell can renew itself and can differentiate to yield some or all of the major specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found.
Scientists also use the term somatic stem cell instead of adult stem cell, where somatic refers to cells of the body (not the germ cells, sperm or eggs). Unlike embryonic stem cells, which are defined by their origin (cells from the preimplantation-stage embryo), the origin of adult stem cells in some mature tissues is still under investigation.
Research on adult stem cells has generated a great deal of excitement. Scientists have found adult stem cells in many more tissues than they once thought possible. This finding has led researchers and clinicians to ask whether adult stem cells could be used for transplants. In fact, adult hematopoietic, or blood-forming, stem cells from bone marrow have been used in transplants for more than 40 years. Scientists now have evidence that stem cells exist in the brain and the heart, two locations where adult stem cells were not at first expected to reside. If the differentiation of adult stem cells can be controlled in the laboratory, these cells may become the basis of transplantation-based therapies.
The history of research on adult stem cells began more than 60 years ago. In the 1950s, researchers discovered that the bone marrow contains at least two kinds of stem cells. One population, called hematopoietic stem cells, forms all the types of blood cells in the body. A second population, called bone marrow stromal stem cells (also called mesenchymal stem cells, or skeletal stem cells by some), were discovered a few years later. These non-hematopoietic stem cells make up a small proportion of the stromal cellpopulation in the bone marrow and can generate bone, cartilage, and fat cells that support the formation of blood and fibrous connective tissue.
Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. They are thought to reside in a specific area of each tissue (called a “stem cell niche”). In many tissues, current evidence suggests that some types of stem cells are pericytes, cells that compose the outermost layer of small blood vessels. Stem cells may remain quiescent (non-dividing) for long periods of time until they are activated by a normal need for more cells to maintain tissues, or by disease or tissue injury.
Typically, there is a very small number of stem cells in each tissue and, once removed from the body, their capacity to divide is limited, making generation of large quantities of stem cells difficult. Scientists in many laboratories are trying to find better ways to grow large quantities of adult stem cells in cell culture and to manipulate them to generate specific cell types so they can be used to treat injury or disease. Some examples of potential treatments include regenerating bone using cells derived from bone marrow stroma, developing insulin-producing cells for type 1 diabetes, and repairing damaged heart muscle following a heart attack with cardiac muscle cells.
Applications of Stem Cells
Research using human and animal stem cell preparations continues to be an extremely active area. It is developing new research tools, new knowledge about pathways of cell differentiation and opening new vistas of cell transplantation therapy for human diseases.
Heterogeneous human stem cell preparations derived from bone marrow have been in clinical use as treatment (bone marrow transplants) for various forms of cancer for many years. More recently, hematopoietic stem cells have been isolated and purified from bone marrow and are being studied.
Much of the impetus for stem cell research comes from the hope that stem cell preparations, or more differentiated cells derived from them, will one day prove useful in cell transplantation therapies for various human diseases. It may cost too much and take too long to produce a sufficient number of well-characterized cells for therapy if one starts with cells from each individual patient. This suggests that cells derived from the stem cells of one individual will be used to treat multiple other individual patients (allogeneic cell transplantation).
Before stem cell-based therapies are used to treat human diseases, they will have to gain approval through the FDA regulatory process. The first step in this process is filing an Investigational New Drug Exemption (IND) application. The disease indications include: 1) providing MSC support for peripheral blood stem cell transplantation in cancer treatment, 2) providing MSC support for cord blood transplantation in cancer treatment, 3) using MSCs to stimulate regeneration of cardiac tissue post acute myocardial infarction (heart attack), and 4) using MSCs to stimulate regeneration of cardiac tissue in cases of congestive heart failure. Each of the first two applications is currently in Phase II of the regulatory process, with pivotal Phase III trials scheduled to begin in 200450.
Therapeutic cloning refers to the removal of a nucleus, which contains the genetic material, from virtually any cell of the body (a somatic cell) and its transfer by injection into an unfertilised egg from which the nucleus has also been removed. The newly reconstituted entity then starts dividing. After 4-5 days in culture, embryonic stem cells can then be removed and used to create many embryonic stem cells in culture. These embryonic stem cell ‘lines’ are genetically identical to the cell from which the DNA was originally removed. Therapeutic cloning is also known as somatic cell nuclear transfer (SNCT) as the term cloning is frequently misunderstood by the general public. The word ‘cloning’ more often conjures up thoughts and beliefs about reproductive cloning.
