Applications of Bio technology

A rose plant that began as cells grown in a tissue culture

Biotechnology has applications in four major industrial areas, including health care (medical), crop production and agriculture, non food (industrial) uses of crops and other products (e.g.biodegradable plastics, vegetable oil, biofuels), and environmental uses.

For example, one application of biotechnology is the directed use of organisms for the manufacture of organic products (examples include beer and milk products). Another example is using naturally present bacteria by the mining industry in bioleaching. Biotechnology is also used to recycle, treat waste, clean up sites contaminated by industrial activities (bioremediation), and also to produce biological weapons.

A series of derived terms have been coined to identify several branches of biotechnology, for example:

• Bioinformatics is an interdisciplinary field which addresses biological problems using computational techniques, and makes the rapid organization and analysis of biological data possible. The field may also be referred to as computational biology, and can be defined as, "conceptualizing biology in terms of molecules and then applying informatics techniques to understand and organize the information associated with these molecules, on a large scale."^[7] Bioinformatics plays a key role in various areas, such as functional genomics,structural genomics, and proteomics, and forms a key component in the biotechnology and pharmaceutical sector.
• Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology, but its use is relatively rare.
• Green biotechnology is biotechnology applied to agricultural processes. An example would be the selection and domestication of plants via micropropagation. Another example is the designing of transgenic plants to grow under specific environmental conditions or in the presence (or absence) of certain agricultural chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides. An example of this would be Bt corn. Whether or not green biotechnology products such as this are ultimately more environmentally friendly is a topic of considerable debate.
• Red biotechnology is applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures through genomic manipulation.
• White biotechnology, also known as industrial biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals. White biotechnology tends to consume less in resources than traditional processes used to produce industrial goods.
• The investments and economic output of all of these types of applied biotechnologies form what has been described as the bioeconomy.

Evolutionary origin

Evolutionary origin

Plant cells with visible chloroplasts.

Chloroplasts are one of the many different types of organelles in the cell. They are generally considered to have originated as endosymbiotic cyanobacteria (i.e. blue-green algae). This was first suggested by Mereschkowsky in 1905 ^[1] after an observation by Schimper in 1883 that chloroplasts closely resemble cyanobacteria. ^[2] All chloroplasts are thought to derive directly or indirectly from a single endosymbiotic event (in the Archaeplastida), except for Paulinellachromatophora, which has recently acquired a photosynthetic cyanobacterial endosymbiont which is not closely related to chloroplasts of other eukaryotes.^[3] In that they derive from an endosymbiotic event, chloroplasts are similar to mitochondria but chloroplasts are found only inplants and protista. The chloroplast is surrounded by a double-layered composite membrane with an intermembrane space; further, it has reticulations, or many infoldings, filling the inner spaces. The chloroplast has its own DNA which codes for redox proteins involved in electron transport in photosynthesis.

In green plants, chloroplasts are surrounded by two lipid-bilayer membranes. The inner membrane is now believed to correspond to the outer membrane of the ancestral cyanobacterium. Chloroplasts have their own genome, which is considerably reduced compared to that of free-living cyanobacteria, but the parts that are still present show clear similarities with the cyanobacterial genome. Plastids may contain 60-100 genes whereas cyanobacteria often contain more than 1500 genes.^[4] Many of the missing genes are encoded in the nuclear genome of the host. The transfer of nuclear information has been estimated in tobacco plants at one gene for every 16000 pollen grains.^[5]

In some algae (such as the heterokonts and other protists such as Euglenozoa and Cercozoa), chloroplasts seem to have evolved through a secondary event of endosymbiosis, in which a eukaryotic cell engulfed a second eukaryotic cell containing chloroplasts, forming chloroplasts with three or four membrane layers. In some cases, such secondary endosymbionts may have themselves been engulfed by still other eukaryotes, thus forming tertiary endosymbionts. In the alga Chlorella, there is only one chloroplast, which is bell shaped.

