1.
DNA sequencing
–
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, the advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography, following the development of fluorescence-based sequencing methods with a DNA sequencer, DNA sequencing has become easier and orders of magnitude faster. DNA sequencing may be used to determine the sequence of genes, larger genetic regions, full chromosomes or entire genomes. DNA sequencing is also the most efficient way to sequence RNA or proteins, in fact, DNA sequencing has become a key technology in many areas of biology and other sciences such as medicine, forensics, or anthropology. Sequencing is used in biology to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes, associations with diseases and phenotypes, the field of metagenomics involves identification of organisms present in a body of water, sewage, dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in an environment is critical to research in ecology, epidemiology, microbiology. Sequencing enables researchers to determine which types of microbes may be present in a microbiome, medical technicians may sequence genes from patients to determine if there is risk of genetic diseases. This is a form of testing, though some genetic tests may not involve DNA sequencing. DNA sequencing may be used along with DNA profiling methods for forensic identification, the canonical structure of DNA has four bases, thymine, adenine, cytosine, and guanine. DNA sequencing is the determination of the order of these bases in a molecule of DNA. However, there are other bases that may be present in a molecule. In some viruses, cytosine may be replaced by hydroxy methyl or hydroxy methyl glucose cytosine, in mammalian DNA, variant bases with methyl groups or phosphosulfate may be found. Depending on the technique, a particular modification, e. g. the 5mC common in humans. This situation changed after 1944 as a result of experiments by Oswald Avery, Colin MacLeod. This was the first time that DNA was shown capable of transforming the properties of cells, in 1953, James Watson and Francis Crick put forward their double-helix model of DNA, based on crystallized X-ray structures being studied by Rosalind Franklin. According to the model, DNA is composed of two strands of nucleotides coiled around each other, linked together by bonds and running in opposite directions
2.
Fluorescence
–
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence, in most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. Fluorescent materials cease to glow immediately when the radiation source stops, unlike phosphorescence, Fluorescence also occurs frequently in nature in some minerals and in various biological states in many branches of the animal kingdom. An early observation of fluorescence was described in 1560 by Bernardino de Sahagún and it was derived from the wood of two tree species, Pterocarpus indicus and Eysenhardtia polystachya. The chemical compound responsible for this fluorescence is matlaline, which is the product of one of the flavonoids found in this wood. The name was derived from the fluorite, some examples of which contain traces of divalent europium. In a key experiment he used a prism to isolate ultraviolet radiation from sunlight, the specific frequencies of exciting and emitted light are dependent on the particular system. S0 is called the state of the fluorophore, and S1 is its first excited singlet state. A molecule in S1 can relax by various competing pathways and it can undergo non-radiative relaxation in which the excitation energy is dissipated as heat to the solvent. Excited organic molecules can also relax via conversion to a triplet state, relaxation from S1 can also occur through interaction with a second molecule through fluorescence quenching. Molecular oxygen is an extremely efficient quencher of fluorescence just because of its unusual triplet ground state, in most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation, this phenomenon is known as the Stokes shift. The emitted radiation may also be of the wavelength as the absorbed radiation. Molecules that are excited through light absorption or via a different process can transfer energy to a second sensitized molecule, the fluorescence quantum yield gives the efficiency of the fluorescence process. It is defined as the ratio of the number of photons emitted to the number of photons absorbed, Φ = Number of photons emitted Number of photons absorbed The maximum fluorescence quantum yield is 1.0, each photon absorbed results in a photon emitted. Compounds with quantum yields of 0.10 are still considered quite fluorescent, thus, if the rate of any pathway changes, both the excited state lifetime and the fluorescence quantum yield will be affected. Fluorescence quantum yields are measured by comparison to a standard, the quinine salt quinine sulfate in a sulfuric acid solution is a common fluorescence standard. The fluorescence lifetime refers to the time the molecule stays in its excited state before emitting a photon. This is an instance of exponential decay, various radiative and non-radiative processes can de-populate the excited state
3.
