For many science problems of practical interest, researchers must rely on “approximations”—guesses whose accuracy is questionable—to determine or predict the properties of assorted fluids and solids given different conditions. An alternative approach to approximation is to use molecular simulation, in which equations that predict properties of interest are solved by numerical methods, using a supercomputer. Many academic and industrial chemical engineers are finding applications for molecular simulation in a wide range of scientific and technological problems.<br/><br/>

At Cornell University, these techniques have been applied to problems of practical interest in chemical engineering, such as the storage of methane at high density in a porous material. This problem is important for the storage of natural gas, which is 95 percent methane. If the gas can be stored at low pressure but at high density, it may become desirable and practical to use natural gas as a fuel for cars and other vehicles.<br/><br/>

Keith Gubbins, professor of chemical engineering at Cornell, has studied adsorption of methane on microporous materials at a variety of temperatures and pressures. Adsorption is the take-up of a fluid by a usually porous solid material; a familiar example is the take-up of water by a sponge. In this case the pores of the sponge are very large compared to the water molecules, and the process by which the sponge takes up the water can be understood without having to look at the molecular structures of the sponge or the water.<br/><br/>

However, in studying changes in the properties of methane, Gubbins and his colleagues are attempting to determine both an optimum gas pressure and an optimum pore size for storing methane under different conditions. They have found that the optimum pore size is about three molecular diameters, a little more than one nanometer (about 40 billionths of an inch). Determining that involved understanding the more complex phenomena occurring in materials whose pores are only a few times larger than the fluid molecules themselves. Examples include the adsorption of unwanted or poisonous gases by activated carbon in gas masks and the use of silica gel packets to adsorb moisture in shoe boxes.<br/><br/>

A full understanding of those phenomena, coupled with the ability to control the nature of the pores—their size, shape, and interconnectedness— in experiments, would open the way to a new era in the design of many industrial processes that are based on the use of microporous materials. Such an understanding would be difficult to achieve with experimental techniques alone because of the difficulty of observing these phenomena at the molecular level, inside pores. Modeling these processes using molecular simulation and applying statistical mechanical theories can now complement experiments and can suggest the most profitable areas for study.<br/><br/>

One example of an industrial process that uses microporous materials is the catalytic “cracking” of petroleum (the breakup of large petroleum molecules, called hydrocarbons, into smaller hydro-carbon molecules, which are more suitable for use as fuels). In this process the vaporized petroleum mixture is passed through a bed of microporous alumina particles treated with a solution of a catalyst that speeds up the reaction. Microporous materials, such as activated carbons and silicas, are also frequently used industrially to remove pollutants and impurities from mixtures in which the component to be removed is more readily adsorbed than the other components.<br/><br/>

The most important molecular-simulation techniques Gubbins and his colleagues use are those of molecular dynamics and Monte Carlo. These techniques allow the researchers to simulate the movement of molecules, their speed, and their location within a porous material. The researchers can also incorporate into the simulation different conditions—such as changes in temperature, pressure, and density—and “observe” the interaction between the molecules and the porous material. Super-computing resources, such as those found at the Cornell Theory Center, are necessary to complete these simulations, particularly now that the researchers have begun to parallelize the algorithms involved in their simulation. In parallel computations, many parts of the calculation are performed simultaneously on different processors, in contrast to the more conventional procedure of making these calculations in sequence. Only supercomputers supply a tool for such sophisticated investigations of constant and changing properties of complicated systems, such as water confined in narrow graphite pores. An appropriate combination of a parallel algorithm and a supercomputer can speed up the investigations a hundredfold.<br/><br/>

The potential of molecular simulation as a tool for practical scientific research has expanded rapidly with improvements in the speed and availability of supercomputers. In the investigation of adsorption on porous materials, work has been extended to studies of the more complex fluids, such as water, aqueous solutions, and liquid crystals, and to other porous ceramic materials, including silicas and other oxides.<br/><br/>

To identify the specific grounds in some regions of the genome is required sensor capable subnanometrovoe notice the difference between A, T, G and C. The only physical method that has such a high resolution - scanning tunneling spectroscopy. However, the sequencing of the length of the billions of links are most often used, not the physical and chemical sposoby.Genom man was decoded using the technique developed in the late 1970’s. American biochemist Frederick Sanger. This procedure is preceded by cutting sequencing study of the DNA molecule into fragments, cloning them in E. coli and multiple duplication to obtain millions of copies of each fragment. As a result of the last round of duplication, conducted in special circumstances, receive a set of copies of fragments of various lengths, each of which ends with a fluorescently labeled nucleotide. Fragments are separated by length using electrophoresis, register the light signal from each of them as it passes through the detector and obtain the nucleotide sequence of the original circuit.