In some groups of mixotrophic protists such as the dinoflagellates, chloroplasts are separated from a captured alga or diatom and used temporarily. These klepto chloroplasts may only have a lifetime of a few days and are then replaced.^[6]

Structure

Chloroplasts are observable morphologically as flat discs usually 2 to 10 micrometer in diameter and 1 micrometer thick. In land plants they are generally 5 μm in diameter and 2.3 μm thick. The chloroplast is contained by an envelope that consists of an inner and an outer phospholipid membrane. Between these two layers is the intermembrane space. A typical parenchyma cell contains about 10 to 100 chloroplasts.

The material within the chloroplast is called the stroma, corresponding to the cytosol of the original bacterium, and contains one or more molecules of small circular DNA. It also contains ribosomes, although most of its proteins are encoded by genes contained in the host cell nucleus, with the protein products transported to the chloroplast.

Chloroplast ultrastructure:
1. outer membrane
2. intermembrane space
3. inner membrane (1+2+3: envelope)
4. stroma (aqueous fluid)
5. thylakoid lumen (inside of thylakoid)
6. thylakoid membrane
7. granum (stack of thylakoids)
8. thylakoid (lamella)
9. starch
10. ribosome
11. plastidial DNA
12. plastoglobule (drop of lipids)

Within the stroma are stacks of thylakoids, the sub-organelles which are the site of photosynthesis. The thylakoids are arranged in stacks called grana (singular: granum).^[7] A thylakoid has a flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes place on the thylakoid membrane; as in mitochondrial oxidative phosphorylation, it involves the coupling of cross-membrane fluxes withbiosynthesis via the dissipation of a proton electrochemical gradient.

In the electron microscope, thylakoid membranes appear as alternating light-and-dark bands, each 0.01 μm thick. Embedded in the thylakoid membrane is the antenna complex, which consists of the light-absorbing pigments, including chlorophyll and carotenoids, and proteins (which bind the chlorophyll). This complex both increases the surface area for light capture, and allows capture of photons with a wider range of wavelengths. The energy of the incident photons is absorbed by the pigments and funneled to the reaction centre of this complex through resonance energy transfer. Two chlorophyll molecules are then ionised, producing an excited electron which then passes onto the photochemical reaction centre.

Recent studies have shown that chloroplasts can be interconnected by tubular bridges called stromules, formed as extensions of their outer membranes.^[8][9] Chloroplasts appear to be able to exchange proteins via stromules,^[10] and thus function as a network.

Transplastomic plants

Recently, chloroplasts have caught attention by developers of genetically modified plants. In most flowering plants, chloroplasts are not inherited from the male parent, although in plants such as pines, chloroplasts are inherited from males.^[13] Where chloroplasts are inherited only from the female, transgenes in these plastids cannot be disseminated by pollen. This makes plastid transformation a valuable tool for the creation and cultivation of genetically modified plants that are biologically contained, thus posing significantly lower environmental risks. This biological containment strategy is therefore suitable for establishing the coexistence of conventional and organic agriculture. The reliability of this mechanism has not yet been studied for all relevant crop species. However, the research programme Co-Extra recently published results for tobacco plants, demonstrating that the containment of transplastomic plants is highly reliable with a tiny failure rate of 3 in 1,000,000.

Chloroplast

The inside of a chloroplast

Chloroplasts are organelles found in plant cells and eukaryotic algae that conductphotosynthesis. Chloroplasts absorb light and use it in conjunction with water and carbon dioxide to produce sugars, the raw material for energy and biomass production in all green plants and the animals that depend on them, directly or indirectly, for food. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis. The word chloroplast is derived from the Greek words chloros which means green andplast which means form or entity. Chloroplasts are members of a class of organelles known as plastids.

Chloroplast membrane

hloroplasts contain several important membranes, vital for their function. Like mitochondria, chloroplasts have a double-membrane envelope, called the chloroplast envelope. Each membrane is a phospholipid bilayer, between 6 and 8 nm thick, and the two are separated by a gap of 10-20nm, called the intermembrane space. The outer membrane is permeable to most ions and metabolites, but the inner membrane is highly specialised with transport proteins.

The origin of chloroplasts is now largely accepted by the botany community as occurring via endosymbiosis on an ancestral basis with the engulfment of photosynthetic bacterium within the eukaryotic cell. Over millions of years the endosymbiotic cyanobacterium evolved structurally and functionally, retaining its own DNA and cellular mitosis capabilities but losing its ablility to live outside of the host cell.