Radiography
–
To create the image, a heterogeneous beam of X-rays is produced by an X-ray generator and is projected toward the object. A certain amount of X-ray is absorbed by the object, which is dependent on the particular density, the X-rays that pass through the object are captured behind the object by a detector. The detector can then provide a superimposed 2D representation of all the internal structures. Contrast radiography uses a radiocontrast agent, a type of contrast medium, to make the structures of interest stand out visually from their background, each type is best suited to certain indications. In tomography, the X-ray source and detector move to blur out structures not in the focal plane, conventional tomography is rarely used now having been replaced by CT. Computed tomography, unlike flat film tomography, generates 3D representations used for computer-assisted reconstruction, the role of the Radiographer has changed dramatically as a result of more advanced equipment. Röntgen referred to the radiation as X, to indicate that it was a type of radiation. He received the first Nobel Prize in Physics for his discovery and he noticed a faint green glow from the screen, about 1 metre away. Röntgen realized some invisible rays coming from the tube were passing through the cardboard to make the screen glow, Röntgen discovered X-rays medical use when he made a picture of his wifes hand on a photographic plate formed due to X-rays. The photograph of his wifes hand was the first ever photograph of a body part using X-rays. When she saw the picture, she said, I have seen my death, the first use of X-rays under clinical conditions was by John Hall-Edwards in Birmingham, England on 11 January 1896, when he radiographed a needle stuck in the hand of an associate. On 14 February 1896, Hall-Edwards also became the first to use X-rays in a surgical operation, the United States saw its first medical X-ray obtained using a discharge tube of Ivan Pulyuis design. In January 1896, on reading of Röntgens discovery, Frank Austin of Dartmouth College tested all of the tubes in the physics laboratory. This was a result of Pulyuis inclusion of a target of mica, used for holding samples of fluorescent material. Indeed, Marie Curie pushed for radiography to be used to treat wounded soldiers in World War I, initially, many kinds of staff conducted radiography in hospitals, including Physicists, Photographers, Physicians, Nurses, and Engineers. The medical speciality of radiology grew up over many years around the new technology, when new diagnostic tests were developed, it was natural for the Radiographers to be trained in and to adopt this new technology. Radiographers now perform fluoroscopy, computed tomography, mammography, ultrasound, nuclear medicine, although a nonspecialist dictionary might define radiography quite narrowly as taking X-ray images, this has long been only part of the work of X-ray Departments, Radiographers, and Radiologists. Initially, radiographs were known as roentgenograms, while Skiagrapher was used until about 1918 to mean Radiographer, a number of sources of X-ray photons have been used, these include X-ray generators, betatrons, and linear accelerators
4.
Wavelength
–
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the waves shape repeats, and thus the inverse of the spatial frequency. Wavelength is commonly designated by the Greek letter lambda, the concept can also be applied to periodic waves of non-sinusoidal shape. The term wavelength is also applied to modulated waves. Wavelength depends on the medium that a wave travels through, examples of wave-like phenomena are sound waves, light, water waves and periodic electrical signals in a conductor. A sound wave is a variation in air pressure, while in light and other electromagnetic radiation the strength of the electric, water waves are variations in the height of a body of water. In a crystal lattice vibration, atomic positions vary, wavelength is a measure of the distance between repetitions of a shape feature such as peaks, valleys, or zero-crossings, not a measure of how far any given particle moves. For example, in waves over deep water a particle near the waters surface moves in a circle of the same diameter as the wave height. The range of wavelengths or frequencies for wave phenomena is called a spectrum, the name originated with the visible light spectrum but now can be applied to the entire electromagnetic spectrum as well as to a sound spectrum or vibration spectrum. In linear media, any pattern can be described in terms of the independent propagation of sinusoidal components. The wavelength λ of a sinusoidal waveform traveling at constant speed v is given by λ = v f, in a dispersive medium, the phase speed itself depends upon the frequency of the wave, making the relationship between wavelength and frequency nonlinear. In the case of electromagnetic radiation—such as light—in free space, the speed is the speed of light. Thus the wavelength of a 100 MHz electromagnetic wave is about, the wavelength of visible light ranges from deep red, roughly 700 nm, to violet, roughly 400 nm. For sound waves in air, the speed of sound is 343 m/s, the wavelengths of sound frequencies audible to the human ear are thus between approximately 17 m and 17 mm, respectively. Note that the wavelengths in audible sound are much longer than those in visible light, a standing wave is an undulatory motion that stays in one place. A sinusoidal standing wave includes stationary points of no motion, called nodes, the upper figure shows three standing waves in a box. The walls of the box are considered to require the wave to have nodes at the walls of the box determining which wavelengths are allowed, the stationary wave can be viewed as the sum of two traveling sinusoidal waves of oppositely directed velocities. Consequently, wavelength, period, and wave velocity are related just as for a traveling wave, for example, the speed of light can be determined from observation of standing waves in a metal box containing an ideal vacuum. In that case, the k, the magnitude of k, is still in the same relationship with wavelength as shown above
5.
