What Is Molecular Biology?

Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interrelationship of DNA, RNA and protein synthesis and learning how these interactions are regulated.

Writing in Nature, W.T. Astbury described molecular biology as:

"... not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned particularly with the forms of biological molecules and ..... is predominantly three-dimensional and structural - which does not mean, however, that it is merely a refinement of morphology - it must at the same time inquire into genesis and function" [Nature 190, 1124 (1961)]

Researchers in molecular biology use specific techniques native to molecular biology (see Techniques section later in article), but increasingly combine these with techniques and ideas from genetics, biochemistry and biophysics. There is not a hard-line between these disciplines as there once was. The following figure is a schematic that depicts one possible view of the relationship between the fields:

Biochemistry is the study of molecules (e.g. proteins) in the absence of the rest of the organism. Biochemists take an organism or cell and dissect it into its molecular components, such as enzymes, lipids and DNA, and reconstitute them in test tubes (in vitro). Genetics is the study of the effect of genetic differences on organisms. Often this can be inferred by the absence of a normal component (e.g. one gene). The study of "mutants" – organisms which lack one or more functional components with respect to the so-called "wild type" or normal phenotype. Genetic interactions such as epistasis can often confound simple interpretations of such "knock-out" studies. Molecular biology is the study of molecular underpinnings of the process of replication, transcription and translation of the genetic material. The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture of molecular biology, still provides a good starting point for understanding the field. This picture, however, is undergoing revision in light of emerging novel roles for RNA. Much of the work in molecular biology is quantitative, and recently much work has been done at the interface of molecular biology and computer science in bioinformatics and computational biology. As of the early 2000s, the study of gene structure and function, molecular genetics, has been amongst the most prominent sub-field of molecular biology.

Increasingly many other fields of biology focus on molecules, either directly studying their interactions in their own right such as in cell biology and developmental biology, or indirectly, where the techniques of molecular biology are used to infer historical attributes of populations or species, as in fields in evolutionary biology such as population genetics and phylogenetics. There is also a long tradition of studying biomolecules "from the ground up" in biophysics.

Techniques of molecular biology Since the late 1950s and early 1960s, molecular biologists have learned to characterise, 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.

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 is called transformation, and can be effected by several methods, including electroporation, microinjection and chemically. Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. Several different transfection techniques are available, including calcium phosphate transfection, liposome transfection, and proprietary transfection reagents such as Fugene. DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called viral transduction, and the cells are said to be transduced.

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.

Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. Gel Electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules to use other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.

The first part, "gel", refers to the matrix used to separate the molecules. In most cases the gel is a crosslinked polymer whose porosity can be controlled by the scientist. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually made with different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid but porous matrix that looks and feels like clear jello. Polyacrylamide is a neurotoxin and needs to be handled using Good Laboratory Practices (GLP) to avoid poisoning.

The second part, "electrophoresis", refers to the electro-motive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and then applying an electric current, the molecules will be moved through the gel at different rates, from the anode towards the cathode. In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar-phosphate backbone. Double-stranded DNA fragments natually behave as long rods, so their migration through the gel is relative to their radius of gyration, or, roughly, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.

Proteins, on the other hand, can have different charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are ususally denaturated in the presense of a detergent such as sodium dodecyl sulfate/sodium docecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein, so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like they were long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

After the electrophoresis run, when the smallest molecules have almost reached the anode, the molecules in the gel can be stained to make them visible. Silver or Coumassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules luminesce under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an autoradiogram can be recorded of the gel (as in the example shown to the right).

If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.

Bands in different lanes that end up at the same "height" contain molecules that passed through the gel with the same speed, which usually means they are about the same size. There are special markers available, which contain molecules of known sizes. If such a marker was run on one lane in the gel parallel to the mixture(s), the bands it displays can be compared to those of the mixture(s) in order to determine their size.

A Western blot is a method in molecular biology to detect a certain protein in a sample by using antibody specific to that protein. It also gives information about the size of that protein. Its name is a pun off the name Southern blot, a similar technique developed earlier by Edward Southern.

Steps in a Western blot The first step is gel electrophoresis. The proteins of the sample are separated according to size on a gel, usually using SDS-PAGE. Usually the gel has several lanes so that several samples can be tested simultaneously. However, it is also possible to use a 2-D gel which spreads the proteins from a single sample out in two dimensions. Nitrocellulose transfer - The proteins in the gel are then transferred onto a membrane made of nitrocellulose or PVDF, by pressure or by applying a current. This is the actual blotting process and is necessary in order to expose the proteins to antibody (see below). The membrane is "sticky" and binds proteins non-specifically (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions as well as charged interactions between the membrane and protein. PVDF is often used because it is sturdier and can be "stripped" of antibodies and reused. Unlike nitrocellulose, PVDF must be soaked in 100% methanol before using.

Blocking - The membrane is then blocked, in order to prevent non-specific protein interactions between the membrane and the antibody protein (next step, below). This is done by placing the membrane in a solution of Bovine serum albumin (BSA) or non-fat dry milk (Without the blocking, the antibody to be applied in the next step would bind to the nitrocellulose). The first antibody (often called the primary antibody) is incubated with the membrane. "Incubation" is typically accomplished by diluting the antibody in a solution containing a modest amount of a salt such as sodium chloride, some protein (such as BSA) to prevent non-specific binding of the antibody to surfaces and a small amount of a buffer to keep the solution near neutral pH. The diluted antibody solution and the membrane can be sealed in a plastic bag and gently agitated for an "incubation" of about half an hour. The primary antibody recognizes only the protein of interest, and will not bind any of the other proteins on the membrane. It is obtained by immunizing an animal (usually a rabbit or goat) with the protein of interest (i.e., injecting the protein into the animal's body) and collecting the antibodies the animal produces against that protein. Some high affinity monoclonal antibodies can also be used for Western blots. After rinsing the membrane to remove unbound primary antibody a secondary antibody is incubated with the membrane. It binds to the first antibody, and is usually produced by a different animal. For example, goat anti-rabbit antibody might be used if the first antibody was produced by rabbits. This secondary antibody is usually linked to an enzyme that can allow for visual identification of where on the membrane it has bound. As with the ELISPOT and ELISA procedures, the enzyme can be provided with a substrate molecule that will be converted by the enzyme to a colored reaction product that will be visible on the membrane (see the figure below with blue bands). Alternately, the reaction product may produce enough fluorescence to expose a sensitive sheet of film when it is placed against the membrane. An alternative to using an enzyme that is coupled to the secondary antibody is to use a radioactive label. An antibody-binding protein such as Staphylococcus Protein A can be used and labeled with a radioactive isotope of iodine.

The unbound secondary antibodies are washed away, and the enzyme substrate is incubated with the membrane so that the positions of membrane-bound secondary antibodies will become visible. If a radioactive label is used, the radioactive membrane can be placed against a sheet of medical X-ray film. Bands corresponding to the detected protein of interest will appear as dark regions on the developed film (see figure to right). Since the first antibody only recognizes the protein of interest, and the second antibody only recognizes the first antibody, if there is stain present on the membrane then the protein of interest must also be present on the membrane. Thus, the protein bands on the membrane that are stained contain the protein that was to be detected, the other locations on the membrane do not. Size approximations can be done by comparing the stained bands to that of a pre-stained protein size marker.

Usually, the gel is not completely devoid of proteins after blotting. Protein staining solution will show all protein bands on the gel. The stained gel can then be compared with the stained membrane to identify which bands contain the wanted protein and which do not.

In principle, one could bind the chemical signal directly to the first antibody, but production of the antibodies is easier if the two functions recognition and signalling are separated.

Medical diagnostic applications The HIV test known as "Western Blot" uses a variant of the technique, where the goal is to detect the presence of antibody in a sample. Known HIV infected cells are opened and their proteins separated and blotted on a membrane as above. Then the serum to be tested is applied. Free antibody is washed away, and a secondary antibody is added that binds to human antibody and is linked to an enzyme signal. The stained bands then indictate the proteins to which the patient's serum contains antibody.

Molecular biology was established in the 1930s, the term was first coined by Warren Weaver in 1938 however. Warren was director of Natural Sciences for the Rockefeller Foundation at the time and believed that biology was about to undergo a period of significant change given recent advances in fields such as X-ray crystallography. He therefore channeled significant amounts of (Rockefeller Institute) money into biological fields.

In science, a molecule is the smallest particle of a pure chemical substance that still retains its chemical composition and properties. A molecule consists of multiple atoms joined by shared pairs of electrons in a covalent bond. It may consist of atoms of the same chemical element, as with oxygen gas (O2), or of different elements, as with water vapor (H2O). Abstractly, a single atom may be considered a molecule, as it is when referred to collectively with molecules of multiple atoms, but in practice the use of the word molecule is usually confined to chemical compounds, of multiple atoms.

Most molecules are much too small to be seen with the naked eye, but there are exceptions. DNA, a macromolecule, can reach macroscopic sizes.

A property of molecules is the integer ratio of the elements that constitute the compound, the empirical formula. For example, in their pure forms, water is always composed of a 2:1 ratio of hydrogen to oxygen, and ethyl alcohol or ethanol is always composed of carbon, hydrogen, and oxygen in a 2:6:1 ratio. However, this does not determine the kind of molecule uniquely - dimethyl ether has the same ratio as ethanol, for instance. Molecules with the same atoms in different arrangements are called isomers.

Chemical formula on the other hand reflects the exact number of atoms that compose a molecule. The molecular mass is calculated from the chemical formula and is expressed in conventional units equal to 1/12 from the mass of a 12C isotope atom.

Molecules have fixed equilibrium geometries--bond lengths and angles--that are dictated by the laws of quantum mechanics. A pure substance is composed of molecules with the same geometrical structure. The chemical formula and the structure of a molecule are the two important factors that determine its properties, particularly its reactivity. Isomers share a chemical formula but normally have very different properties because of their different structures. Stereoisomers, a particular type of isomers, may have very similar physico-chemical properties and at the same time very different biochemical activities.