[edit]Internal parts

Within the inner membrane, in the region called the stroma, there is a system of interconnecting flattened membrane compartments, called thethylakoids. These are the sites of light absorption and ATP synthesis, and contain many proteins, including those involved in the electron transport chain. Photosynthetic pigments such as chlorophyll α and B, and some others e.g. xanthophylls and carotenoids are also located within this space. These are responsible for the conversion of light energy to chemical energy as described below:

[edit]Functions of Thylakoids

The membranes of the chloroplasts contain photosystems I and II which harvest solar energy in order to excite electrons which travel down theelectron transport chain. This exergonic fall in potential energy along the way is used to pump H+ ions from the stroma into the thylakoid space. A concentration gradient is formed, which allows chemiosmosis to occur, where the protein ATP synthase harvests the potential energy of the Hydrogen ions and uses it to combine ADP and a phosphate group to form ATP.

Experiments have shown that the pH within the stroma is about 7.8, while that of the thylakoid space is about 5. This corresponds to a thousandfold difference in concentration of H- ions.

Chromosome

A chromosome is an organized structure of DNA and protein that is found in cells. A chromosome is a single piece of DNA that contains many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. The word chromosome comes from the Greekχρῶμα (chroma, color) and σῶμα (soma, body) due to their property of being stained very strongly by some dyes. Chromosomes vary extensively between different organisms. The DNA molecule may be circular or linear, and can contain anything from 10,000 to 1,000,000,000^[1]nucleotides in length. Typically eukaryotic cells (cells with nuclei) have large linear chromosomes and prokaryotic cells (cells without defined nuclei) have smaller circular chromosomes, although there are many exceptions to this rule. Furthermore, cells may contain more than one type of chromosome; for example, mitochondria in most eukaryotes andchloroplasts in plants have their own small chromosomes. In eukaryotes, nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. The structure of chromosomes and chromatin varies through the cell cycle. Chromosomes may exist as either duplicated or unduplicated—unduplicated chromosomes are single linear strands, whereas duplicated chromosomes (copied during synthesis phase) contain two copies joined by a centromere. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right).

"Chromosome" is a rather loosely defined term. In prokaryotes, a small circular DNA molecule may be called either a plasmid or a small chromosome. These small circular genomes are also found in mitochondria and chloroplasts, reflecting their bacterial origins. The simplest chromosomes are found in viruses: these DNA or RNA molecules are short linear or circular chromosomes that often lack any structural proteins.

Human Genome project

The Human Genome Project is an initiative of the U.S. Department of Energy (“DOE”) that aims to generate a high-quality reference sequence for the entire human genome and identify all the human genes.

The DOE and its predecessor agencies were assigned by the U.S. Congress to develop new energy resources and technologies and to pursue a deeper understanding of potential health and environmental risks posed by their production and use. In 1986, the DOE announced its Human Genome Initiative. Shortly thereafter, the DOE and National Institutes of Health developed a plan for a joint Human Genome Project (“HGP”), which officially began in 1990.

The HGP was originally planned to last 15 years. However, rapid technological advances and worldwide participation accelerated the completion date to 2003 (making it a 13 year project). Already it has enabled gene hunters to pinpoint genes associated with more than 30 disorders.

Cloning

Cloning involves the removal of the nucleus from one cell and its placement in an unfertilized egg cell whose nucleus has either been deactivated or removed.

There are two types of cloning:

1. Reproductive cloning. After a few divisions, the egg cell is placed into a uterus where it is allowed to develop into a fetus that is genetically identical to the donor of the original nucleus.
2. Therapeutic cloning. The egg is placed into a Petri dish where it develops into embryonic stem cells, which have shown potentials for treating several ailments.

In February 1997, cloning became the focus of media attention when Ian Wilmut and his colleagues at the Roslin Institute announced the successful cloning of a sheep, named Dolly, from the mammary glands of an adult female. The cloning of Dolly made it apparent to many that the techniques used to produce her could someday be used to clone human beings. This stirred a lot of controversy because of its ethical implications.