Cloning
–
In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments, cells, the term also refers to the production of multiple copies of a product such as digital media or software. The term clone, invented by J. B. S. Haldane, is derived from the Ancient Greek word κλών klōn, twig, referring to the process whereby a new plant can be created from a twig. In horticulture, the spelling clon was used until the twentieth century, since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively. In botany, the term lusus was traditionally used, Cloning is a natural form of reproduction that has allowed life forms to spread for more than 50 thousand years. It is the method used by plants, fungi, and bacteria. Examples of these organisms include blueberry plants, hazel trees, the Pando trees, the Kentucky coffeetree, Myricas, molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes and it is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence, initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a procedure is used where the amplified fragment is inserted into a vector. The vector is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase, following ligation the vector with the insert of interest is transfected into cells. A number of techniques are available, such as chemical sensitivation of cells, electroporation, optical injection. Finally, the cells are cultured. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained, further investigation of the resulting colonies must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing, Cloning a cell means to derive a population of cells from a single cell. In the case of organisms such as bacteria and yeast. However, in the case of cell cultures from multi-cellular organisms, a useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings
6.
Thermal cycler
–
The thermal cycler is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction. Thermal cyclers may also be used in laboratories to facilitate other temperature-sensitive reactions, the device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, the earliest thermal cyclers were designed for use with the Klenow fragment of DNA polymerase I. Since this enzyme is destroyed during each heating step of the amplification process and this led to a cumbersome machine based on an automated pipettor, with open reaction tubes. Later, the PCR process was adapted to the use of thermostable DNA polymerase from Thermus aquaticus, while in some old machines the block is submerged in an oil bath to control temperature, in modern PCR machines a Peltier element is commonly used. Quality thermal cyclers often contain blocks to achieve fast temperature changes. Other cyclers have multiple blocks with high capacity, each of which is kept at a constant temperature. Miniaturized thermal cyclers have been created in which the reaction mixture moves via channel through hot, thermal cyclers designed for quantitative PCR have optical systems which enable fluorescence to be monitored during reaction cycling. Modern thermal cyclers are equipped with a lid that presses against the lids of the reaction tubes. This prevents condensation of water from the reaction mixtures on the insides of the lids, traditionally, a layer of mineral oil was used for this purpose. Some thermal cyclers are equipped with multiple blocks allowing several different PCR reactions to be carried out simultaneously, some models also have a gradient function to allow for different temperatures in different parts of the block. This is particularly useful when testing suitable annealing temperatures for PCR primers, thermal cyclers are traditionally very expensive, and typically cost in the thousands of dollars. However, mass production and recent developments in hardware designs
7.