Biochemistry is the chemistry of life. Biochemists study the elements, compounds and chemical reactions that are controlled by enzymes and take place in all living organisms.

Biochemistry is focused on the structure and function of cellular components, such as proteins, carbohydrates, lipids, nucleic acids, and other biomolecules. Recently biochemistry has focused more specifically on the chemistry of enzyme-mediated reactions, and on the properties of proteins.

The biochemistry of cell metabolism has been extensively described. Other areas of biochemistry include the genetic code (DNA, RNA), protein synthesis, cell membrane transport, signal transduction and energy decomposition cycles.

Development of biochemistry The dawn of biochemistry may have been the discovery of the first enzyme, diastase, in 1833 by Anselme Payen. In 1828, Friedrich Wöhler published a paper about the synthesis of urea, proving that organic compounds can be created artificially, in contrast to the common belief of the time that organic compounds can only be made by living organisms. Since then, biochemistry has advanced, especially since the mid-20th century, with the development of new techniques such as chromatography, X-ray diffraction, NMR, radioisotopic labelling, electron microscopy and molecular dynamics simulations. These techniques allowed for the discovery and detailed analysis of many molecules and metabolic pathways of the cell, such as glycolysis and the Krebs cycle.

Today, the findings of biochemistry are used in many areas, from genetics to molecular biology and from agriculture to medicine. The first application of biochemistry was probably the making of bread using yeast, about 5000 years ago.

Biophysics (also biological physics) is an interdisciplinary science that applies theories and methods of the physical sciences to questions of biology. Biophysics research today comprises a number of specific biological studies, which do not share a unique identifying factor, or subject themselves to clear and concise definitions. This is the result of biophysics' relatively recent appearance as a scientific discipline. The studies included under the umbrella of biophysics range from sequence comparison to neural networks. In the recent past, biophysics included creating mechanical limbs and nanomachines to regulate biological functions. Nowadays, these are more commonly referred to as belonging to the fields of bioengineering and nanotechnology respectively. We may expect these definitions to further refine themselves.

Traditional studies in biology are conducted using statistical ensemble experiments, using molar concentrations of macromolecules. Because the molecules that comprise living cells are so small, techniques such as PCR amplification, gel blotting, fluorescence labeling and in vivo staining are used so that experimental results are observable with an unaided eye or, at most, optical magnification. Using these techniques, biologists attempt to elucidate the complex systems of interactions that give rise to the processes that make life possible. Biophysics typically addresses biological questions similar to those in biology, but the questions are asked at a molecular (i.e. low Reynolds number) level. By drawing knowledge and experimental techniques from a wide variety of disciplines (as described below), biophysicisists are able to indirectly observe or model the structures and interactions of individual molecules or complexes of molecules. In addition things like solving a protein structure or measuring the kinetics of single molecule interactions, biophysics is also understood to encompass research areas that apply models and experimental techniques derived from physics (e.g. electromagnetism and quantum mechanics) to larger systems such as tissues or organs (hence the inclusion of basic neuroscience as well as more applied techniques such as fMRI).

Biophysics often does not have university-level departments of its own, but have presence as groups across departments within the fields of biology, biochemistry, chemistry, computer science, mathematics, medicine, pharmacology, physiology, physics, and neuroscience. What follows is a list of examples of how each department applies its efforts toward the study of biophysics. This list is hardly all inclusive. Nor does each subject of study belong exclusively to any particular department. Each academic institution makes its own rules and there is much mixing between departments.

Viruses are not typically considered to be organisms because they are not capable of independent reproduction or metabolism. However, according to the United States Code, they are considered to be microorganisms in the sense of biological weaponry and malicious use. This controversy is problematic, though, since some parasites and endosymbionts are incapable of independent life either. Although viruses do have enzymes and molecules characteristic of living organisms, they are incapable of surviving outside a host cell and most of their metabolic processes require a host and its 'genetic machinery'. The origin of such parasites is uncertain, but it appears most likely that they are derived from their hosts.

Life span One of the basic parameters of organism is its life span. Some animals live as short as one day, while some plants can live thousands of years. Aging is important when determining life span of most organisms, bacterium, a virus or even a prion.

An enzyme is a protein, or protein complex, that catalyzes a chemical reaction. Like any catalyst, enzymes work by lowering the activation energy of a reaction, thus allowing the reaction to proceed to its steady state or completion much faster than it otherwise would; the enzyme (again, as with any catalyst) remains unaltered by the completed reaction and can therefore continue catalysis.

It is important to note that, as with all catalysts, all reactions catalyzed by enzymes must be 'spontaneous' (containing a net negative Gibbs free energy), i.e. with the enzyme, they run in the same direction as they would without the enzyme, just more quickly; the concept is similar to the likelihood of a ball rolling down a hill versus the likelihood of it rolling up the hill. This is required by the Law of Conservation of Energy, which would be violated by the possibility of a cycle of moving down a pathway releasing less net energy and back up a different pathway with higher net energy, or vice versa. Given a particular starting set of conditions, the end products of a particular reaction (including net energy), once steady state is reached, must always be identical, independent of the specific individual pathway taken from beginning point to end point. An enzyme can, however, run a normally nonspontaneous reaction 'backwards' by coupling it to a spontaneous one, as long as the net free energy from the total of both reactions is negative.

Enzymes are necessary because within biological cells, many chemical reactions would occur too slowly to sustain life; oxidation of organic food compounds to provide energy, for instance. Enzymes speed up reactions by a factor of one thousand times or more. They also provide a means to control the reactions, by modulating enzymatic activity.

An enzyme can be a monomeric protein made up of about hundred amino acids or more, or an oligomeric protein consisting of several monomers, different or identical, that act together as a unit. As with any protein, each monomer is actually produced as a long, linear chain of amino acids, which then folds up in a particular fashion to produce the correct three-dimensional product. The factors that go into ensuring that the chain folds correctly and maintains its shape are complex, and still not completely identified, although the general principles seem to be understood.

As a consequence of this basic structure, the amino acid chain of an enzyme tends to consist of one or more active regions, separated by stretches whose purpose is mainly to position the active regions correctly. Because the precise structure of each region tends to be fairly critical to correct function, and because the frequency of a mutation which would produce a nonfunctional active region is proportional to the length of the chain separating the amino acids involved, evolution works against having the active amino acids from an active region dispersed along the protein chain, instead tending to keep the amino acids involved in each active region compacted fairly close together in the chain and tightly folded, separating these regions by long stretches of 'spacer' amino acids where mutation is not critical (although exceptions occur). This has the additional effect of making each region act, relative to mutation, somewhat like an independent subunit which as a unit can be duplicated, deleted, moved, or 'mixed-and-matched' with other such regions, generating new proteins to be tested for evolutionary success. This would seem to be a more efficient process in terms of successfully generating new, functional, enzymes than would having the functionally related amino acids of the active site dispersed throughout the chain, with random mutation occurring anywhere in the amino acid chain frequently disrupting the function of the active site.

An enzyme contains an active site, consisting of the catalytic amino acids and one or more binding sites that bind the substrate(s). Enzymes also frequently have binding sites that serve regulatory functions, which increase or inhibit the enzyme's activity. These typically bind metals or small molecules.

Enzymes are usually specific as to the reactions they catalyze and the substrates that are involved in these reactions. Complementary structural properties of the enzyme and substrate are responsible for this specificity (Fig. 2).

Enzymes can perform up to several million catalytic reactions per second. To determine the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is achieved. This is the maximum velocity (Vmax) of the enzyme. In this state, all enzyme active sites are saturated with substrate. This was proposed in 1913 by Leonor Michaelis and Maud Menten. Since the substrate concentration at Vmax cannot be measured exactly, enzymes are characterized by the substrate concentration at which the rate of reaction is half its maximum. This substrate concentration is called the Michaelis-Menten constant (KM). Many enzymes obey Michaelis-Menten kinetics.

The efficiency of an enzyme can be expressed in terms of kcat/Km. The quantity kcat, also called the turnover number, incorporates the rate constants for all steps in the reaction, and is the product of Vmax and the total enzyme concentration. kcat/Km is a useful quantity for comparing different enzymes against each other, or the same enzyme with different substrates, because it takes both affinity and catalytic ability into consideration. The theoretical maxium for kcat/Km is about 108 to 109 (mol/L)-1s-1. At this point, every collision of the enzyme with its substrate will result in catalysis. Some enzymes, such as fumarase, actually approach this limit.

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. The end product(s) of such a pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathway.

Quantum-mechanical model of enzyme catalysis The lecture "Quantum Theory of some Biochemical Reactions", presented to the IV International Biophysical Congress (Moscow, 1972) by R.R. Dogonadze and Z.D. Urushadze, formulated the first quantum mechanical model of the simplest form of enzyme catalysis. In 1972-1973, in the works of M.V. Volkenshtein, R.R. Dogonadze, A.K. Madumarov, Z.D. Urushadze and Yu.I. Kharkats were formulated the quantum-mechanical (physical) model of Enzyme Catalysis. These works demonstrated the role of conformational transformations in catalytic reactions.

Ribozymes An RNA enzyme or "ribozyme" is made of RNA instead of protein. Generally, ribozymes only catalyze RNA splicing, though one notable exception is the RNA portion of the ribosome, which catalyzes the formation of peptide bonds in growing protein chains.

Enzymes and health Enzymes are essential to living organisms, and a malfunction of even a single enzyme out of approximately 2,000 present in our bodies can lead to severe or lethal illness. An example of a disease caused by an enzyme malfunction in humans is phenylketonuria (PKU). The enzyme phenylalanine hydroxylase, which usually converts the essential amino acid phenylalanine into tyrosine does not work, resulting in a buildup of phenylalanine that leads to mental retardation. Enzymes in the human body can also be influenced by inhibitors. Aspirin, for example, inhibits an enzyme that produces prostaglandins (inflammation messengers), thus suppressing pain and inflammation. Enzymes are also used in everyday products such as washing detergents, where they speed up chemical reactions involved in cleaning the clothes (for example, breaking down blood stains).