DNA -Physical definitions

The vast majority of living organisms encode their genes in long strands of DNA. DNA consists of a chain made from four types of nucleotide subunits: adenine, cytosine,guanine, and thymine. Each nucleotide subunit consists of three components: aphosphate group, a deoxyribose sugar ring, and a nucleobase. Thus, nucleotides in DNA or RNA are typically called 'bases'; consequently they are commonly referred to simply by their purine or pyrimidine original base components adenine, cytosine, guanine, thymine. Adenine and guanine are purines and cytosine and thymine are pyrimidines. The most common form of DNA in a cell is in a double helix structure, in which two individual DNA strands twist around each other in a right-handed spiral. In this structure, the base pairing rules specify that guanine pairs with cytosine andadenine pairs with thymine (each pair contains one purine and one pyrimidine). The base pairing between guanine and cytosine forms three hydrogen bonds, while the base pairing between adenine and thymine forms two hydrogen bonds. The two strands in a double helix must therefore be complementary, that is, their bases must align such that the adenines of one strand are paired with the thymines of the other strand, and so on.

Due to the chemical composition of the pentose residues of the bases, DNA strands have directionality. One end of a DNA polymer contains an exposed hydroxyl group on the deoxyribose, this is known as the 3' end of the molecule. The other end contains an exposed phosphate group, this is the 5' end. The directionality of DNA is vitally important to many cellular processes, since double helices are necessarily directional (a strand running 5'-3' pairs with a complementary strand running 3'-5') and processes such as DNA replication occur in only one direction. All nucleic acid synthesis in a cell occurs in the 5'-3' direction, because new monomers are added via a dehydration reaction that uses the exposed 3' hydroxyl as a nucleophile.

The expression of genes encoded in DNA begins by transcribing the gene into RNA, a second type of nucleic acid that is very similar to DNA, but whose monomers contain the sugar ribose rather than deoxyribose. RNA also contains the base uracil in place of thymine. RNA molecules are less stable than DNA and are typically single-stranded. Genes that encode proteins are composed of a series of three-nucleotidesequences called codons, which serve as the "words" in the genetic "language". The genetic code specifies the correspondence during protein translation between codons and amino acids. The genetic code is nearly the same for all known organisms.

DNA sequence

A DNA sequence or genetic sequence is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, with the capacity to carryinformation as described by the central dogma of molecular biology.

The possible letters are A, C, G, and T, representing the four nucleotide bases of a DNA strand — adenine, cytosine, guanine, thymine — covalently linked to a phosphodiesterbackbone. In the typical case, the sequences are printed abutting one another without gaps, as in the sequence AAAGTCTGAC, read left to right in the 5' to 3' direction. Short sequences of nucleotides are referred to as oligonucleotides and are used in a range of laboratory applications in molecular biology. With regard to biological function, a DNA sequence may be considered sense or antisense, and either coding or noncoding. DNA sequences can also contain "junk DNA."

Sequences can be derived from the biological raw material through a process called DNA sequencing.

In some special cases, letters besides A, T, C, and G are present in a sequence. These letters represent ambiguity. Of all the molecules sampled, there is more than one kind of nucleotide at that position. The rules of the International Union of Pure and Applied Chemistry (IUPAC) are as follows:

Controversial questions

Several issues have been raised regarding the use of genetic testing:

1. Absence of cure. There is still a lack of effective treatment or preventive measures for many diseases and conditions now being diagnosed or predicted using gene tests. Thus, revealing information about risk of a future disease that has no existing cure presents an ethical dilemma for medical practitioners.
2. Ownership and control of genetic information. Who will own and control genetic information, or information about genes, gene products, or inherited characteristics derived from an individual or a group of people like indigenous communities? At the macro level, there is a possibility of a genetic divide, with developing countries that do not have access to medical applications of biotechnology being deprived of benefits accruing from products derived from genes obtained from their own people. Moreover, genetic information can pose a risk for minority population groups as it can lead to group stigmatization.