The advantages of the method of Sanger include its relative simplicity and high precision, but despite subsequent improvements, it remains expensive and time consuming. The challenge the founders of alternative ways of sequencing was to increase the speed of the procedure and its cheaper. For this it was necessary to exclude the stages of separation, time-consuming, miniaturized the entire system, while preserving the opportunity to read the sequence millions of fragments.

Many research groups have based their developments biosynthesis - a process that uses living organisms when playing its genome and removing it damage. Thus, in the cell prepares to divide, untwist the DNA double helix, its constituent chains apart, and then on each of them is synthesized by a new chain (one - continuously, on the other - is intermittent, with the formation of separate fragments). The process of successive additions to a growing chain of nucleotides is catalyzed by specific enzyme DNA polymerase. Another enzyme, ligase, binds the fragments. The result is two new full length polynucleotide chain complementary to the DNA matrix in which they are synthesized.

Methods for sequencing using the biosynthesis taken as a basis for the stages that process, which occur on a single chain sekveniruemoy DNA. Record the time of accession to the primer hybridized with the DNA-matrix, the complementary nucleotide (extension chain), or the moment matching ligase primer with oligonucleotide probe containing known nucleotide in a certain position.

There are different ways of data acquisition processes, but usually use one of two types of signals. If the label is attached to a nucleotide fluorophores, then recorded it emits light of a certain wavelength. Fluorescence detection is used for sequencing as a method of lengthening the chains, and the method of ligation. It is used by many researchers, among them - Metsker, Michael (Michael Metzker) from the University of Baylor, Robi Mitra (Robi Mitra) from the University of Washington, and led my laboratory at Harvard Medical School and the Corporation Agencourt Bioscience.

However, worship times, and resentment gave way to apprehension. The Old Testament forbade have internal fat of animals - it was supposed to be sacrificed. We have declared war on body fat - just look at the popular women’s and health magazines. They do not prohibit, but advised not to have a lot of fat pets, and promote useful lipids of fish and marine invertebrates of shrimp, sea cucumber, and others. People who eat plenty of seafood, rarely suffer from cardiovascular and other diseases than those whose diet contains a lot of meat and dairy dishes. Nutritionists believe that the whole matter of fat. It would be nice if polyunsaturated fatty acids dovozili seafood to continental cities unoxidised, but, unfortunately, in the air, they quickly and easily oxidized. People living far from the sea, it is necessary to diversify and improve fat menu, lard, nuts and seeds, eggs, birds, fish roe or grape snails and of course, oil.

Modern technology, who know what elements and quantitative analysis, have engaged in fats and oils in the late XVIII century. Antoine Laurent Lavoisier found that these substances are composed mainly of carbon and hydrogen. After that, it became clear why fats contain more energy per unit weight than carbohydrates - the first less oxidized. Karl Scheele discovered that fats contained glycerin. Owner lipidologii Michel Eugène Chevrel since 1811 identified several fatty acids - from oil to stearic acid. In 1812 he opened in gallstones cholesterol and fats in the divided Unsaponifiable and saponificable which are esters of fatty acids and glycerol. In 1823 came his essay “Chemical studies of animal fats.”

In 1884 the English physician John Tudikum published a book “Guide to the chemical composition of the brain”, which wrote that the phospholipids (phosphatides) are the soul of any chemical bioplasm, animal or plant. They are able to perform various functions due to the fact that combine highly contrasting properties. Tudikum explained the universality of the phospholipids of their ability to form colloids, and colloid chemistry had just evolved, and she waited for solving the mystery of life.