The precise replication of the hereditary information in the DNA leads to the question of how this information actually influences the activities of the cell. A major step forward in understanding this was the discovery that the DNA is copied, in a process called transcription, into a single-stranded molecule of the related ribonucleic acid (RNA). As in the replication of DNA, the information in the bases of DNA is precisely copied by base pairing to produce the RNA. After further processing the so-called messenger RNA (mRNA) moves to subcellular particles called ribosomes where it is translated into protein. This translation is governed by the genetic code in which each combination of three bases, or triplet, directs the addition of a particular amino acid on to the protein chain: ACC directs addition of threonine, CCC of proline, and so on. Hence the genetic information contained in the linear array of bases in the DNA directs the production of a linear array of amino acids within a protein. Thus genetic changes in the bases in the DNA result in changes in the protein produced. For example, an A to C change in an ACC triplet would lead to the addition of a proline instead of a threonine. As specific proteins have particular biological effects, changes which affect the function of the protein will lead to an alteration in the appearance or function of an organism. l, c. In this way differences in the information in the DNA are observed as inherited differences between individuals, such as eye colour, or genetic diseases, such as haemophilia. The conclusion that DNA makes RNA makes protein has been referred to as the “central dogma of molecular biology”.

Digestive and metabolic enzymes Nutrition in animals relies on digestive enzymes such as salivary amylase, trypsin and chymotrypsin. Their primary role is for the digestion of food and making nutrients available to all of the body processes which need them. Another class of enzymes is called metabolic enzymes. Their role is to catalyze chemical reactions involving every process in the body, including the participation of oxygen. Most of our cells (an exception being erythrocytes), would literally starve for oxygen even with an abundance of oxygen without the action of the enzyme, cytochrome oxidase. Enzymes are also necessary for muscle contraction and relaxation. The fact is, without both of these classes of enzymes, (digestive and metabolic) life could not exist.

A protein complex is a group of two or more associated proteins. Protein complexes are a form of quaternary structure. Understanding the functional interactions of proteins is an important research focus in biochemistry, often referred to as proteomics.

Many protein complexes are established, particular in the model organism Saccharomyces cerevisiae, a yeast. The discovery of protein complexes is now performed genome wide; the elucidation of most protein complexes of the yeast is undergoing.

Lipids are fatty acid esters, a class of relatively water-insoluble organic molecules, which are the "basic" components of biological membranes. There are three forms of lipids: phospholipids, steroids. and triglycerides.

Lipids consist of a polar or hydrophilic (attracted to water) head and one to three nonpolar or hydrophobic (repelled by water) tails (Fig. 1). Since lipids have both functions, they are called amphiphilic. The hydrophobic tail consists of one or two (in triglycerides, three) fatty acids. These are unbranched chains of carbon atoms (with the correct number of H atoms), which are connected by single bonds alone (saturated fatty acids) or by both single and double bonds (unsaturated fatty acids). The chains are usually 14-24 carbon groups long.

For lipids present in biological membranes, the hydrophilic head is from one of three groups:

Glycolipids, whose heads contain an oligosaccharide with 1-15 saccharide (sugar) residues. Phospholipids, whose heads contain a positively charged group that is linked to the tail by a negatively charged phosphate group. Sterols, whose heads contain a planar steroid ring, for example, cholesterol (only in animals). In an aqueous environment, the heads of lipids are turned towards the environment, and the tails are turned towards a hydrophobic region of another molecule. a, e, b. With lots of lipids present, the tails "prefer" to turn toward each other, forming a hydrophobic region. This can be a bilayer or a micelle (Fig. 2). Micelles are spheres and can only reach a certain size, whereas bilayers have no limit to their extension. They can also form tubules.

Genetic material is the material used to store genetic information for a living organism. For all currently known living organisms, with the exception of prions, the genetic material is almost exclusively DNA. This is supplemented with cytoplasmic inheritance factors, often proteins. For some forms of artificial life, the genetic material is computer memory or other digital data storage media.

The central dogma of molecular biology (sometimes Crick's central dogma after Francis Crick who coined the term and discovered some of the principles) states that the flow of genetic information is "DNA to RNA to protein". With a few notable exceptions, all biological cells conform to this rule.

It can be stated in a very short and oversimplified manner as "DNA makes RNA makes proteins, which in turn facilitate the previous two steps as well as the replication of DNA", or simply "DNA → RNA → protein". This process is therefore broken down into three steps: transcription, translation, and replication. By new knowledge of the RNA processing, a fourth step must be included: splicing.

In genetics, transcription is the first process in gene expression. In transcription, DNA is copied to RNA by an enzyme called RNA polymerase (RNAP). Transcription to yield an mRNA is the first step of protein biosynthesis.

The followings steps occur upon initiation:

The RNAP recognizes the promoter region of the gene and binds to the DNA at that specific location. At this stage, the DNA is still double-stranded and called closed complex. Promoter binding is a two step process. Binding is much tighter above 15°C The DNA is unwound and becomes single-stranded at the initiation site (the -10 promoter region). This is called open complex. The RNA polymerase attempts to transcribe the DNA but produces about 10 abortive transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the σ-factor. At some point, the σ-factor dissociates from the holoenzyme, and elongation continues. RNAP prefers to start transcripts with ATP, and to a lesser extent GTP (purine nucleotide triphosphates). UTP, and CTP are disfavoured (pyrimidine nucleotide triphosphates).

Regulation Selective transcription is mainly responsible for the differential protein synthesis among various types of cells in the same organism.

Elongation The RNAP runs along the DNA, synthesizing mRNA in the process. In bacteria, the nascending mRNA is processed right away by ribosomes.

Termination Two termination mechanisms are well known:

Intrinsic termination involves terminator sequences within the RNA as it is being made that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic DNA sequence that forms a hairpin. Rho-dependent termination uses a termination factor called ρ factor to stop RNA sysnthesis at specific sites. This protein binds and runs along the mRNA towards the RNAP. When ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription. Other termination mechanisms include the fact that transcription will terminate if the RNAP comes across a region with repetitious base pairs (for example, TTTTTT).

Eukaryotic transcription Gene expression in eukaryotes is also controlled by complex interactions between cis-acting sites within the regulatory regions of the DNA, and trans-acting factors that include transcription factors and the basal transcription complex, but eukaryotes have evolved a much more complex system for regulation of transcription. For example, eukaryotes have three RNA polymerases, in contrast to prokaryotes, which only have one.

RNA Polymerase I is located in the nucleolus and transcribes only rRNAs. RNA Polymerase II transcribes messenger RNA. RNA Polymerase III transcribes tRNAs and other small RNAs. The C-terminus of all RNAPs is highly conserved and binds to two enzyme factors which sense a poly-adenylation sequence. These factors bind to the DNA and attach approximately 200 adenines to the 3' end of the mRNA.

Although the major advances described above were made in the 1950s and 1960s, the explosion in molecular biology began in the 1970s with the development of gene-cloning techniques which allowed the isolation of large amounts of a pure DNA fragment free of all the other DNA sequences that together constitute the genome (all the genes in the chromosomes) of the organism. This enabled a DNA fragment (perhaps representing a particular gene) to be characterized. This was coupled with the development of hybridization procedures in which a cloned DNA molecule is radioactively labelled and then made single-stranded. This molecule can now be used as a probe since it will bind by base pairing to any DNA or RNA that contains the same linear order of the four bases. Thus this technique can be used to investigate the structure of a gene by hybridizing a probe, derived from the same gene, to DNA fragments that have been separated on the basis of their size and bound to an inert filter. l, d, b, b, j. This procedure is known as Southern blotting after its inventor Ed Southern. Similarly, in the related technique of Northern blotting, DNA from the gene of interest is hybridized to the RNA prepared from different tissues, which allows the RNA homologous (corresponding) to the gene to be detected and quantified in different tissues. These techniques have produced much information on gene structure and expression.

The basal transcription complex includes the RNA polymerase and additional proteins that are necessary for correct initiation and elongation of RNA synthesis. Eukaryotes have evolved more complex regulatory mechanisms than prokaryotes. For instance, in eukaryotes the genetic material (DNA) is synthesized in the nucleus, separated from the site of translation, the cytoplasm, by the nuclear membrane. This allows temporal regulation of gene expression by sequestration of the RNA in the nucleus, and allows for selective transport of RNAs to the cytoplasm, where the translation machinery resides. Primary transcripts in eukaryotic cells are also synthesized as a larger precursor RNAs that must be processed by splicing out non-coding sequences (introns) and ligating non-contiguous coding sequences (exons) into the mature mRNA. Primary transcripts for some genes can be quite large. The primary transcripts of the neurexin genes, for instance, are as large as 1.7 megabases (1,700,000 bases), while the mature neurexin mRNAs are under 10 kilobases (10,000 bases), with as many as 24 exons and thousands of possible alternative splice variants that produce proteins with different activities.

Initiation The core promoter of eukaryotic genes, where the core transcription complex, including RNA polymerase, is usually a region within 50 bases upstream of the transcription intitiation site. Additionally, there can be an upstream control element usually present within 2000 bases upstream of the transcription initiation site. Some genes use enhancer elements that can be thousands of bases upstream or downstream of the transcription initiation site. Combinations of these upstream elements regulate and amplify the formation of the basal transcription complex. This UCE usually contains a TATA box, a highly conserved DNA sequence that reads

T A T A T/A A A similar sequence, thus not that highly conserved, is found in the INR element (initiator element, part of the complex core promoter).

Elongation Elongation in eukaryotes is identical to elongation in prokaryotes.

Termination A major difference between prokaryotic and eukaryotic transcription is that the latter have splicing of the primary transcript, modifying the mRNA created during transcription.