At the individual level, the absence of privacy and anti-discrimination legal protections in most countries can lead to discrimination in employment or insurance or other misuse of personal genetic information. This raises questions such as whether genetic privacy is different from medical privacy.^[13]

1. Reproductive issues. These include the use of genetic information in reproductive decision-making and the possibility of genetically altering reproductive cells that may be passed on to future generations. For example, germline therapy forever changes the genetic make-up of an individual’s descendants. Thus, any error in technology or judgment may have far-reaching consequences. Ethical issues like designer babies and human cloning have also given rise to controversies between and among scientists and bioethicists, especially in the light of past abuses with eugenics.
2. Clinical issues. These center on the capabilities and limitations of doctors and other health-service providers, people identified with genetic conditions, and the general public in dealing with genetic information.
3. Effects on social institutions. Genetic tests reveal information about individuals and their families. Thus, test results can affect the dynamics within social institutions, particularly the family.
4. Conceptual and philosophical implications regarding human responsibility, free will vis-à-vis genetic determinism, and the concepts of health and disease.

Genetic testing

Genetic testing involves the direct examination of the DNA molecule itself. A scientist scans a patient’s DNA sample for mutated sequences.

There are two major types of gene tests. In the first type, a researcher may design short pieces of DNA (“probes”) whose sequences are complementary to the mutated sequences. These probes will seek their complement among the base pairs of an individual’s genome. If the mutated sequence is present in the patient’s genome, the probe will bind to it and flag the mutation. In the second type, a researcher may conduct the gene test by comparing the sequence of DNA bases in a patient’s gene to disease in healthy individuals or their progeny.

Genetic testing is now used for:

• Carrier screening, or the identification of unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest;
• Confirmational diagnosis of symptomatic individuals;
• Determining sex;
• Forensic/identity testing;
• Newborn screening;
• Prenatal diagnostic screening;
• Presymptomatic testing for estimating the risk of developing adult-onset cancers;
• Presymptomatic testing for predicting adult-onset disorders.

Some genetic tests are already available, although most of them are used in developed countries. The tests currently available can detect mutations associated with rare genetic disorders like cystic fibrosis, sickle cell anemia, and Huntington’s disease. Recently, tests have been developed to detect mutation for a handful of more complex conditions such as breast, ovarian, and colon cancers. However, gene tests may not detect every mutation associated with a particular condition because many are as yet undiscovered, and the ones they do detect may present different risks to different people and populations.^[12]

Genomics

New Initiative to Study Societal Issues Associated with Synthetic Biology -- A Rapidly Developing Field where Novel Organisms are Constructed from The

NEW YORK, Dec. 18 -- The Alfred P. Sloan Foundation announces a new initiative to study societal issues associated with synthetic biology -- a rapidly developing scientific field where researchers are constructing novel organisms from the building blocks of DNA. This new effort brings together leading scientists, ethicists and public policy specialists to explore the field's potential benefits and risks, as well as ethical questions and regulatory issues. The new initiative launches with three grants totaling more than $1.6 million to The Hastings Center, the J. Craig Venter Institute, and the Woodrow Wilson International Center for Scholars.

"The Foundation has a long and rich tradition of funding scientific research," said Dr. Paul Joskow, President Alfred P. Sloan Foundation. "With synthetic biology, scientists have gone from reading to writing the genetic code; it's imperative that we take a carefully reasoned and systematic approach to understanding the full spectrum of ethical and policy issues that may arise as research and applications in this field develop."

At the Hastings Center (http://www.thehastingscenter.org/), Foundation funding will allow for in-depth investigation into ethical issues that may arise in connection with developments in synthetic biology. The project aims to make serious contributions to scholarly literature, produce a base for further scholarship, and inform public policymaking.

Alfred P. Sloan Foundation funding will allow the J. Craig Venter Institute (http://www.jcvi.org/) to examine potential societal concerns associated with developments in synthetic genomics. The project will both inform the scientific community about these issues while also educating the policy and journalistic communities about the science. As a result, scientists, journalists and policymakers will be able to engage in informed discussions.

A grant to the Woodrow Wilson International Center for Scholars (http://www.wilsoncenter.org/) will analyze evolving public perceptions of potential societal risks that may arise related to research in and applications of synthetic biology, clarify whether our existing regulatory systems can address relevant risks that may be associated with the science, and inform and educate policymakers.