While the procedure of therapeutic cloning employs aspects of cloning technology, researchers today are interested in therapeutic cloning as a means of deriving human embryonic stem cell lines for use in research and, ultimately, therapy.
The capacity of therapeutic cloning to re-program adult nuclei is extraordinary and unique. Cells of particular tissues generally express a characteristic set of genes. Whether they are primitive stem cells, fully-differentiated (i.e. mature) cells, or something in between these extremes. Particularly for more mature cells, the tissue-specific patterns of gene expression are quite stable through many rounds of cell division. Upon transfer to an enucleated egg, the adult nucleus becomes re-programmed in the environment of the egg. That is, genes that were not used before (switched off) become reactivated. A poorly-understood process, re-programming involves dramatic changes in the pattern of genes which are active in the nucleus. Instead of the adult nucleus causing the egg to behave like an adult cell, the egg causes the nucleus to go backwards along a differentiation sequence, resulting in an embryonic type cell. As a result of therapeutic cloning, the previously unfertilised egg takes on the properties of a fertilised egg and begins the first stages of development into an embryo.
In broad terms, embryos arising from therapeutic cloning are the same as embryos from fertilisation of an egg by a sperm. In agricultural research, therapeutic cloning has been used to create embryos in the laboratory which have been transferred into animals and given rise to offspring. However, there are also differences between a normally fertilised egg and one produced by therapeutic cloning, which are not generally understood. In animal studies clones appear to have increased abnormality and decreased pregnancy rates.
The various OMICS of Biotechnology
Omics has become the new mantra in molecular research. “Omics” technologies include genomics, transcriptomics, proteomics and metabolomics. Genomics had revealed the static sequences of genes and proteins and focus has now been shifted to their dynamic functions and interactions. Transcriptomics, proteomics and metabolomics reveal the biological function of the gene product.
The “-omic-” technologies are high-throughput technologies and they increase substantially the number of proteins/genes that can be detected simultaneously to relate complex mixtures to complex effects in the form of gene/protein expression profiles. The primary aim of omic technologies is the nontargeted identification of all gene products (transcripts, proteins, and metabolites) present in a specific biological sample. The powerful “omics” technologies have opened new avenues towards biomarker discovery, identification of signaling molecules associated with cell growth, cell death, cellular metabolism and early detection of cancer. Omics will not only have an impact on our understanding of biological processes, but the prospect of more accurately diagnosing and treating disease will soon become a reality.
Within the field of molecular biology, genomics is the study of genomes, the complete set of genetic material within an organism. Genomics involves the sequencing and analysis of genomes. Genomics is also concerned with the structure, function, comparison, and evolution of genomes.
In contrast to genetics, which refers to the study of individual genes and their roles in inheritance, genomics uses high throughput DNA sequencing and bioinformatics to assemble, and analyze the function and structure of entire genomes. The field also includes studies of intragenomic (within the genome) phenomena such as heterosis (hybrid vigour), epistasis (effect of one gene on another), pleiotropy (one gene affecting more than one trait) and other interactions between loci and alleles within the genome.
Advances in genomics have triggered a revolution in systems biology which facilitates the understanding of complex biological systems such as the brain.
Structural genomics is a term that refers to high-throughput three-dimensional structure determination and analysis of biological macromolecules, at this stage primarily individual protein domains. The determination of the three-dimensional structures of proteins has for many years come under the classification of “curiosity” or “hypothesis driven” research.
Structures were generally determined because they could be expected to teach us something new about a biological problem; for example, the details of an enzyme mechanism, the nature of a molecular recognition process, or the energetic basis of energy transduction processes. An important spinoff of structural biology has been the discovery of new relationships between amino acid sequences and protein structures, and among different protein structures. New computational tools have been developed to exploit the information that has become available, and many remarkable and unexpected relationships have been uncovered.