In the first half of the XIX and XX century fats or their derivatives were separated, given the different solubility in organic solvents. These methodologies were not accurate, and German scientists called lipidologiyu dirty chemistry. Only 50 years of XX century, chemists have learned to separate the lipids with chromatographic methods, which enabled them to describe systematically and reliably identify.

Two functions of fats, fuel and construction, for a long time obscured the rest. First, scientists realized that fats are the fuel. Directly in turn, give energy, fatty acids are involved - the hydrocarbon tails, similar to the molecules of gasoline and kerosene, only longer and with acid-head (Figure 1). And for transport and storage in adipose tissue fatty acids coupled to a hydrophilic molecule of glycerol. These are called neutral fats, or triglycerides. Along with proteins and other lipids, they form a transport form - lipoproteins, which are located on the periphery of the polar groups, and in the center - fat tails.

Fat, in contrast to carbohydrates, - long-play fuel. Prolonged stress, exhausting physical work a person begins to use less carbohydrates and more lipids. When work is finished or the marathon run, the chambers are beginning to enter fats obtained from food and re-synthesized. Stockpiles are not always pleased with their owners, and the struggle with excess - an exhausting exercise, but popular.

One of the most important developments in physiology in recent years - the opening of the peptide hormone leptin, which is responsible for fat deposition and appetite. It regulates not quick reaction to saturation and slow - the overall amount of adipose tissue. When pantries are full of body fat cells of adipose tissues (adipocytes) and overblown generate a lot of leptin. It binds to receptors in the hypothalamus (if they are in the order), that produces some other substance, and is not hungry. This fat is not delayed, and fatty acids are oxidized rapidly. Leptin increases within a few hours after a meal in rodents and after several days of abundant food in humans, but decreases for several hours of fasting in both.

Ob gene is leptin, discovered in 1994. Mice homozygous for the mutant gene, obese and binge-eating, spending little energy, hard to bear the cold, were often fruitless.

Injections of leptin lead only to a decrease in adipose tissue, muscle and internal organs without atrophy. Obese regulation is violated. The level of leptin in their blood increased, but the function of the brakes it does not perform, and I’m hungry anyway. Probably, appetite control and other regulators. Nevertheless, in 1998, reported the successful treatment of leptin ozhirevshih mice.

Fuel - is certainly important, but the lipids are needed for the body and other problems. One of them - the formation of external and internal cell membranes. The tails of lipid molecules do not interact with the water, gather together, unite the ranks, turning his head toward the water, and form a film (Fig. 2). This is done not only triglycerides but also the polar phospholipids, carry negatively charged phosphate group (Fig. 3), and cholesterol and other substances. Cholesterol though not related to lipids in the strict sense, but is usually seen with them. It gives the necessary rigidity of the membranes.

In disputes about how to begin life, lipids occupy a special place. Until molecules floating freely in open water, talking about life and the organisms do not have to. When the membrane appeared, they separated the contents of cells from the outer space, inside began to accumulate the necessary ions and small molecules whirled cycles of reactions, and life arose. Several important steps in its evolution was associated with membranes, which divided the cells into compartments. Hereditary material segregated and formed a nucleus having organelles - mitochondria, chloroplasts, lysosomes, Golgi apparatus, endoplasmic reticulum. Supplied to all these economies are called eukaryotic cells - have this kernel.

The membranes made of lipids and proteins, perform several functions. They regulate the penetration of substances into the cell and back, and transmit signals that are responsible for conducting nerve impulses, energy production in mitochondria and chloroplasts. For the work of membrane proteins is very important to lipid environment, so it is carefully regulated. When the rising or falling temperature, the properties of the membrane are also changing: there is a phase transition. Therefore, to life in cold water, lipid composition have to adjust to the membrane does not become too rigid. With this and the associated large number of polyunsaturated fatty acids in marine animals.

In the 70 years scientists have learned to do from the lipids of microscopic bubbles and fill them with stuffing, such as medicine. Such capsules called liposomes - fatty calves. They can be equipped with molecules that target certain cells, and they will deliver the stuffing only there. And you can do from liposomes cosmetic cream to enter into the skin useful for her substance.