"This program builds on the Foundation's biosecurity work and will establish a community of scientists, ethicists and policy specialists who can work synergistically on these issues," said Paula Olsiewski, Program Director, Alfred P. Sloan Foundation. "Ethical and policy discussions must be informed by the realities of the science and similarly the science must take into consideration societal concerns so that synthetic biology can be applied both inventively and wisely."

About the Alfred P. Sloan Foundation

The Alfred P. Sloan Foundation, established in 1934, makes grants to support original research and broad-based education related to science, technology, and economic performance; and to improve the quality of American life. The Foundation believes that a carefully reasoned and systematic understanding of the forces of nature and society, when applied inventively and wisely, can lead to a better world for all. Please visit the Foundation's Web site at www.sloan.org.

Human Genome project

Human Genome Project

DNA Replication image from the Human Genome Project (HGP)

The Human Genome Project is an initiative of the U.S. Department of Energy (“DOE”) that aims to generate a high-quality reference sequence for the entire human genome and identify all the human genes.

The DOE and its predecessor agencies were assigned by the U.S. Congress to develop new energy resources and technologies and to pursue a deeper understanding of potential health and environmental risks posed by their production and use. In 1986, the DOE announced its Human Genome Initiative. Shortly thereafter, the DOE and National Institutes of Health developed a plan for a joint Human Genome Project (“HGP”), which officially began in 1990.

The HGP was originally planned to last 15 years. However, rapid technological advances and worldwide participation accelerated the completion date to 2003 (making it a 13 year project). Already it has enabled gene hunters to pinpoint genes associated with more than 30 disorders.

Cloning

Cloning involves the removal of the nucleus from one cell and its placement in an unfertilized egg cell whose nucleus has either been deactivated or removed.

There are two types of cloning:

1. Reproductive cloning. After a few divisions, the egg cell is placed into a uterus where it is allowed to develop into a fetus that is genetically identical to the donor of the original nucleus.
2. Therapeutic cloning. The egg is placed into a Petri dish where it develops into embryonic stem cells, which have shown potentials for treating several ailments.

In February 1997, cloning became the focus of media attention when Ian Wilmut and his colleagues at the Roslin Institute announced the successful cloning of a sheep, named Dolly, from the mammary glands of an adult female. The cloning of Dolly made it apparent to many that the techniques used to produce her could someday be used to clone human beings. This stirred a lot of controversy because of its ethical implications.

Intermembrane space

The intermembrane space is the region between the inner membrane and theouter membrane of a mitochondrion or a chloroplast. The main function of the intermembrane space is oxidative phosphorylation.

Channel proteins called porins in the outer membrane allow free movement of ionsand small molecules into the intermembrane space. This means that it is essentially continuous with the cytosol in terms of the solutes relevant for the functioning of these organelles. Enzymes destined for the mitochondrial matrix or the stroma can pass through the intermembrane space via transport throughtranslocators. These are known as translocase of the outer mitochondria membrane (TOM) and translocase of the inner mitochondrial membrane (TIM) in mitochondriaand translocase of the outer chloroplast membrane (TOC) and translocase of the inner chloroplast membrane (TIC) in chloroplasts. It tends to have a low pH because of the proton gradient which results when protons are pumped from themitochondrial matrix into the intermembrane space during electron transport. The structures responsible for this are coenzyme Q, NADH coenzyme Q oxidoreductasecomplex (complex I), succinate-coenzyme Q oxidoreductase complex (complex II), and coenzyme Q-cytochrome c oxidoreductase complex (complex III).

ELECTRON MICROSCOPES

An electron microscope uses electrons to “illuminate” an object. Electrons have a much smaller wavelength than light, so they can resolve much smaller structures. The smallest wavelength of visible light is about 4000 angstroms (40 millionths of a meter). The wavelength of electrons used in electron microscopes is usually about half an angstrom (50 trillionths of a meter).