Gene expression (also protein expression or often simply expression) is the process by which a gene's information is converted into the structures and functions of a cell.

Gene expression is a multi-step process that begins with transcription and translation and is followed by folding, post-translational modification and targeting. The amount of protein that a cell expresses depends on the tissue, the developmental stage of the organism and the metabolic or physiologic state of the cell.

Indirectly, the expression of particular genes may be assessed with DNA microarray technology, which can provide a rough measure of the cellular concentration of different mRNAs; often thousands at a time. While the name of this type of assessment is actually a misnomer, it is often referred to as expression profiling. (The expression of many genes is known to be regulated after transcription, so an increase in mRNA concentration need not always increase expression.)

The protein encoded for by a gene can be expressed in increased quantity. This can come about by:

increasing the number of copies of the gene increasing the binding strength of the promoter region Gene networks and expression Main article: Gene regulatory network

Genes have sometimes been regarded as nodes in a network, with inputs being proteins such as transcription factors, and outputs being the level of gene expression. The node itself performs a function, these and the operation of these functions have been interpreted as performing a kind information processing within cell and determine cellular behaviour.

Transfer RNA (abbreviated tRNA) is a small RNA chain (74-93 nucleotides) that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino-acid attachment and codon (a particular sequence of 3 bases) recognition. The codon recognition is different for each tRNA and is determined by the anticodon region, which contains the complementary bases to the ones encountered on the mRNA. Each tRNA molecule binds only one type of amino acid, but because the genetic code is degenerate, more than one codon exists for each amino acid.

Transfer RNA is the "adaptor" molecule hypothesized by Francis Crick, which mediates recognition of the codon sequence in mRNA and allows its translation into the appropriate amino acid.

Aminoacylation is the process of adding an aminoacyl group to a compound.

Each tRNA is aminoacylated (or charged) with a specific amino acid by an aminoacyl tRNA synthetase. There is often just one aminoacyl tRNA synthetase for each amino acid, despite the fact that there can be more than one tRNA, and more than one anticodon, for an amino acid. Recognition of the approriate tRNA by the synthetases in not mediated solely by the anticodon, and the acceptor stem often plays a prominent role.

Reverse transcriptase is an enzyme used by all retroviruses and retrotransposons that transcribes the genetic information from the virus or retrotransposon from RNA into DNA, which can integrate into the host genome. Eukaryotes with linear DNA uses a variant of reverse transcriptase, called telomerase, with the RNA template contained in the enzyme itself. The enzyme collectively referred to as reverse transcriptase generally includes an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase, which work together to perform transcription in the reverse of the standard direction. Usually, transcription only runs from DNA to RNA, catalyzed by RNA polymerase. In addition to the transcription function, retroviral reverse transcriptase carries a RNase domain, which belongs to the RNase H family. An example of a reverse transcriptase is the reverse transcriptase from the human immunodeficiency virus type 1

Reverse transcriptase is commonly used in the field of research to be able to apply the polymerase chain reaction technique to RNA. The classical PCR technique can only be applied to DNA strands, but with the help of reverse transcriptase, RNA can be transcribed into DNA making PCR analysis of RNA molecules possible. The technique is collectively called: Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Reverse transcriptase is also used to create cDNA libraries from mRNA.

Since HIV use reverse transcriptase, together with integrase, to infect human DNA with viral DNA, reverse transcriptase inhibitors are used to prevent this.

Molecular biology is the branch of biology that seeks to interpret biological phenomena in terms of the molecules within the cell. The ultimate goal of molecular biology is to understand the molecular basis of all biological phenomena. The border between molecular biology and other branches of biology is thin and hazy and is apt to be adjusted as molecular biology is extended to fresh territory. Molecular biology is also an inherently logical discipline that crosses traditional boundaries between genetics, biochemistry, cell biology, physics, organic chemistry, and biophysical chemistry. Hence, molecular biology can no longer be thought of as an indivisible subject. The recombinant DNA technology, or genetic engineering, was developed in the late 1970s. It became an integral part of molecular biology as these techniques provided one of the most powerful experimental tools in the study of molecular biological phenomena.

When we are born we are given a name, and most of us can easily live with the one our parents chose for us. Only occasionally do we come across an unfortunate soul, obviously named in a rush of exuberance, who must have a challenge for life—the 'boy named Sue' syndrome. Then there are those names that are fashionable, and we all know examples that move down the charts with time, showing that the individual was born in a certain era—the 'Elvis' syndrome.

The world of science is not very different. Once in a while, our research is renamed in order to reflect new fashions, or if some biologists feel that their work deserves a better or more catchier name. And this is not unique to our times. Nearly 40 years ago, the founders of what became known as the European Molecular Biology Organization had similar discussions when they faced the problem of finding a name that reflected the organisation's scope and mission. Younger readers are probably not aware of the debates that revolved around the name 'molecular biology' at that time. Nowadays, we are also witnessing a surge of trendy names, such as functional genomics, proteomics—indeed, the whole 'omics' family is perhaps only a fashion and, 10 years from now, will be a reminder of the current flurry of redefining or renaming biological research activities. It seems there are new skirmishes afoot, and 'molecular biology' is being redefined again.

But, despite all the descriptive or catchy names, we should not forget that a constant aspect of our work is the fact that we study biology, living matter as opposed to astronomy where, excuse my prejudice, I do not expect us to run into life. If we look back in time, we find that the original biologists were purely descriptive scientists. Animals and plants were picked up, examined, drawn and classified to impose order on the living world. What these species did and how they did it was described, as were their innards and their general physiology. Later, professors of anatomy, physiology and related specialities joined their colleagues from botany and zoology around the faculty table to discuss the relationships between these organisms and their evolution.

The use of Southern blotting to probe gene structure led to the biggest surprise obtained in molecular biology studies so far. This was the finding that in eukaryotes (plants and animals) the regions of the DNA that contain the information coding for the protein, known as exons, are interrupted by other DNA sequences, known as introns. These introns are transcribed into a single RNA molecule with the exons and are then removed by the process of RNA splicing. This happens inside the cell nucleus, producing the mRNA molecule in which the exons are appropriately joined together without any intervening DNA. This mRNA is then transported to the cytoplasm and translated into protein on the ribosomes. Although the significance of introns is unclear, their existence does allow different combinations of the exons present in the initial transcript to be joined together in different cell types. This process, known as alternative splicing, results in the production of different but related proteins from the same gene. Northern blotting can be used to investigate the presence of mRNA molecules derived from different genes in extracts of whole tissues. Such studies can be complemented by in situ hybridization that can identify the mRNA within individual cells, allowing its distribution within a tissue to be characterized. h, l, e, i, k. These studies lead to the conclusion that, in the vast majority of cases, the mRNA encoding a particular protein is only present in tissues and cells that express the protein. Similarly unspliced precursor RNAs still containing introns are not detectable in tissues that do not contain the spliced mRNA or the protein.

These groups were joined by scientists who showed less respect for the organism and wanted to understand life by breaking it apart into smaller and more manageable units. The biochemists added the ambiguous aspect of chemistry to the description of life. They were fundamentally reductionistic, believing that one could understand the Ferrari of life by examining each screw, piston and cylinder in isolation. They—and I should include myself in this category as I was trained as a biochemist—were aware that they studied life with a precision that earlier biologists were not able to achieve. And they had an influence on the more traditional departments, such as botany that mutated, in name at least, to plant science.

In the 1950s, a new sect of biologists entered the laboratories. In an exaggerated form they worked with the molecules of life, but aimed to integrate them into the biology from whence they came. If the biochemists stressed the inanimate chemistry in the molecules they obtained from living sources, the molecular biologists tried to put the puzzle back together by demonstrating their consequences for life. They saw the need to work across the traditional boundaries in order to obtain a better understanding of living matter. Nonetheless, the founders of the molecular biology movement originated from the departments of biochemistry, microbiology, virology, botany, etc. The overlap with the biochemists is obvious and to some the difference is not worth a new name.

The term molecular biology went through a transition when, in the 1970s, recombinant DNA technologies found their way into the laboratory. Strangely, many people today equate molecular biology with cloning and related procedures. Historically this makes no sense, and it has a strangling limitation. Molecular remains an adjective defining the scale at which biologists study their objects, and it is not a pseudonym for a collection of related techniques.

Now, just as peoples' names can date, we are running into a situation where 'our' name is less likely to be used for a new department in a new institute. We are also seeing the consequences of growth, which inevitably leads to subdivisions. Today colleagues define themselves as cell biologists, immunologists, neurobiologists, developmental biologists, cellular microbiologists, etc. But they all look at life at the same level and they all use common research tools. So the challenge is to find a name that represents the unifying features behind all this diversity. One wit suggested that EMBO should change its name to the European Modern Biology Organization, and while this is a rather flippant contribution to the debate, such a move might avoid a problem that we may have to face in the future. We are currently engaged in establishing a European Research Council in the area of biology. But whether it should be called the European Life Sciences or the European Biosciences or the European Molecular Biology Research Council remains a trickier question than anticipated. What is 'molecular biology' today, and is it a term that should be tagged on to a new entity born at the start of this new century? Ultimately, as for an individual, the name should not be the basis for any a priori judgement. Rather we should judge the aims and aspirations of the enterprise, its track record and then decide if it would be a beneficial addition to the crèche of the life sciences/biosciences/biotechnology/biology community?

Molecular Biology is the study of how life works at the molecular level. It is the study of the molecular mechanisms responsible for converting genotype to phenotype. In multicellular organisms, different cell types have specialized structures and functions within the same organism. For example, the cone cells in your retina look very different and function very differently from your muscle cells. This cellular phenotype is a direct consequence of the proteins made in that cell. Since all cells in any given organism have the same genotype, the differences between them are due to the selective use of a subset of the genes held in common. It is the selection of which genes are expressed and which repressed and how this occurs that is the essence of the sub-discipline of Molecular Biology.