Lipids and related substances on them are also present in the nucleus. Some of them (especially cardiolipin (Fig. 4), and cholesterol) are associated with DNA and are involved in the regulation of genes. The composition of these lipids varies depending on the activity of the genome, the phases of the cell cycle transition of DNA from supercoiled to relaxed form, as well as pathology - when a cell becomes malignant.

This is just one example of the involvement of lipids in the information processes. Another, much more famous - conducting a nerve impulse spikes of neurons. Supporting this process in the nervous tissue and especially in the brain involved in many complex and interesting lipids: sphingomyelin, cerebrosides, gangliosides, glycolipids, and others. However, the membrane conduct and other signals. Thus, the flow of calcium into the muscle cells start to diminish through the membrane proteins in the cell are transmitted hormonal messages. The work of all these and many other mechanisms depends on the composition of lipids

• Find something that contains a lot of DNA. For example, green peas (but may be chicken liver, herring roe, or onion).
• Put in a blender about 100 ml (half a cup) of this product, add 1 / 8 teaspoon of salt and 200 ml (cup) of cold water. Whips within 15 seconds. Mixer «cook» you pea-cell soup.
• Strain the mixture through a strainer or a piece of nylon (stockings quite fit). In the obtained pulp, add 1 / 6 of its number (it will be about 2 tablespoons) of liquid detergent (for dishes, for example) and stir well. Leave on for 5-10 minutes.
• Pour the liquid in test tube or other glass vessel, so that each was filled with no more than a third volume.
• Add to each tube for a bit, or the juice squeezed out of a pineapple, a contact lens solution and gently shake it, turning and bending the tube (if you shake too vigorously, break up DNA and did not see it).
• Tilt the tube and slowly pour into it a little bit of ethyl alcohol, that it formed a layer on top of pea mixture. Leite, while a mixture of alcohol and not be a tie. DNA pops up in the form of flakes.
• Wooden stick (pencil) to catch them and examine under a microscope.

Of course, for researchers this instruction - to some extent a joke and none of them in this way the DNA does not evolve, but in the meantime if you really use it, then everything will turn out! Output DNA is, however, is small, and the substance - not particularly clean, but seen through a microscope long thin filaments - crystals of DNA technology - it is quite possible.

What happens with green peas or chicken liver in the process described manipulation and why eventually the DNA is separated from all other substances, which in a great many cells?

1. Select object

DNA is known to have in each cell, and thus make it possible from any fabric - even from animal bones, fish scales, or wood, where the cells are not so much in comparison with the volume of extracellular substances.

In all tissues of both animals and plants, DNA is usually the same. Differences between the tissues that in some of them more than the substance of heredity almost nothing (herring roe), while in others, such as bone tissue, DNA content is relatively low. In addition, there are tissues in which cells have a doubled set of chromosomes (tetraploid to include, in particular, liver cells), and therefore the DNA in them two times more than all the rest. In seed plants relative, DNA content higher than in the stem, and from the young growing shoots it is possible to allocate substantially more than the same volume of a piece of lignified stem.

In general, if the researcher is not worth a special problem, it tries to select for tissue in which little intercellular substance and a lot of cells themselves. It is desirable that the fabric easily fall into these components, and the cells were not overloaded with protein (like muscle), lipids (as lipid) or polysaccharides (such as brain cells).

2. Fractions tissue cells

In blender fabric from which we are going to get the substance of heredity, is divided into separate cells: in order to mechanically break the connection between them is usually required much less effort than to damage the cell itself. And as with our method of DNA isolation require more or less whole, intact cells, it is clear that canned peas or salted herring for such experiments do not work - better to take something frozen, if you are sure that the product is thawed during storage several times.

A little salt should be added to the solution so that the cells do not burst ahead of time: the internal pressure of the contents of the cell membrane to equalize the pressure inside a saline solution from the outside.

3. Macromolecule releases

As for filtration, it is necessary to mechanically remove from the cell suspension of various impurities, including large pieces of fabric - all the same substances that we are going to handle the mixture will not be able to penetrate deep into these conglomerates, and to extract DNA will be useless.

A process must be received by the cells in the first place, some detergent. Tool “Ferry”, capable, according to advertising, it is easy to clean the most greasy dishes, fit and in order to make many holes in the lipid membrane of both the cell and its nucleus. If no liquid detergent, you can make a concentrated solution of detergent - is also nice.