Electron microscopes have an electron gun that emits electrons, which then strike the specimen. Conventional lenses used in optical microscopes to focus visible light do not work with electrons; instead, magnetic fields are used to create “lenses” that direct and focus the electrons. Since electrons are easily scattered by air molecules, the interior of an electron microscope must be sealed at a very high vacuum. Electron microscopes also have systems that record or display the images produced by the electrons.

There are two types of electron microscopes: the transmission electron microscope (TEM), and the scanning electron microscope (SEM). In a TEM, the electron beam is directed onto the object to be magnified. Some of the electrons are absorbed or bounce off the specimen, while others pass through and form a magnified image of the specimen. The sample must be cut very thin to be used in a TEM, usually no more than a few thousand angstroms thick. A photographic plate or fluorescent screen beyond the sample records the magnified image. Transmission electron microscopes can magnify an object up to one million times.

In a scanning electron microscope, a tightly focused electron beam moves over the entire sample to create a magnified image of the surface of the object in much the same way an electron beam scans an image onto the screen of atelevision. Electrons in the tightly focused beam might scatter directly off the sample or cause secondary electrons to be emitted from the surface of the sample. These scattered or secondary electrons are collected and counted by an electronic device. Each scanned point on the sample corresponds to a pixel on a television monitor; the more electrons the counting device detects, the brighter the pixel on the monitor is. As the electron beam scans over the entire sample, a complete image of the sample is displayed on the monitor.

An SEM scans the surface of the sample bit by bit, in contrast to a TEM, which looks at a relatively large area of the sample all at once. Samples scanned by an SEM do not need to be thinly sliced, as do TEM specimens, but they must be dehydrated to prevent the secondary electrons emitted from the specimen from being scattered by water molecules in the sample.

Scanning electron microscopes can magnify objects 100,000 times or more. SEMs are particularly useful because, unlike TEMs and powerful optical microscopes, they can produce detailed three-dimensional images of the surface of objects.

The scanning transmission electron microscope (STEM) combines elements of an SEM and a TEM and can resolve single atoms in a sample.

The electron probe microanalyzer, an electron microscope fitted with an X-ray spectrum analyzer, can examine the high-energy X rays emitted by the sample when it is bombarded with electrons. The identity of different atoms or molecules can be determined from their X-ray emissions, so the electron probe analyzer not only provides a magnified image of the sample, but also information about the sample's chemical composition.

OPTICAL MICROSCOPES

The most widely used microscopes are optical microscopes, which use visiblelight to create a magnified image of an object. The simplest optical microscope is the double-convex lens with a short focal length. Double-convex lenses can magnify an object up to 15 times.

The compound microscope uses two lenses, an objective lens and an ocular lens, mounted at opposite ends of a closed tube, to provide greater magnification than is possible with a single lens. The objective lens is composed of several lens elements that form an enlarged real image of the object being examined. The real image formed by the objective lens lies at the focal point of the ocular lens. Thus, the observer looking through the ocular lens sees an enlarged virtual image of the real image. The total magnification of a compound microscope is determined by the focal lengths of the two lens systems and can be more than 2000 times.

Optical microscopes have a firm stand with a flat stage to hold the material examined and some means for moving the microscope tube toward and away from the specimen to bring it into focus. Ordinarily, specimens are transparent and are mounted on slides—thin, rectangular pieces of clear glass that are placed on the stage for viewing. The stage has a small hole through which light can pass from a light source mounted underneath the stage—either a mirror that reflects natural light or a special electric light that directs light through the specimen.

Microbiology

An agar plate streaked withmicroorganisms

Microbiology (from Greek μῑκρος, mīkros, "small"; βίος, bios, "life"; and -λογία, -logia) is the study ofmicroorganisms, which are unicellular or cell-cluster microscopic organisms. This includes eukaryote such as fungi and protists, and prokaryotes, which are bacteria and archaea. Viruses, though not strictly classed as living organisms, are also studied. In short; microbiology refers to the study of life and organisms that are too small to be seen with the naked eye.

Microbiology is a broad term which includes virology, mycology, parasitology, bacteriology and other branches. A microbiologist is a specialist in microbiology.