Simply put, molecular biologyis "the biochemical study of the genetic basis for phenotype". It allows us to understand how and why physiological events occur, what controls them, and what has gone wrong in disease states. It is not to be confused with the technical procedures used to study molecular biology (for example: hybridizations, Southern blots, dot blots, polymerase chain reaction, etc.). These are just methods used to study molecular genetics. On the other hand, technological spinoffs of molecular biology allow the improved detection and diagnosis of certain infectious agents and genetic defects, sometimes even before they cause clinical signs or are expressed as a phenotype.

Ever since the creation of living beings on this planet, Man has struggled to understand Life. However, the phenomenon of Life has remained shrouded in mystery. Philosophers in the times of Aristotle and Omar Khayyam discussed Life in abstract terms. However, recent breakthroughs in Biology such as unravelling the structure of DNA (1953), the isolation of DNA polymerase (1958), reverse transcriptase (1969) and restriction enzymes (1970), the practical uses of these enzymes in mapping (1971), cloning (1972) and sequencing of genes (1977) and the use of plasmids as vectors for cell transformation (1983) have enabled biologists to penetrate deep into the intricate web of life. As a consequence, a scientific concept of life has emerged: that biological activity is the expression of structural and functional information stored in gigantic organic molecules such as DNA. The study of biologically active molecules is called "Molecular Biology."

The largest of all biologically active molecules, "DNA", determines the characteristics of an organism. The DNA of any living thing __ be it a tiny bacterium, a giant elephant or a mammonth banyan tree __ is made up of the same four nitrogenous bases, adenine, guanine, thymine and cytosine. It is the order in which these four types of organic molecules are arranged on a strand of DNA (the nucleotide sequence) that distinguishes the genetic characteristics of one living being from another. Recent progress in DNA enzymology and chemistry has made it possible to make new DNA (recombinant DNA) in the test tube by polymerizing individual nucleotides or joining together pieces of existing DNAs from different species and has thus produced the molecular equivalent of a chimera, the legendary animal (part lion, part goat and part snake) of Greek mythology. Such capabilities have helped biologists in not only learning more about how a living cell does what it does but also in harnessing living organisms to do more useful work for mankind. This has made molecular biology the most exciting of sciences and has lead to the pursuit of a new endeavour -- "Biotechnology."

This indicates that, in most cases, the production of different proteins by different tissues is determined by controlling which genes are transcribed in each tissue with the subsequent stages of intron removal and translation following automatically. This has been confirmed by studies in which the transcription rate of a specific gene was directly measured in different tissues where the corresponding protein is either present or absent. Therefore the production of different proteins, which is central to the functional differences between different tissues, is controlled at the level of gene transcription. In turn gene transcription is regulated by proteins known as transcription factors that bind to specific DNA sequences in the regulatory regions of the gene and stimulate transcription. Such transcription factors may be present in only one tissue producing tissue-specific transcription of the genes that they activate. c, f, f, l, d. Alternatively, they may be present in all cells in an inactive form, being activated by specific signals which result in their post-translational modification, for example, by the addition of phosphate residues (phosphorylation). This, in turn, leads to transcription of their target genes in response to the signal.

Biology has been harnessed since antiquity to fulfill humanity's most fundamental needs -- from increasing food supplies to improving health care. The availability of new and novel methodologies has greatly expanded the scope of applications of Molecular Biology which is limited only by the imagination of the people using it. For the first time in the history of mankind, it seems within reach of human endeavour to custom design plants that can grow normally under stressed conditions, drive off pests, produce unusually high yields and nutritionally improved foods; remediate underground soil and water; and even synthesize new types of cotton fibers, biodegradable plastics, industrial lubricants, feedstocks for soaps and detergents, and new pharmaceuticals and vaccines. The identification of human genes and their expression in bacteria and yeast has enabled the production of wonder drugs, vaccines and other therapeutic materials to fight against cancer, hepatitis, leprosy, AIDS and cardiovascular disease; eliminate viral and parasitic diseases; diagnose and correct (via gene therapy) human genetic disorders even before birth. The capability of plants and bacteria to produce industrial and pharmaceutical products is expected to give new directions to industry and the sectors of energy and the environment. The vast panorama of applications of molecular biology is just unfolding. According to various international Resource Development research firms, the economic impact of molecular biology during the next century will be equal to, if not more than, that of plastics in the 1940's, transistors in the 1950's, computers in the 1960's, electronics in the 1970's and microelectronics in the 1980's.

In view of such glowing hopes for making large profits, it is not surprising that private DNA companies have proliferated in Europe and USA. Most of these have focused attention on the areas in which there are obviously high profits to be made. Such developments will certainly result in substantial reductions in the overall demand for primary products from developing countries with serious economic consequences for their exports of raw materials which generate most of their foreign exchange earnings. In a background of such developments taking place at the international scene, it is imperative for a country like Pakistan to build indigenous capability in this new field.

Clearly, the challenge of applying innovative biological approaches to solve unique and specific problems of national economic importance will require the talents of a soundly trained manpower which can define problems and devise strategies that will work in the local setting. In order to develop a researcher having "hands-on" experience of the various molecular biological techniques, training should be carried out in local situations to see how many things can go wrong and how to get around those hurdles and solve problems. The quality of the training will depend on the condition of the training laboratories. Traditionally, our university biology laboratories, that are the breeding grounds for future biotechnologists, have been even more deficient than the laboratories of non-university research institutes in the country. As a consequence, the university training in Pakistan lacks laboratory experience that is so vitally important for building the intellectual maturity of future researchers. It is exceedingly important, therefore, to improve the local laboratory infrastructure if the quality of our training is to be improved.

Research in molecular biology is vitally dependent on a regular and reliable provision of specialized enzymes and fine chemicals that form the heart of the technology. Specialized chromatography media, centrifuges, protein and DNA sequencing and electrophoresis equipment and the provision of a constant source of electricity and air-conditioning are essential components of a molecular biology lab. Evidently, the development of molecular biology capability will be expensive and it will inevitably involve a firm political commitment.

Molecular Biology is the interdisciplinary use of genetics, biochemistry and structural biology to solve problems of biological function. DNA; its structure and properties.

Molecular genetics is the study of the agents that pass information from generation to generation. These molecules, our genes, are long polymers of deoxyribonucleic acid, or DNA. Just four chemical building blocks—guanine (G), adenine (A), thymine (T), and cytosine (C)—are placed in a unique order to code for all of the genes in all living organisms.

Genes determine hereditary traits, such as the color of our hair or our eyes. They do this by providing instructions for how every activity in every cell of our body should be carried out. For example, a gene may tell a liver cell to remove excess cholesterol from our bloodstream. How does a gene do this? It will instruct the cell to make a particular protein. It is this protein that then carries out the actual work. In the case of excess blood cholesterol, it is the receptor proteins on the outside of a liver cell that bind to and remove cholesterol from the blood. The cholesterol molecules can then be transported into the cell, where they are further processed by other proteins.

Many diseases are caused by mutations, or changes in the DNA sequence of a gene. When the information coded for by a gene changes, the resulting protein may not function properly or may not even be made at all. In either case, the cells containing that genetic change may no longer perform as expected. We now know that mutations in genes code for the cholesterol receptor protein associated with a disease called familial hypercholesterolemia. The cells of an individual with this disease end up having reduced receptor function and cannot remove a sufficient amount of low density lipoprotein (LDL), or bad cholesterol, from their bloodstream. A person may then develop dangerously high levels of cholesterol, putting them at increased risk for both heart attack and stroke.

How do scientists study and find these genetic mutations? They have available to them a variety of tools and technologies to compare a DNA sequence isolated from a healthy person to the same DNA sequence extracted from an afflicted person. Advanced computer technologies, combined with the explosion of genetic data generated from the various whole genome sequencing projects, enable scientists to use these molecular genetic tools to diagnose disease and to design new drugs and therapies. Below is a review of some common laboratory methods that geneticists— scientists who study the inheritance pattern of specific traits—can use to obtain and work with DNA, followed by a discussion of some applications.

Laboratory Tools and Techniques

The methods used by molecular geneticists to obtain and study DNA have been developed through keen observation and adaptation of the chemical reactions and biological processes that occur naturally in all cells. Many of the enzymes that copy DNA, make RNA from DNA, and synthesize proteins from an RNA template were first characterized in bacteria. These basic research results have become fundamental to our understanding of the function of human cells and have led to immense practical applications for studying a gene and its corresponding protein. For example, large-scale protein production now provides an inexpensive way to generate abundant quantities of certain therapeutic agents, such as insulin for the treatment of diabetes. As science advances, so do the number of tools available that are applicable to the study of molecular genetics.

Obtaining DNA for Laboratory Analysis

Isolating DNA from just a single cell provides a complete set of all a person's genes, that is, two copies of each gene. However, many laboratory techniques require that a researcher have access to hundreds of thousands of copies of a particular gene. One way to obtain this many copies is to isolate DNA from millions of cells grown artificially in the laboratory. Another method, called cloning, uses DNA manipulation procedures to produce multiple copies of a single gene or segment of DNA. The polymerase chain reaction (PCR) is a third method whereby a specific sequence within a double-stranded DNA is copied, or amplified. PCR amplification has become an indispensable tool in a great variety of applications.

Isolating DNA and mRNA from Cells

Cell Culture Cell culture involves growing cells under artificial conditions, such as in the laboratory, either attached to some type of artificial surface or suspended in a special solution. In both cases, the cells are bathed in fluids containing nutrients that are either synthetically produced or extracted from related organisms. Certain cell types are more amenable to being grown in culture than others. For example, fibroblasts, a type of skin cell, have been cultured in the lab for decades, whereas the nuances of growing other cell types, such as nerve cells and stem cells, have only recently been elucidated. Conditions that serve to sustain one cell type may not apply to other cell types, or even the same cell type from another species. The conditions necessary for growing cells from humans, and many other mammals and plants upon which we depend, have been generally determined, whereas the conditions for culturing cells from exotic animals and plants still require experimentation with each new species.