As a result of processing all the cellular contents and would be hung out in solution, which is done with this very sticky, viscid and significantly more transparent than was the cell suspension. Changing the consistency of the solution - a sure sign that the lysis was successful.

4. Everything is clear and no comments —

do not pour too much mud in a container, there is still much to pour, in addition, if the mixture will be in excess, it will be difficult to mix.

5. Freed from proteins

Why just not in our mix! However, the proteins here - most of all, and they form the most stable complexes with DNA. There are techniques where proteins are removed from the solution in several stages. For example, some of them easily denatures and precipitates by adding concentrated solutions of salts. Under laboratory conditions, these methods work fine, but researchers are exempt from the draft, putting the tube for several minutes in a centrifuge. After that, all more or less large cell debris, denatured proteins and other impurities are at the bottom, forming a very dense cake, and poured into another test tube supernatant containing mainly nucleic acids - DNA and RNA - is not working. But at home, this purification step we have to miss, we are interested in donating part of the substance - it will remain a “prisoner of the protein.

We will immediately proceed to the purification of DNA from residual proteins with special enzymes that can destroy these molecules. It is such a substance contains pineapple juice. They themselves - the same protein, so the pineapple, from which the pressed juice must be fresh: the enzyme is not the slightest chance to survive intact in compote or canned food. As a solution for cleaning lenses, if you intend to use it - do not forget to put a pill to remove protein deposits! Themselves, the solutions for storing contact lenses are no active ingredients do not contain - otherwise our eyes and not be faint.

The fact that the enzymes worked, you can try to reduce the viscosity of the solution. If not, place the mixture in a warm place (about 37 ° C) for half an hour, sometimes you may need to add more pineapple juice or a solution for cleaning lenses.

6. DNA precipitated from solution

Now DNA floating in the solution by itself. Proteins do not cling to it, though the wreckage of various molecules in the mixture is still a lot. Under laboratory conditions, these unwanted parts removed, thoroughly mixing the solution with phenol and / or chloroform. Organic solvents that can pick up proteins on a “heavier than water, and therefore the subsequent stratification of the mixture in a centrifuge, they sink to the bottom. After centrifugation the bottom of the tube are phenol and / or chloroform with dissolved proteins in them, and at the top - the water phase containing DNA. The aqueous phase is collected in a separate tube and more are already working with a relatively clean solution.

In the absence of the centrifuge and organic solvents, which requires work to the same special security measures, this stage of treatment at home have missed and to precipitate the DNA directly from the “dirty” solution.

We note at once - to replace the ethyl alcohol vodka or spirits can not be: if the concentration of alcohol is low and falls when mixed with an aqueous phase up to 60-65% of DNA in the crystalline state can not pass. Partly for this reason to pour alcohol into a test tube with the DNA-containing mixture should be carefully layering it on top. Then the lower layers of partially mixed with alcohol solution of DNA, will begin the process of crystallization of nucleic acids, and they come to the surface (where alcohol is more concentrated) in the form of flakes.

If you pour alcohol on top do not get all hopelessly mixed up, then the small amount of ethanol you do not succeed, but when a large starts to crystallize not only DNA: a precipitate fall out and the remnants of proteins, and something else from the original content of the cells.

7. What have we got?

Pure crystals of DNA are like balls of tangled fibers, but we must not forget that you can see the crystals of substance rather than its macromolecules, and say to their appearance, which contains genes isolated nucleic acid by you, of course, impossible. To find out, would have to dissolve the DNA. However, “read” the sequence of nucleotides in the home, alas, is impossible: it requires not only special devices, but also expensive reagents.

However, if you are already well-reviewed crystals and they managed to dry out, you can observe how the DNA is dissolved. It is the beginning swells and becomes jelly-like jellyfish, and only a few days later, the solution is homogeneous. The process can be accelerated if the tube often shake.