Microbiology is researched actively, and the field is advancing continually. We have probably only studied about one percent of all of the microbe species on Earth. Although microbes were first observed over three hundred years ago, the field of microbiology can be said to be in its infancy relative to older biological disciplines such as zoology and botany.

Mitochondrion

a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells.^[1] These organelles range from 1–10 micrometers (μm) in size. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply ofadenosine triphosphate (ATP), used as a source of chemical energy. ^[2] In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth.^[3] Mitochondria have been implicated in several human diseases, including mitochondrial disorders^[4] and cardiac dysfunction,^[5] and may play a role in the aging process. The word mitochondrion comes from the Greek μίτος or mitos, thread + χονδρίον orkhondrion, granule. Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria.^[6][7] The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissues and species. In human, 615 distinct types of proteins were identified from cardiac mitochondria;^[8] whereas in murinae (rats), 940 proteins encoded by distinct genes were reported.^[9] The mitochondrial proteome is thought to be dynamically regulated.^[10] Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

Techniques of molecular biology

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.

For more extensive list on protein methods, see protein methods.

For more extensive list on nucleic acid methods, see nucleic acid methods.

[edit]Expression cloning

Main article: Expression cloning

One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid.

This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation (via uptake of naked DNA), conjugation (via cell-cell contact) or by transduction (via viral vector). Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection,electroporation, microinjection and liposome transfection. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers, the latter is sometimes called bactofection and in particular uses Agrobacterium tumefaciens. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection.

In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.

[edit]Polymerase chain reaction (PCR)

Main article: Polymerase chain reaction

The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA, the latter is a method referred to as "Quick change". PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.

[edit]Gel electrophoresis

Main article: Gel electrophoresis

Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel. Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis.

[edit]Southern blotting

Main article: Southern blot

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Most original protocols used radioactive labels, however non-radioactive alternatives are now available. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number intransgenic mice, or in the engineering of gene knockout embryonic stem cell lines.

[edit]Northern blotting

Main article: northern blot

The northern blot is used to study the expression patterns a specific type of RNA molecule as relative comparison among of a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.

[edit]Western blotting

Main article: western blot

Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques.

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with an enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis.

Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. However, theseimmunostaining methods, such as FISH, are used more often in cell biology research.

[edit]Blotting jokes

The terms "western" and "northern" are molecular biology jokes that play on the term southern blot. The first blots were with DNA, and since they were done by Ed Southern, they came to be known as Southerns. Patricia Thomas, inventor of the RNA blot, which became known as a "northern", actually didn't use the term. ^[2]. To carry the joke further, one can find references in the literature to "southwesterns" (protein-DNA interactions), "northwesterns" (protein-RNA interactions) and "farwesterns" (protein-protein interactions).

[edit]Arrays

Main article: DNA microarray

A DNA array is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragment. Arrays make it possible to put down a large quantity of very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence (similar to Southern blotting). A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified (expression profiling). In this technique the RNA in a tissue is isolated and converted to labeled cDNA. This cDNA is then hybridized to the fragments on the array and visualization of the hybridization can be done. Since multiple arrays can be made with the exact same position of fragments they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors. For instance, the common baker's yeast,Saccharomyces cerevisiae, contains about 7000 genes; with a microarray, one can measure qualitatively how each gene is expressed, and how that expression changes, for example, with a change in temperature. There are many different ways to fabricate microarrays; the most common are silicon chips, microscope slides with spots of ~ 100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array.

Arrays can also be made with molecules other than DNA. For example, an antibody array can be used to determine what proteins or bacteriaare present in a blood sample.

[edit]Allele Specific Oligonucleotide

Allele specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis. Short (20-25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA. Hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the labeled probes that didn't hybridize are removed. The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation.

[edit]Abandoned technology

As new procedures and technology become available, the older technology is rapidly abandoned. A good example is methods for determining the size of DNA molecules. Prior to gel electrophoresis (agarose or polyacrylamide) DNA was sized with rate sedimentation in sucrose gradients, a slow and labor intensive technology requiring expensive instrumentation; prior to sucrose gradients, viscometry was used.

Aside from their historical interest, it is worth knowing about older technology as it may be useful to solve a particular problem.