As well as these gene structure and expression studies, it is also possible to read the linear order of the bases in the DNA using a process known as DNA sequencing. The most common method for this is that described by Fred Sanger in 1977 and this was used in the Human Genome Project to sequence the entire human genome. By using DNA sequencing a gene can be characterized in terms of a linear sequence of AGCT bases that, in turn, can be used to predict the amino acid sequence of the corresponding protein using the genetic code. Indeed, DNA sequencing is so much easier than direct sequencing of the protein itself that protein sequences are now normally determined indirectly by sequencing the corresponding gene. Similarly, by sequencing a gene involved in a specific disease from normal and diseased individuals, it is possible to characterize the alteration in the corresponding protein which results in the disease. g, h, b, d, l. This may involve, for example, a base change which leads to a single amino acid change in the protein or a loss of a DNA segment leading to the loss of a corresponding portion of the protein.

Cell culture is a useful technique because it provides a renewable source of cells for isolating DNA. In addition, scientists can use cells grown in culture to study how various chemicals and drugs affect certain cells and by extrapolation, the whole organism. The process of growing cells outside a living organism, such as in a test tube, is referred to as in vitro. Once the effects of an agent on a cell have been thoroughly evaluated in vitro, the search for safe and effective treatments can be tested within a living organism, a process called in vivo testing.

Some cells tend to lose valuable characteristics or may even die out if kept in culture too long. To remedy this problem, researchers freeze down a cell line so that it can be thawed at a later date for subsequent use. This process requires the use of a chemical "cryopreservative" that protects and prevents the cell from bursting during the freezing and thawing process.

DNA Isolation DNA isolation refers to the process of extracting DNA from a cell in a relatively pure form. It involves separating DNA from other cellular components, such as proteins, RNA, and lipids. The cells used to obtain and isolate the DNA could come directly from tissue or could be cultured laboratory cell lines obtained using the methods described earlier. Whatever the source, the DNA is isolated by placing the cells in a tube containing a special solution, called a "cocktail", and mechanically or chemically breaking them open. This causes the cell to release its contents into the cocktail containing enzymes, chemicals, and salts. Enzymes are used to chew up the proteins; chemicals to destroy any RNA present; and salts to help pull the DNA out of solution. At this point, the DNA will exist in long strands that form a mucous-like glob within the solution. The DNA is then harvested by spinning the tube in a machine called a centrifuge. During spinning, the DNA collects in the bottom of the tube. The solution is then poured off, and the DNA is dissolved, or resuspended, in a second solution that will make it easy to work with in subsequent procedures. The result is a concentrated DNA sample containing many thousands of copies of each gene. For large-scale DNA analysis methods, such as those required to sequence the human genome, DNA isolation is performed using robots.

mRNA Isolation Many researchers want to work with what is called expressed DNA, or DNA that codes directly for the synthesis of a protein. This special type of DNA is obtained by first isolating messenger RNA (mRNA), an intermediate between the expressed portions of DNA and the protein product. Laboratory methods for mRNA isolation take advantage of a normal cellular modification of mRNA—the addition of up to 200 adenine nucleotides to one end of the mRNA molecule—called a poly(A) tail. In the first step of mRNA isolation, a cell is ruptured, and the cellular contents are exposed to synthetic beads coated with strings of thymine nucleotides.

Because adenine and thymine readily bind to each other, poly(A) mRNA is selectively retained on the beads while the other cellular components are washed away. Once isolated, purified mRNA is converted to single-stranded DNA using the enzyme reverse transcriptase and is then made into a stable double-stranded DNA using the enzyme DNA polymerase. DNA produced in this way is called complementary DNA (cDNA) because its sequence, at least the first strand, is complementary to that of the mRNA from which it was made. Why do researchers go to the trouble of making cDNA? cDNA is a much more stable compound than mRNA and, more importantly, because it was generated from an mRNA in which the non-coding regions have been removed, cDNA represents only expressed DNA sequence.

Reverse transcriptase, an enzyme required for forming a complementary DNA sequence from a RNA sequence, is vital for the survival of a group of viruses called the retroviruses. Retroviruses contain RNA, instead of DNA, as their genetic material. Reverse transcriptase is used to make a DNA copy of the virus' genetic material, a necessary component for integrating into the host organism's genome. Although most retroviruses are not considered beneficial to humans, reverse transcriptase is an invaluable laboratory tool for studying and treating some of the ailments caused by these viruses.

Methods for Amplifying DNA

Cloning DNA in Bacteria Cloning revolutionized biological research in the 1970s by making it possible to study individual genes.

The word "cloning" can be used in many ways. In this document, it refers to making multiple, exact copies of a particular sequence of DNA. To make a clone, a target DNA sequence is inserted into what is called a cloning vector. A cloning vector is a DNA molecule originating from a virus, plasmid, or the cell of a higher organism into which another DNA fragment of appropriate size can be integrated without interfering with the vector's capacity for self-replication. The target and vector DNA fragments are then ligated, or joined together, to create what is called a recombinant DNA molecule. Recombinant DNA molecules are usually introduced into Escherichia coli, or E. coli—a common laboratory strain of a bacterium— by transformation, the natural DNA uptake mechanism possessed by bacteria. Within the bacterium, the vector directs the multiplication of the recombinant DNA molecule, producing a number of identical copies. The vector replication process is such that only one recombinant DNA molecule can propagate within a single bacterium; therefore, each resulting clone contains multiple copies of just one DNA insert. The DNA can then be isolated using the techniques described earlier.

A restriction enzyme is a protein that binds to a DNA molecule at a specific sequence and makes a double-stranded cut at, or near, that sequence. Restriction enzymes have specialized applications in various scientific techniques, such as manipulating DNA molecules during cloning. These enzymes can cut DNA in two different ways. Many make a simple double-stranded cut, giving a sequence what are called blunt or flush ends. Others cut the two DNA strands at different positions, usually just a few nucleotides apart, such that the resulting DNA fragments have short single-stranded overhangs, called sticky or cohesive ends. By carefully choosing the appropriate restriction enzymes, a researcher can cut out a target DNA sequence, open up a cloning vector, and join the two DNA fragments to form a recombinant DNA molecule.

More on Cloning Vectors In general, a bacterial genome consists of a single, circular chromosome. They can also contain much smaller extrachromosomal genetic elements, called plasmids, that are distinct from the normal bacterial genome and are nonessential for cell survival under normal conditions. Plasmids are capable of copying themselves independently of the chromosome and can easily move from one bacterium to another. In addition, some plasmids are capable of integrating into a host genome. This makes them an excellent vehicle, or vector, for shuttling target DNA into a bacterial host. By cutting both the target and plasmid DNA with the same restriction enzyme, complementary base pairs are formed on each DNA fragment. These fragments may then be joined together, creating a new circular plasmid that contains the target DNA. This recombinant plasmid is then coaxed into a bacterial host where it is copied, or replicated, as though it were a normal plasmid.

Bacterial plasmids were the first vectors used to transfer genetic information and are still used extensively. However, their use is sometimes limited by the amount of target DNA they can accept, approximately 15,000 bases, or 15 Kb. With DNA sequences beyond this size, the efficiency of the vector decreases because it now has trouble entering the cell and replicating itself. However, other vectors have been discovered or created that can accept larger target DNA including: bacteriophages, bacterial viruses that accept inserts up to 20 Kb; cosmids, recombinant plasmids with bacteriophage components that accept inserts up to 45 Kb; bacterial artificial chromosomes (BACs) that accept inserts up to 150 Kb; and yeast artificial chromosomes (YACs) that accept inserts up to 1000 kb. Many viruses have also been modified for use as cloning vectors.

Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) is an amazingly simple technique that results in the exponential amplification of almost any region of a selected DNA molecule. It works in a way that is similar to DNA replication in nature. The primary materials, or reagents, used in PCR are:

DNA nucleotides, the building blocks for the new DNA Template DNA, the DNA sequence that you want to amplify Primers, single-stranded DNAs between 20 and 50 nucleotides long that are complementary to a short region on either side of the template DNA Taq polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA

Taq polymerase was first isolated from a bacterium that lives in the hot springs in Yellowstone National Park. The Taq polymerase enzyme has evolved to withstand the extreme temperatures in which the bacteria live and can therefore remain intact during the high temperatures used in PCR.

Protein Structure and Function These ideas lead us to consider how the functional activity of a protein is related to its properly folded structure as determined by its amino acid sequence. In the 1960s, the British biochemist and recipient of the 1962 Nobel Prize for Chemistry John Kendrew determined the structure of myoglobin using purified protein and X-ray crystallography. His colleague and joint prizewinner Max Perutz subsequently determined the more complex structure of haemoglobin, which consists of four myoglobin-like units. However, in the same way that protein sequence analysis is now performed using DNA-based procedures, structural analysis is now normally carried out on protein which has been produced by artificially expressing the equivalent gene, for example in bacteria. This allows the protein to be obtained in large amounts. Moreover, specific changes can be introduced into the DNA by site-directed mutation and the altered protein being expressed in bacteria. b, a, e, h, i. Studies on the activity and structure of the altered protein can then be used to relate changes in amino acid sequence to their effect on the structure and functional activity of the resulting protein. In this way, the goal of relating structure to function can be achieved.

The PCR reaction is carried out by mixing together in a small test tube the template DNA, DNA nucleotides, primers, and Taq polymerase. The primers must anneal, or pair to, the template DNA on either side of the region that is to be amplified, or copied. This means that the DNA sequences of these borders must be known so that the appropriate primers can be made. These oligonucleotides serve to initiate the synthesis of the new complementary strand of DNA. Because Taq polymerase, a form of DNA polymerase that catalyzes the synthesis of new DNA, is incredibly heat stable (thermostable), the reaction mixture can be heated to approximately 90 degrees centigrade without destroying the molecules' enzymatic activity. At this temperature, the newly created DNA strands detach from the template DNA.