Hour
–
An hour is a unit of time conventionally reckoned as 1⁄24 of a day and scientifically reckoned as 3, 599–3,601 seconds, depending on conditions. The seasonal, temporal, or unequal hour was established in the ancient Near East as 1⁄12 of the night or daytime, such hours varied by season, latitude, and weather. It was subsequently divided into 60 minutes, each of 60 seconds, the modern English word hour is a development of the Anglo-Norman houre and Middle English ure, first attested in the 13th century. It displaced the Old English tide and stound, the Anglo-Norman term was a borrowing of Old French ure, a variant of ore, which derived from Latin hōra and Greek hṓrā. Like Old English tīd and stund, hṓrā was originally a word for any span of time, including seasons. Its Proto-Indo-European root has been reconstructed as *yeh₁-, making hour distantly cognate with year, the time of day is typically expressed in English in terms of hours. Whole hours on a 12-hour clock are expressed using the contracted phrase oclock, Hours on a 24-hour clock are expressed as hundred or hundred hours. Fifteen and thirty minutes past the hour is expressed as a quarter past or after and half past, respectively, fifteen minutes before the hour may be expressed as a quarter to, of, till, or before the hour. Sumerian and Babylonian hours divided the day and night into 24 equal hours, the ancient Egyptians began dividing the night into wnwt at some time before the compilation of the Dynasty V Pyramid Texts in the 24th century BC. By 2150 BC, diagrams of stars inside Egyptian coffin lids—variously known as diagonal calendars or star clocks—attest that there were exactly 12 of these. The coffin diagrams show that the Egyptians took note of the risings of 36 stars or constellations. Each night, the rising of eleven of these decans were noted, the original decans used by the Egyptians would have fallen noticeably out of their proper places over a span of several centuries. By the time of Amenhotep III, the priests at Karnak were using water clocks to determine the hours and these were filled to the brim at sunset and the hour determined by comparing the water level against one of its twelve gauges, one for each month of the year. During the New Kingdom, another system of decans was used, the later division of the day into 12 hours was accomplished by sundials marked with ten equal divisions. The morning and evening periods when the failed to note time were observed as the first and last hours. The Egyptian hours were closely connected both with the priesthood of the gods and with their divine services, by the New Kingdom, each hour was conceived as a specific region of the sky or underworld through which Ras solar bark travelled. Protective deities were assigned to each and were used as the names of the hours, as the protectors and resurrectors of the sun, the goddesses of the night hours were considered to hold power over all lifespans and thus became part of Egyptian funerary rituals. The Egyptian for astronomer, used as a synonym for priest, was wnwty, the earliest forms of wnwt include one or three stars, with the later solar hours including the determinative hieroglyph for sun
8.
DNA polymerase
–
In molecular biology, DNA polymerases are enzymes that synthesize DNA molecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase reads the existing DNA strands to create two new strands that match the existing ones. Every time a cell divides, DNA polymerases are required to duplicate the cells DNA. In this way, genetic information is passed down generation to generation. Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form and this opens up or unzips the double-stranded DNA to give two single strands of DNA that can be used as templates for replication. In 1956, Arthur Kornberg and colleagues discovered DNA polymerase I and they described the DNA replication process by which DNA polymerase copies the base sequence of a template DNA strand. Kornberg was later awarded the Nobel Prize in Physiology or Medicine in 1959 for this work, DNA polymerase II was also discovered by Thomas Kornberg and Malcolm E. Gefter in 1970 while further elucidating the role of Pol I in E. coli DNA replication. The main function of DNA polymerase is to synthesize DNA from deoxyribonucleotides, the DNA copies are created by the pairing of nucleotides to bases present on each strand of the original DNA molecule. This pairing always occurs in specific combinations, with cytosine along with guanine, by contrast, RNA polymerases synthesize RNA from ribonucleotides from either RNA or DNA. When synthesizing new DNA, DNA polymerase can add free nucleotides only to the 3 end of the newly forming strand and this results in elongation of the newly forming strand in a 5-3 direction. No known DNA polymerase is able to begin a new chain, it can add a nucleotide onto a pre-existing 3-OH group. Primers consist of RNA or DNA bases, in DNA replication, the first two bases are always RNA, and are synthesized by another enzyme called primase. It is important to note that the directionality of the newly forming strand is opposite to the direction in which DNA polymerase moves along the template strand. Since DNA polymerase requires a free 3 OH group for initiation of synthesis, hence, DNA polymerase moves along the template strand in a 3-5 direction, and the daughter strand is formed in a 5-3 direction. This difference enables the resultant double-strand DNA formed to be composed of two DNA strands that are antiparallel to each other, the function of DNA polymerase is not quite perfect, with the enzyme making about one mistake for every billion base pairs copied. Error correction is a property of some, but not all DNA polymerases and this process corrects mistakes in newly synthesized DNA. When an incorrect base pair is recognized, DNA polymerase moves backwards by one pair of DNA
9.