Different layers of the skin vary considerably in its content: the share of the epidermis is about 10%, papillary layer - 71-72%. net dermis - about 61%. Normal cells activity is under constant ionic composition and pH of body fluids. In the water, readily soluble chemical compounds containing polar groups and capable of entering into the dipole-dipole interactions with water molecules or form with hydrogen bonds (-OH,-NH, C = 0): non-polar hydrocarbon molecules poorly or not soluble in it.

On the mineral science components of the skin accounts for between 0.7 to 1% of dry weight of the skin, and subcutaneous tissue - about 0,5% of its dry weight. The skin is an important depot of cations - sodium, potassium, calcium and magnesium. Sodium is the major extracellular cation in the human body. However, potassium ions, it participates in the regulation of water-electrolyte and acid-base balance. In the skin of sodium contained mainly in the intercellular space, and potassium (75%>) - in the cell cytoplasm. Calcium is contained mainly in the dermis and is involved in the activation of synthesis of prostaglandins. Magnesium also is an intracellular cation, they are the richest in the epidermis. Magnesium is involved in the activation of kinases in phosphorylation reactions.
Important role in biological systems of the body are phosphates. Phosphorus is in the cell mainly in the form of organic compounds - phosphoric, nucleoproteins, adenosine phosphates, etc. Sulfur is part of the cysteine and methionine - amino acids involved in the formation of keratin, and is contained mainly in the stratum corneum of the skin, hair and nails.

For dermatology great interest elements such as copper, zinc and iron that make up enzymes, vitamins, and play the role of activators of biological processes. For example, a tyrosinase, copper is involved in the synthesis of melanin, a lysyl oxidase - the exchange of elastin and collagen, through tioloksidazu - in the process of keratinization, iron is an integral part of hemoglobin, myoglobin, peroxidase and cytochrome providing cellular respiration.

The most important chemical component of the skin is a protein - a polypeptide formed by the condensation of amino acids. In the skin contains the structural proteins: collagen, retikulin, elastin and keratin. The main structural protein of skin collagen is contained mainly in the dermis (about 70% devoid of water of the skin), and retikulin and elastin contained in the skin in much smaller quantities, form the basis of the reticular and elastic fibers of the dermis, the connective tissue membranes of the sebaceous and sweat glands, are of the hair follicles. Keratin is the basis of the stratum corneum. Its synthesis begins in the basal keratinocytes in the form prekeratina, which has a lower molecular weight compared with mature keratin. He has no intra-and mezhtsepochechnyh disulfide bonds, giving the molecules of keratin strength and insolubility. In the lower ranks of the stratum corneum prekeratin under the influence of specific enzymes into a mature keratin. In this case between the individual molecules within them formed disulfide bonds at the expense of the keratin acquires strength and loses solubility. Special protein - filagrin - causes the aggregation of keratin filaments. As the synthesis filagrin keratogialinovyh accumulates in the form of granules, and there are up to as long as tightly packed keratin stabilizes strong disulfide bonds. Then filagrin in korneotsitah decomposes to free amino acids.

Contents of the decay products of protein (urea, uric acid, amino acids, ammonia, etc.) in the skin, almost 3 times higher than their levels in the blood, it becomes even higher in the pathologically changed skin areas with the prevalence of decay processes. Much of the skin cells, like other cells of the body (especially their nuclei), are nucleoproteins and nucleic acids (DNA, RNA). In the skin of DNA and RNA are contained mainly in the epidermis.

The share of the skin accounts for about 20% of the total carbohydrate metabolism. In the epidermis the concentration of glucose is around 30-60 mg%, and glycogen - 70-80 mg%. Despite the small number (approximately 0,1%), glycogen is an important source of energy for the processes of cell division and keratinization. In the skin of adult human glycogen is contained mainly in the thorny and basal layers of the epidermis. Glycosaminoglycans (mukopolisaharily), having a high viscosity, promote cells binding together. Structure and functions of the skin play a major role acidic mucopolysaccharides: hyaluronic, chondroitinsulphuric acid and heparin. When depolymerization of mucopolysaccharides (eg, with an increase in hyaluronidase activity) reduces the viscosity of gels formed by them, and thereby increases the permeability of the skin tissues to microbes and various toxic products. Heparin is formed in the skin and accumulates in fat cells and plays an important role in the regulation of microcirculatory processes.