The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA–DNA matches to occur in the reaction.

The reaction mixture is then cooled again, allowing more primers to anneal to the template DNA and also to the newly created DNA. The Taq polymerase can now carry out a second cycle of DNA synthesis. This cycle of heating, cooling, and heating is repeated over and over. Because each cycle doubles the amount of template DNA in the previous cycle, one template DNA molecule rapidly becomes hundreds of thousands of molecules in just a couple of hours.

PCR has many applications in biology. It is used in DNA mapping, DNA sequencing, and molecular phylogenetics. A modified version of PCR can also be used to amplify DNA copies of specific RNA molecules. Because PCR requires very little starting material, or template DNA, it is frequently used in forensic science and clinical diagnosis.

Preparing DNA for Experimental Analysis

Gel Electrophoresis: Separating DNA Molecules of Different Lengths Originally, proteins were separated on a gel made from potato starch. Today, gels are made from agarose or synthetic polymers such as polyacrylamide.

Gels are usually made from agarose—a chain of sugar molecules extracted from seaweed—or some other synthetic molecule. Purified agarose is generally purchased in a powdered form and is dissolved in boiling water. While the solution is still hot, it is poured into a special gel casting apparatus that contains three basic parts: a tray, a support, and a comb. The tray serves as the mold that will provide the shape and size for the gel. The support prevents the liquid agarose from leaking out of the mold during the solidification process. As the liquid agarose starts to cool, it undergoes what is known as polymerization. Rather than staying dissolved in the water, the sugar polymers crosslink with each other, causing the solution to gel into a semi-solid matrix much like Jello, only more firm. The support also allows the polymerized gel to be removed from the mold without breaking. The job of the comb is to generate small wells into which a DNA sample will be loaded.

Once a gel has polymerized, it is lifted from the casting tray, placed into a running tank, and submerged in a special aqueous buffer, called a running buffer. The gel apparatus is then connected to a power supply via two plugs, or electrodes. Each plug leads to a thin wire at opposite ends of the tank. Because one electrode is positive and the other is negative, a strong electric current will flow through the tank when the power supply is turned on.

Next, DNA samples of interest are dissolved in a tiny volume of liquid containing a small amount of glycerol. Because glycerol has a density greater than water, it serves to weight down the sample and stops it from floating away once the sample has been loaded into a well. Also, because it is helpful to be able to monitor a DNA sample as it migrates across a gel, charged molecules, called dyes, are also added to the sample buffer. These dyes are usually of two different colors and two different molecular weights, or sizes. One of the dyes is usually smaller than most, if not all, of the sample DNA fragments and will migrate faster than the smallest DNA sample. The other dye is usually large and will migrate with the larger DNA samples. It is assumed that most of the DNA fragments of interest will migrate somewhere in between these two dyes. Therefore, when the small dye reaches the end of the gel, electrophoresis is usually stopped.

Once the gel has been prepared and loaded, the power supply is turned on. The electric current flowing through the gel causes the DNA fragments to migrate toward the bottom, or positively charged end, of the gel. This is because DNA has an overall negative charge because of the combination of molecules in its structure. Smaller fragments of DNA are less impeded by the crosslinks formed within the polymerized gel than are larger molecules. This means that smaller DNA fragments tend to move faster and farther in a given amount of time. The result is a streak, or gradient, of larger to smaller DNA pieces. In those instances where multiple copies of DNA all have the same length, a concentration of DNA occurs at that position in the gel, called a band. Bands can result from a restriction enzyme digest of a sample containing thousands of copies of plasmid DNA, or PCR amplification of a DNA sequence. The banded DNA is then detected by soaking the gel briefly in a solution containing a dye called ethidium bromide (EtBr). EtBr is an intercalating agent, which means that it is capable of wedging itself into the grooves of DNA, where it remains. The more base pairs present within a DNA fragment, the greater the number of grooves available for EtBr to insert itself. EtBr also fluoresces under ultraviolet (UV) light. Therefore, if a gel soaked in a solution containing EtBr is placed under a UV source, a researcher can actually detect DNA by visualizing where the EtBr fluoresces. Because a scientist always loads and runs a "control" sample that contains multiple fragments of DNA with known sizes, the sizes of the sample DNA fragments can be estimated by comparing the control and sample bands.

DNA Blotting The porous and thin nature of a gel is ideal for separating DNA fragments using electrophoresis, but as we mentioned earlier, these gels are delicate and rarely usable for other techniques. For this reason, DNA that has been separated by electrophoresis is transferred from a gel to an easy-to-handle inert membrane, a process called blotting. The term "blotting" describes the overlaying of the membrane on the gel and the application of a pad to ensure even contact, without disturbing the positions of the DNA fragments. In the first step, the DNA trapped in the gel is denatured—the double-stranded DNA is broken into single strands by soaking the gel in an alkaline solution. This readies the DNA for hybridization with a probe, a piece of DNA that is complementary to the sequence under investigation. A membrane, usually made of a compound called nitrocellulose, is then placed on top of the gel and compressed with a heavy weight. The DNA is transferred from the gel to the membrane by simple capillary action. This procedure reproduces the exact pattern of DNA captured in the gel on the membrane. The membrane can then be probed with a DNA marker to verify the presence of a target sequence.

The generation of large amounts of specific proteins by expressing the corresponding DNA has been used to produce large amounts of proteins for therapeutic use in individuals who suffer from diseases caused by the non-production of a specific protein. This makes the purification of such proteins from animal sources or human cadavers redundant. This approach has been successfully used to produce insulin to treat diabetics and growth hormone to treat dwarfism. Recently, attempts have been made to bypass the protein production stage and treat individuals with genetic diseases by supplying the functional gene itself so that the corresponding protein is synthesized in the affected individual. Such gene therapy approaches are in their infancy but offer considerable hope for the future. Molecular biology has achieved much in the 50 years since the structure of DNA was characterized. This progress offers real hope that the complex processes underlining embryonic development and the functioning of the adult organism may one day be sufficiently understood in molecular terms to allow the effective treatment of human diseases. j, i, e, g, h. Although much remains to be done, molecular biology has rapidly achieved such importance today that 20 years ago it would not have merited an article in a general encyclopedia.

Southern blotting is the name of the procedure for transferring denatured DNA from an agarose gel to a solid support membrane. This procedure takes advantage of a special property of nitrocellulose, its ability to bind very strongly to single-stranded DNA but not double-stranded DNA. On the other hand, Northern blotting refers to any blotting procedure in which electrophoresis is performed using RNA.

Creating a DNA Library To track the millions, or even billions, of nucleotides in the genome of an organism, scientists often create what is called a DNA library, or a large collection of DNA fragments. There are many different kinds of libraries. A genomic library contains all of the different types of DNA sequences found in a genome— introns, exons, and non-coding and repetitive DNA sequences. Scientists also make libraries exclusively of genes that are expressed, or those genes that get transcribed into messenger RNA and then translated into protein. This library is called a complementary DNA (cDNA) library and is often made from mRNA expressed in a particular tissue type. A library is called chromosome specific if the starting DNA came from just one chromosome.

Methods for Analyzing DNA

Once DNA has been isolated and purified, it can be further analyzed in a variety of ways, such as to identify the presence or absence of specific sequences or to locate nucleotide changes, called mutations, within a specific sequence.

Autoradiography: Probing DNA To locate a specific DNA sequence, scientists rely on the base-pairing, or hybridization, of a short piece of DNA that is complementary to the sequence of interest. This short, single-stranded piece of DNA is called a "probe" and can be tagged with either mildly radioactive nucleotides or nucleotides that are linked to a substance that emits light when exposed to certain chemicals.

If we refer back to the blotting procedure described earlier, we mentioned that after the target DNA becomes trapped in the nylon membrane, the membrane is incubated in a solution that contains a probe. In this case, the probe would be radioactively labeled. Wherever the probe sequence complements a sequence on the membrane, it will anneal, or join together, to form a region of double-stranded DNA. The membrane is then washed to remove all unbound probe and then exposed to a piece of X-ray film. The detection of radioactively labeled molecules by exposure to an X-ray-sensitive photographic film is referred to as autoradiography. Wherever the radioactively labeled probe has annealed to the test DNA, a black spot will be appear on the film.

This method is useful for a variety of applications. For example, suppose you know the DNA sequence of a particular gene (allele) that causes a disease. Now you want to know if a certain individual carries that allele. You can do this by following the steps outlined above. Isolate some of their DNA. Separate it out on a gel. Then, perform a Southern blot followed by autoradiography. If a black spot appears on the film, it indicates the presence of the disease-causing allele in that individual.

RFLP Analysis: Detecting Disease Genes Every individual has slight differences or sequence polymorphisms that make their DNA sequence unique. These differences are often single base-pair changes that occur in regions of DNA that do not encode a gene but which are recognized and bound by restriction enzymes. Restriction enzymes are proteins that bind to a DNA molecule at a specific sequence and make a double-stranded cut at, or near, that sequence. Thus, when DNA from two different individuals is cut with a single restriction enzyme, DNA of different lengths will usually be produced. This is because not all restriction sites will exist within everyone's DNA. These variations in fragment length are referred to as restriction fragment length polymorphisms (RFLPs), and the pattern of fragments is unique for each person. If a restriction polymorphism can be linked to a particular phenotype, such as eye color, it is called a restriction marker. Restriction markers are important because they offer a diagnostic procedure for detecting a disease and can help a researcher isolate a gene.

DNA Sequencing The process of determining the order of the nucleotide bases along a DNA strand is called sequencing. In 1977, 24 years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination method and the chemical degradation method. Both methods were equally popular to begin with, but, for many reasons, the chain termination method is the method more commonly used today. This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis, described earlier.