Microsatellite
–
A microsatellite is a tract of repetitive DNA in which certain DNA motifs are repeated, typically 5–50 times. Microsatellites occur at thousands of locations within a genome, additionally. Microsatellites and their cousins, the minisatellites, together are classified as VNTR DNA. The name satellite refers to the observation that centrifugation of genomic DNA in a test tube separates a prominent layer of bulk DNA from accompanying satellite layers of repetitive DNA. Microsatellites are often referred to as tandem repeats by forensic geneticists. They are widely used for DNA profiling in kinship analysis and in forensic identification and they are also used in genetic linkage analysis/marker assisted selection to locate a gene or a mutation responsible for a given trait or disease. Microsatellites are also used in genetics to measure levels of relatedness between subspecies, groups and individuals. A microsatellite is a tract of tandemly repeated DNA motifs that range in length from two to five nucleotides, and are typically repeated 5-50 times, for example, the sequence TATATATATA is a dinucleotide microsatellite, and GTCGTCGTCGTCGTC is a trinucleotide microsatellite. Repeat units of four and five nucleotides are referred to as tetra- and pentanucleotide motifs, microsatellites are distributed throughout the genome. Many are located in non-coding parts of the genome and therefore do not produce proteins, however they can also be located in regulatory regions. The telomeres at the ends of the chromosomes, thought to be involved in ageing/senescence, consist of repetitive DNA and they are thus classified as minisatellites, although insects for example have shorter repeat motifs in their telomeres which therefore could arguably be considered microsatellites. Unlike point mutations, which only a single nucleotide, microsatellite mutations lead to the gain or loss of an entire repeat unit. Thus, the rate at microsatellite loci is expected to differ from other mutation rates. The actual cause of mutations in microsatellites is debated, one proposed cause of such length changes is replication slippage, caused by mismatches between DNA strands while being replicated during meiosis. DNA polymerase, the responsible for reading DNA during replication, can slip while moving along the template strand. DNA polymerase slippage is more likely to occur when a sequence is replicated. Because microsatellites consist of repetitive sequences, DNA polymerase may make errors at a higher rate in these sequence regions. Several studies have found evidence that slippage is the cause of microsatellite mutations, typically, slippage in each microsatellite occurs about once per 1,000 generations
10.
DNA sequencer
–
A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample of DNA, a DNA sequencer is used to determine the order of the four bases and this is then reported as a text string, called a read. Some DNA sequencers can be also considered optical instruments as they analyze light signals originating from fluorochromes attached to nucleotides, the first automated DNA sequencer, invented by Lloyd M. Smith, was introduced by Applied Biosystems in 1987. It used the Sanger sequencing method, a technology which formed the basis of the “first generation” of DNA sequencers and this first generation of DNA sequencers are essentially automated electrophoresis systems that detect the migration of labelled DNA fragments. Therefore, these sequencers can also be used in the genotyping of genetic markers where only the length of a DNA fragment needs to be determined. The Human Genome Project catalysed the development of cheaper, high throughput and these include the 454, SOLiD and Illumina DNA sequencing platforms. Next generation sequencing machines have increased the rate of DNA sequence substantially compared with previous Sanger methods, DNA samples can be prepared automatically in as little as 90 mins, while a human genome can be sequenced at 15 times coverage in a matter of days. More recent, third-generation DNA sequencers such as SMRT and Oxford Nanopore measure the addition of nucleotides to a single DNA molecule in real time. Because of limitations in DNA sequencer technology these reads are compared to the length of a genome therefore the reads must be assembled into longer contigs. The data may contain errors, caused by limitations in the DNA sequencing technique or by errors during PCR amplification. DNA sequencer manufacturers use a number of different methods to detect which DNA bases are present, the specific protocols applied in different sequencing platforms have an impact in the final data that is generated. Therefore, comparing data quality and cost across different technologies can be a daunting task, each manufacturer provides their own ways to inform sequencing errors and scores. However, errors and scores between different platforms cannot always be compared directly, since these systems rely on different DNA sequencing approaches, choosing the best DNA sequencer and method will typically depend on the experiment objectives and available budget. The first DNA sequencing