Concepts such as protein family, fold, and superfamily have been introduced, and detailed taxonomies have been developed that help us understand the complex three-dimensional shapes of proteins. Structural genomics represents a new direction in structural biology in that it is based on the goal of determining as many structures as possible, even in advance of a well-defined biological question. Nevertheless, the field is ultimately “curiosity driven” but the questions being asked now relate to the discovery of complex relationships in sequence and structure space and, ultimately, to a deeper understanding of many biological problems once these relationships are understood.
Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic and transcriptomic projects (such as genome sequencing projects and RNA sequencing) to describe gene (and protein) functions and interactions. Unlike structural genomics, functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures.
Functional genomics attempts to answer questions about the function of DNA at the levels of genes, RNA transcripts, and protein products. A key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional “gene-by-gene” approach.
Proteomics is the large-scale study of proteomes. A proteome is a set of proteins produced in an organism, system, or biological context. We may refer to, for instance, the proteome of a species (for example, Homo sapiens) or an organ (for example, the liver). The proteome is not constant; it differs from cell to cell and changes over time. To some degree, the proteome reflects the underlying transcriptome. However, protein activity (often assessed by the reaction rate of the processes in which the protein is involved) is also modulated by many factors in addition to the expression level of the relevant gene.
Proteomics is used to investigate:
1. When and where proteins are expressed;
2. Rates of protein production, degradation, and steady-state abundance;
3. How proteins are modified (for example, post-translational modifications (PTMs) such as phosphorylation);
4. The movement of proteins between subcellular compartments;
5. The involvement of proteins in metabolic pathways;
6. How proteins interact with one another.
Several high-throughput technologies have been developed to investigate proteomes in depth. The most commonly applied are mass spectrometry (MS)-based techniques such as Tandem-MS and gel-based techniques such as differential in-gel electrophoresis (DIGE). These high-throughput technologies generate huge amounts of data. Databases are critical for recording and carefully storing this data, allowing the researcher to make connections between their results and existing knowledge.
Proteomic experiments generally collect data on three properties of proteins in a sample: location, abundance/turnover and post-translational modifications. Depending on the experimental design, researchers may be directly interested in these data, or may use them to infer additional information. For example, it may be possible to infer a protein’s interaction partners among others that are colocalised with it, or to assess whether a protein is active from its post-translational modifications.
Bioinformatics is the application of Information technology to store, organize and analyse the vast amount of biological data which is available in the form of sequences and structures of proteins – the building blocks of organisms and nucleic acids – the information carrier. The biological information of nucleic acids is available as sequences while the data of proteins is available as sequences and structures. Sequences are represented in single dimension where as the structure contains the three dimensional data of sequences.
Computers and software tools are extensively used for creating these databases and to predict the function of proteins, model the structure of proteins, determine the coding(useful) regions of nucleic acid sequences, find suitable drug compounds from a large pool and optimise the drug development process by predicting possible targets.
As the analysis involves a lot of computational effort, it is impossible to do it manually. Some of the software tools which are handy in the analysis include: BLAST –Commonly used for comparing sequences; Annotator – an interactive genome (nucleic acid sequence) analysis tool; GeneFinder – Tool to identify coding regions and splicesites.
The sequence information generated by the human genome research, initiated in 1988 has now been stored as a primary information source for future applications in medicine. The available data is so huge that if compiled in books, the data would run into 200 volumes of 1000 pages each and reading alone(ignoring understanding factor) would require 26 years working around the clock. For the population of about 5 billion human beings with two individuals differing in three million bases, the genomic sequence difference database would have about 15,000,000 billion entries. The present challenge to handle such a huge volume of data is to improve database design, develop software for database access and manipulation, and device data-entry procedures to compensate for the varied computer procedures and systems used in different laboratories.
Synthetic biology aims to make biology easier to engineer. Synthetic biology is the convergence of advances in chemistry, biology, computer science, and engineering that enables us to go from idea to product faster, cheaper, and with greater precision than ever before. It can be thought of as a biology-based “toolkit” that uses abstraction, standardization, and automated construction to change how we build biological systems and expand the range of possible products. A community of experts across many disciplines is coming together to create these new foundations for many industries, including medicine, energy and the environment.