The DNA to be sequenced, called the template DNA, is first prepared as a single-stranded DNA. Next, a short oligonucleotide is annealed, or joined, to the same position on each template strand. The oligonucleotide acts as a primer for the synthesis of a new DNA strand that will be complementary to the template DNA. This technique requires that four nucleotide-specific reactions—one each for G, A, C, and T—be performed on four identical samples of DNA. The four sequencing reactions require the addition of all the components necessary to synthesize and label new DNA, including:

A DNA template A primer tagged with a mildly radioactive molecule or a light-emitting chemical DNA polymerase, an enzyme that drives the synthesis of DNA Four deoxynucleotides (G, A, C, and T) One dideoxynucleotide, either ddG, ddA, ddC, or ddT After the first deoxynucleotide is added to the growing complementary sequence, DNA polymerase moves along the template and continues to add base after base. The strand synthesis reaction continues until a dideoxynucleotide is added, blocking further elongation. This is because dideoxynucleotides are missing a special group of molecules, called a 3'-hydroxyl group, needed to form a connection with the next nucleotide. Only a small amount of a dideoxynucleotide is added to each reaction, allowing different reactions to proceed for various lengths of time until by chance, DNA polymerase inserts a dideoxynucleotide, terminating the reaction. Therefore, the result is a set of new chains, all of different lengths.

To read the newly generated sequence, the four reactions are run side-by-side on a polyacrylamide sequencing gel. The family of molecules generated in the presence of ddATP is loaded into one lane of the gel, and the other three families, generated with ddCTP, ddGTP, and ddTTP, are loaded into three adjacent lanes. After electrophoresis, the DNA sequence can be read directly from the positions of the bands in the gel.

Variations of this method have been developed for automated sequencing machines. In one method, called cycle sequencing, the dideoxynucleotides, not the primers, are tagged with different colored fluorescent dyes; thus, all four reactions occur in the same tube and are separated in the same lane on the gel. As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded, and the sequence is reconstructed from the pattern of colors representing each nucleotide in the sequence.

Chromosome Analysis

Cytogenetics is the field of science that deals with the relationship between human cells—and their chemical building blocks—and heredity. Key to connecting chromosomes to symptoms and traits is the karyotype, a size-order alignment of chromosome pairs in a chart. The first such efforts to align the chromosome pairs, however, were quite crude. By 1959, about all that could be discerned was an extra or missing chromosome. Throughout the 1960s, pioneering cytogeneticists amassed techniques for capturing chromosomes at their most visible state. For most of a cell's existence, the chromosomal material is unwound and unable to absorb dyes. It is only during cell division that the chromosomes condense and become detectable. Researchers learned that treating cells with a hypotonic solution would cause them to swell, spreading apart the tangle of chromosomes. Another chemical agent, colchicine, was found to stop cell division when the chromosomes were at their most striking state. A third chemical, phytohemagglutinin, was found to entice lymphocytes, the blood cells most accessible for chromosomal study, to divide. With these tools in hand, the art of karyotyping was soon transformed into true science.

But still, chromosome pairs could not always be distinguished very well, and researchers had to rely on such large-scale and subjective clues as chromosome size and position of the centromere, a characteristically located constriction in each chromosome. Even staining the chromosomes distinguished unequivocally only 4 of the 23 chromosome pairs. These pairs were then grouped crudely by size, and only large sections of extra or missing chromosomal material could be discerned.

By the 1970s, combining stains with digestive enzymes yielded far more subtle shading patterns, revealing the distinctive characteristic of each chromosome. Several different treatments were also developed that allowed researchers to further define the patterns of each chromosome. Now, tiny inversions (reversals in the banding pattern), duplications, deficiencies, and translocations (chromosomes that swap parts) could be detected. But building a karyotype required many hours of skilled work. The karyotyping procedure involved obtaining blood or some other appropriate tissue, separating out dividing cells, growing them in culture, fixing them, and then dropping them onto a microscope slide. Then, using a light microscope, a researcher had to find a cell in which all of the untangled chromosomes were present and a photograph was taken. A print was then developed, and the individual chromosomes were cut out and arranged in pairs by size order into a chart, referred to as the karyotype. It is literally a scissors-and-tape operation and, believe it or not, many cytogenetics laboratories still depend chiefly on this method of chromosome analysis. But now an automatic chromosome analyzer—a system that includes a camera, a computer, and a microscope—may radically speed and improve the accuracy of the chromosome views.

Fluorescence in Situ Hybridization Fluorescence in situ hybridization (FISH), a newer method for analyzing chromosomes, uses fluorescent molecules, called dyes, to "paint" genes on a chromosome. This technique is particularly useful for gene mapping and for detecting various chromosomal abnormalities. In this procedure, short sequences of DNA complementary to the sequence of interest, called probes, are hybridized to the sample DNA. Because the probes are labeled with fluorescent tags, a researcher can see the exact location of the DNA sequence of interest on a chromosome. An additional advantage of FISH is that it can be performed on nondividing cells, making it much more versatile than traditional karyotyping.

Scientists can actually create three types of FISH probes, each of which has a different application. Locus-specific probes hybridize to a particular region of a chromosome and are useful for detecting the location of a gene on a chromosome. Alphoid, or centromeric repeat probes, are generated from repetitive sequences found at the centromeres of chromosomes. Because each chromosome can be painted a different color, researchers use these probes to determine whether an individual has the correct number of chromosomes. Whole chromosome probes are actually collections of smaller probes, called libraries, that each hybridize to a different sequence along the same chromosome. Using these libraries, researchers can paint an entire chromosome with various colors, generating what is called a spectral karyotype. These types of probes are useful for examining both large- and small-scale chromosomal abnormalities.

Spectral Karyotyping A new karyotyping method, called spectral karyotyping, uses fluorescent dyes that bind to specific regions of chromosomes. By using a series of specific DNA probes, each with various amounts of the fluorescent dyes attached, different pairs of chromosomes demonstrate unique spectral characteristics. A special feature of this technology is the use of a device called an interferometer, similar to the device used by astronomers for measuring light spectra emitted by stars. Slight variations in color, normally not visible to the human eye, can be detected using a computer program that then reassigns an easy-to-distinguish color to each pair of chromosomes. The result is a digital image in full color, rather than just a photograph. Pairing the chromosomes is now much simpler because homologous pairs are the same color. In addition, chromosomal aberrations are more easily recognizable.

Somatic Cell Hybridization The term "somatic" cell refers to all the cells in an organism that have differentiated into a specific cell type, excluding germ cells, stem cells, and gametes. Somatic cell hybridization is the technique of combining two cells from different tissues or species in a cell culture, typically human and rodent, with the intent of deriving various cell lines, each with a different combination of chromosomes. The hybridized cells fuse and coalesce, but their nuclei generally remain separate. However, during cell division, a single spindle is formed so that each daughter cell has a single nucleus containing sets of chromosomes from each parental line. As hybrid cells grow and divide, they tend to randomly lose many of their chromosomes until they reach a stable point. From there on out, the cell will maintain the same number and species of chromosomes in subsequent divisions. Little is known about the mechanisms behind this process, but for some reason, hybrids between humans and rodents typically shed most of the human chromosomes until only 8 to 12 of the original 46 human chromosomes remain. Yet somehow, these cells can still survive. Through the careful isolation and culture of different hybrid cell lines, researchers can create a whole set of somatic cell hybrids which, together, contain the entire complement of human chromosomes. Researchers can then use these cell lines to screen for the presence or absence of a gene or gene product (protein). For example, a researcher may test the cells' ability to metabolize a particular substance or study traits of antibiotic resistance. If a cell line demonstrates an effect, the researcher can then study the chromosomes present in that particular cell line to identify the gene that confers the desired effect.

Most sequencing and analysis technologies were developed from studies of nonhuman genomes, notably those of the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, the fruit fly Drosophila melanogaster, the roundworm Caenorhabditis elegans, and the laboratory mouse Mus musculus. These simpler systems provide excellent models for developing and testing the procedures needed for studying the much more complex human genome.

A large amount of genetic information has already been derived from these organisms, providing valuable data for the analysis of normal human gene regulation, genetic diseases, and evolutionary processes. For example, researchers have already identified single genes associated with a number of diseases, such as cystic fibrosis. As research progresses, investigators will also uncover the mechanisms for diseases caused by several genes or by single genes interacting with environmental factors. Genetic susceptibilities have been implicated in many major disabling and fatal diseases including heart disease, stroke, diabetes, and several kinds of cancer. The identification of these genes and their proteins will pave the way to more effective therapies and preventive measures. Investigators determining the underlying biology of genome organization and gene regulation will also begin to understand how humans develop, why this process sometimes goes awry, and what changes take place as people age.

Molecular biology is the study of how cells work. It is a branch of biological science that studies the biology of a cell at the molecular level. Molecular biological studies are directed at studying the structure and function of biological macromolecules and the relationship of their functioning to the structure of a cell and its internal components: including nuclei, cell membranes and mitochondria.

Molecular Biology is a research that seeks to understand the molecular basis of life. In particular it relates the structure of specific molecules of biological importance—such as proteins, the nucleic acids DNA and RNA, and enzymes—to their functional role in the intact cell and organism.

The discovery that represents the effective beginning of molecular biology was the discovery of the structure of DNA (deoxyribonucleic acid) by Francis Crick and James Watson in 1953. This was of importance not only because DNA is the molecule that transmits hereditary information from generation to generation (see Genetics: Gene Action), but also because its structure immediately provided an insight into how this was achieved. DNA is a double-stranded helical molecule in which the two single-stranded helices are joined together by bonds between the bases adenine (A), guanine (G), cytosine (C), and thymine (T). In this structure an A in one strand always pairs with a T in the other and a G always pairs with a C. When DNA replicates, the two single strands separate and the information is precisely reproduced as each single strand becomes double-stranded by an A being inserted in the new strand to pair with a T in the old strand, a G being inserted to pair with a C, and so on. In this way the hereditary information, which controls the properties of the cell and organism, is transmitted to daughter cells when a cell divides and to the offspring when an organism reproduces.

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Last modified: May 25, 2005