methods were developed by Gilbert and Sanger, Gilbert introduced a sequencing method based on chemical modification of DNA followed by cleavage at specific bases whereas Sanger’s technique is based on dideoxynucleotide chain termination. The Sanger method became popular due to its efficiency and low radioactivity. The first automated DNA sequencer was the AB370A, introduced in 1986 by Applied Biosystems, the AB370A was able to sequence 96 samples simultaneously,500 kilobases per day, and reaching read lengths up to 600 bases. The next major advance was the release in 1995 of the AB310 which utilized a linear polymer in a capillary in place of the gel for DNA strand separation by electrophoresis. These techniques formed the base for the completion of the human genome project in 2001, the human genome project catalysed the development of cheaper, high throughput and more accurate platforms known as Next Generation Sequencers
11.
Gel electrophoresis
–
Gel electrophoresis is a method for separation and analysis of macromolecules and their fragments, based on their size and charge. Nucleic acid molecules are separated by applying a field to move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel, proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of nanoparticles, Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. In simple terms, electrophoresis is a process which enables the sorting of molecules based on size, using an electric field, molecules can be made to move through a gel made of agar or polyacrylamide. The electric field consists of a charge at one end which pushes the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material, the gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel, the term gel in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a polymer whose composition and porosity is chosen based on the specific weight. When separating proteins or small nucleic acids the gel is usually composed of different concentrations of acrylamide, when separating larger nucleic acids, the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix, acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Electrophoresis refers to the force that is used to move the molecules through the gel matrix. Species that are positively charged will migrate towards the cathode which is negatively charged, if the species are negatively charged they will migrate towards the positively charged anode. If several samples have been loaded into adjacent wells in the gel, 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 distance from the top contain molecules that passed through the gel with the same speed. There are molecular weight size markers available that contain a mixture of molecules of known sizes
12.
Polyacrylamide gel electrophoresis
–
Mobility is a function of the length, conformation and charge of the molecule. For nucleic acids, urea is the most commonly used denaturant, for proteins, sodium dodecyl sulfate is an anionic detergent applied to protein samples to linearize proteins and to impart a negative charge to linearized proteins. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, samples may be any material containing proteins or nucleic acids. These may be derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental samples. Synthetic biomolecules such as oligonucleotides may also be used as analytes, the sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids. SDS is a detergent that denatures secondary and non–disulfide–linked tertiary structures. Urea breaks the bonds between the base pairs of the nucleic acid, causing the constituent strands to separate. Heating the samples to at least 60 °C further promotes denaturation and this is known as reducing SDS-PAGE. A tracking dye may be added to the solution and this typically has a higher electrophoretic mobility than the analytes to allow the experimenter to track the progress of the solution through the gel during the electrophoretic run. The gels typically consist of acrylamide, bisacrylamide, the optional denaturant, the solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. Alternatively, butanol may be added to the gel after it is poured. A source of radicals and a stabilizer, such as ammonium persulfate. The polymerization reaction creates a gel because of the added bisacrylamide, the ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part in 35. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%, lower percentage gels are better for resolving very high molecular weight molecules, while much higher percentages are needed to resolve smaller proteins. Gels are usually polymerized between two plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis, various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different, an electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel away from the negative electrode and towards the positive electrode. Depending on their size, each biomolecule moves differently through the gel matrix, small molecules more easily fit through the pores in the gel, after the set amount of time, the biomolecules have migrated different distances based on their size