The skin is rich in proteoglycans, consisting of polysaccharide (95%) and protein (5%) components. As polyanions, they bind water and cations, forming the main substance of connective tissue.

As in the skin, and on its surface contains a variety of lipids. Lipids of the epidermis contain 20% of free fatty acids, triglycerides 17%, 6% mono-and diglycerides, 16% cholesterol. The bulk of the subcutaneous adipose tissue are neutral fats. They dominated most low-melting triglycerides - triolein (70%), in connection with which human fat has the lowest melting point.

Other lipids (sterols, steroids and phospholipids) are contained in the cells of the epidermis and connective tissue in the walls of blood vessels and in smooth muscles and especially in the secretion of sebaceous glands. The skin surface lipids are mixed to form sebum. The fat content increases after puberty and decreases with age.

The skin contains a large number of enzymes, the most important of which are amylase, phosphorylase, aldolase, lactic acid dehydrogenase, succinic acid dehydrogenase, cytochrome oxidase, transaminase, arginase, lipase, tyrosinase, etc.
Human skin contains a large number of antigens (some types of collagen, nuclear antigens, antigens of endothelial cells, fibroblasts and structure, etc.). In common diseases and skin diseases in relation to them can produce antibodies or autoantibodies. Their detection is used for diagnosis and prognosis of the disease.

Linear RCAT (L-RCAT)
One end of a DNA probe is joined to the desired target.
The other end is hybridized to a generic DNA circle.

An enzyme catalyst is added. Starting at the probe for the circle, the enzyme catalyst makes a copy of the DNA circle, returns to the starting point and continues to copy the circle.

A single product is thus generated that is composed of copy after copy of the circle like a thread unraveling from a spool.

Within several hours RCAT produces thousands of copies of the circle as a single ribbon of DNA at a rate of 55 nucleotides per second.

The newly synthesized, long ribbon of linked-up copies of the circle that is the product of the RCAT reaction folds itself up into a tiny speck that is attached to the target. When fluorescent detector molecules are used, the result is an intensely bright signal at the location of each single target biomolecule.

Copies of the circle remain linked to the target, not dispersed in solution or distributed throughout samples.

Exponential RCAT (E-RCAT)

A second short DNA probe, identical in sequence to part of the DNA circle is added.

Upon incubation, each newly formed linear copy of the circle is recognized by this second DNA probe, which, in turn makes copies of the RCAT product.

In this way, amplification and displacement take place both around the circle and on the RCAT product.

The resultant amplified material is a so-called hyper-branched product that is produced in exponentially increasing amounts.

Multiply Primed RCAT (M-RCAT)

Oligonucleotide primers complementary to the amplification target circle are hybridized to a circular DNA template generating multiple replication forks.

Polymerase is added and RCAT proceeds by displacing the non-template strand. Product strands are rolled off the template as tandem copies of the circle.

Priming allows synthesis of both strands resulting in double-stranded product.

A cascade of priming events results in exponential (or hyper-branched) amplification.

Alliances
We are actively pursuing partnerships in the in vitro diagnostic industry as well as key additional strategic areas. These alliances will enable us to rapidly expand the applications for Rolling Circle Amplification Technology and the other technology platforms at Molecular Staging.

Financing
MSI has received financing from Domain Associates, Biotechnology Investments Limited, and Collinson, Howe & Lennox, LLC as well as from Amersham Pharmacia Biotech. For more information,
please contact:

Molecular Staging Inc.
Business Development
300 George Street
info@molecularstaging.com

Voice (203)772-5000
FAX (203)776 5276

We seek experienced and motivated Research Scientists and Research Associates with training in the areas of Immunoassay Development, Microarray & Robotics, DNA Diagnostics & In Situ Hybridization, Protein & Nucleic Acid Chemistry, and Molecular Biology.

Successful candidates will have excellent research, interpersonal and communication skills. We are located in New Haven, Connecticut near several major centers for biological research and biotechnology including the Yale University School of Medicine in Connecticut. For confidential consideration, please send your resume by e-mail to jobs@molecularstaging.com.

“This procedure, which removes the need for lengthy growth periods and traditional DNA isolation procedures, will deliver operational efficiency gains for DNA sequencing centers and genomics companies” said Dr. Stephen Kingsmore, COO of Molecular Staging Inc.

The method is very simple, involving only a few steps, namely heat lysis of the organism containing the DNA of interest and an isothermal amplification step.

“Amersham Pharmacia Biotech have already enabled huge productivity gains in preparation of template DNA for the DNA sequencing market with their new TempliPhiTM DNA Sequencing Template Amplification Kit, which is based on this procedure. We believe this procedure will also deliver additional first time benefits across a wide range of applications.”

The paper, Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification, published in the June issue of Genome Research 11:1095-1099, describes a rapid, scalable method for the amplification of circular DNA directly from bacterial cells or from viral plaques, generating high quality templates for use in DNA sequencing, probe generation or cloning.

“Sample preparation procedures for all manner of circular DNA such as plasmid, phage and mitochondrial DNA, can now be made significantly simpler, faster and more cost effective” said Dr. Kingsmore.

Use of a high fidelity DNA polymerase, Phi29 from Amersham Pharmacia Biotech, ensures a very low error rate of 1 in 106 – 107 bases. This feature will be valuable for amplification of genomic DNA or cDNA where accuracy is paramount.

The procedure effectively amplifies even large DNA circles such as bacterial artificial chromosomes (BACs) and cosmids. Unlike PCR this method does not appear to be limited by target length. In addition, the procedure holds promise for obtaining DNA sequencing templates from unculturable organisms as well as in vitro propagation of unclonable circular templates. This will greatly expand the number of organisms in the world from which new genes can be isolated and studied.

The authors of the Genome Research paper are Molecular Staging Inc. scientists Frank Dean and Roger Laskin, together with John Nelson and Theresa Giesler of Amersham Pharmacia Biotech.

Molecular Staging Inc. (MSI) is a life science company developing a portfolio of technologies for detection and measurement of proteins and nucleic acids across a broad range of applications, including proteomics, genomics, pharmacogenomics and diagnostics. MSI’s core technology is Rolling Circle Amplification Technology TM (RCATTM), a proprietary amplification process that has significant advantages in terms of sensitivity, multiplexing, dynamic range and scalability. Additional information about MSI can be found at www.molecularstaging.com.

Motorola Inc. and MSI entered into a license option agreement related to the use of RCAT™ on clinical diagnostic microarrays, November 7, 2000, whereby Motorola Inc. paid certain research • fees and made an equity investment in MSI. The agreement provided for an evaluation period during which Motorola Inc and MSI would work together to meet specific technical milestones.

Nick Naclerio, Vice President and General Manager of Motorola BioChip Systems, a division of Motorola Inc. confirmed that the technical work performed by MSI was outstanding.

Torben Christensen, President and CEO of Molecular Staging said, “We are delighted with the outcome of the evaluation program undertaken by teams of scientists from MSI and Motorola Biochip Systems. MSI’s technology achieved and in some cases surpassed the milestones set for the program. The decision by the Motorola Life Science division to pursue Rolling Circle Amplification Technology for a number of research applications once again validates the substantial potential of this technology for on chip amplification”.

Both Dr. Naclerio and Mr. Christensen stated that they would welcome an opportunity for the two organizations to work together in other areas of common interest.

About Molecular Staging Inc.
Molecular Staging Inc. (MSI) is a life science company developing a portfolio of technologies for detection and measurement of proteins and nucleic acids across a broad range of applications, including proteomics, genomics, pharmacagegenomics and diagnostics. MSI’s core technology is Rolling Circle Amplification Technology (RCAT), a proprietary amplification process that has significant advantages in terms of sensitivity, multiplexing, dynamic range and scalability. Additional information about MSI can be found at www.molecularstaging.com

About Motorola Inc.
Motorola Life Sciences was established in 1998 to develop products which enable the delivery of better healthcare through the understanding and practical application of genomics. The products under development at Motorola Life Sciences will enable scientists and healthcare professionals to quickly and accurately analyze the DNA, RNA and proteins in living cells. In the future, the outcomes from this work will lead to the delivery of faster and more individualized healthcare. For more information, visit Motorola at www.motorola.com/biochipsystems