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Polymerase Chain Reaction Optimization
The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure. Contamination and PCR The PCR method is extremely sensitive, requiring only a few DNA molecules in a single reaction for amplification across several orders of magnitude. Therefore, adequate measures to avoid contamination from any DNA present in the lab environment (bacteria, viruses, or human sources) are required. Because products from previous PCR amplifications are a common source of contamination, many molecular biology labs have implemented procedures that involve dividing the lab into separate areas. One lab area is dedicated to preparation and handling of pre-PCR reagents and the setup of the PCR reaction, and another area to post-PCR processing, such as gel electrophoresis or PCR product purification. For the setup of PCR reactions, ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993. PCR is fundamental to many of the procedures used in genetic testing and research, including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease. In both archaea and eukaryotes, one of the main routes of RNA degradation is performed by the multi-protein exosome complex, which consists largely of 3′ to 5′ exoribonucleases. Significance to polymerase RNA polymerase II is known to be in effect during transcriptional termination; it works with a 5' exonuclease (human gene Xrn2) to degrade the newly formed tra ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Topoisomerase
DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues in DNA arise due to the intertwined nature of its double-helical structure, which, for example, can lead to overwinding of the DNA duplex during DNA replication and transcription. If left unchanged, this torsion would eventually stop the DNA or RNA polymerases involved in these processes from continuing along the DNA helix. A second topological challenge results from the linking or tangling of DNA during replication. Left unresolved, links between replicated DNA will impede cell division. The DNA topoisomerases prevent and correct these types of topological problems. They do this by binding to DNA and cutting the sugar-phosphate backbone of either one (type I topoisomerases) or both (type II topoisomerases) of the DNA strands. This transien ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Polymerase Chain Reaction Inhibitors
PCR inhibitors are any factor which prevent the amplification of nucleic acids through the polymerase chain reaction (PCR). PCR inhibition is the most common cause of amplification failure when sufficient copies of DNA are present. PCR inhibitors usually affect PCR through interaction with DNA or interference with the DNA polymerase. Inhibitors can escape removal during the DNA purification procedure by binding directly to single or double-stranded DNA. Alternatively, by reducing the availability of cofactors (such as Mg2+) or otherwise interfering with their interaction with the DNA polymerase, PCR is inhibited. In a multiplex PCR reaction, it is possible for the different sequences to suffer from different inhibition effects to different extents, leading to disparity in their relative amplifications. Types of inhibitors Inhibitors may be present in the original sample, such as blood, fabrics, tissues and soil but may also be added as a result of the sample processing and DNA extr ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Fusion Protein
Fusion proteins or chimeric (kī-ˈmir-ik) proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this ''fusion gene'' results in a single or multiple polypeptides with functional properties derived from each of the original proteins. ''Recombinant fusion proteins'' are created artificially by recombinant DNA technology for use in biological research or therapeutics. '' Chimeric'' or ''chimera'' usually designate hybrid proteins made of polypeptides having different functions or physico-chemical patterns. ''Chimeric mutant proteins'' occur naturally when a complex mutation, such as a chromosomal translocation, tandem duplication, or retrotransposition creates a novel coding sequence containing parts of the coding sequences from two different genes. Naturally occurring fusion proteins are commonly found in cancer cells, where they may function as oncoprotei ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Stochastic
Stochastic (, ) refers to the property of being well described by a random probability distribution. Although stochasticity and randomness are distinct in that the former refers to a modeling approach and the latter refers to phenomena themselves, these two terms are often used synonymously. Furthermore, in probability theory, the formal concept of a '' stochastic process'' is also referred to as a ''random process''. Stochasticity is used in many different fields, including the natural sciences such as biology, chemistry, ecology, neuroscience, and physics, as well as technology and engineering fields such as image processing, signal processing, information theory, computer science, cryptography, and telecommunications. It is also used in finance, due to seemingly random changes in financial markets as well as in medicine, linguistics, music, media, colour theory, botany, manufacturing, and geomorphology. Etymology The word ''stochastic'' in English was originally used as ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Magnesium Chloride
Magnesium chloride is the family of inorganic compounds with the formula , where x can range from 0 to 12. These salts are colorless or white solids that are highly soluble in water. These compounds and their solutions, both of which occur in nature, have a variety of practical uses. Anhydrous magnesium chloride is the principal precursor to magnesium metal, which is produced on a large scale. Hydrated magnesium chloride is the form most readily available. Production Magnesium chloride can be extracted from brine or sea water. In North America, it is produced primarily from Great Salt Lake brine. In the Jordan Valley, it is obtained from the Dead Sea. The mineral bischofite () is extracted (by solution mining) out of ancient seabeds, for example, the Zechstein seabed in northwest Europe. Some deposits result from high content of magnesium chloride in the primordial ocean. Some magnesium chloride is made from evaporation of seawater. In the Dow process, magnesium chloride is ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are rarely considered to be proteins and are commonly called peptides. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
EDTA
Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes even at neutral pH. It is thus used to dissolve Fe- and Ca-containing scale as well as to deliver iron ions under conditions where its oxides are insoluble. EDTA is available as several salts, notably disodium EDTA, sodium calcium edetate, and tetrasodium EDTA, but these all function similarly. Uses Textile industry In industry, EDTA is mainly used to sequester (bind or confine) metal ions in aqueous solution. In the textile industry, it prevents metal ion impurities from modifying colours of dyed products. In the pulp and paper industry, EDTA inhibits the ability of metal ions, especially Mn2+, from catalysing the disproportionation of hydrogen peroxide, which is used in chlorine-free bleaching. In a similar manner, EDTA is added ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Chelating Agents
Chelation is a type of bonding of ions and molecules to metal ions. It involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom. These ligands are called chelants, chelators, chelating agents, or sequestering agents. They are usually organic compounds, but this is not a necessity, as in the case of zinc and its use as a maintenance therapy to prevent the absorption of copper in people with Wilson's disease. Chelation is useful in applications such as providing nutritional supplements, in chelation therapy to remove toxic metals from the body, as contrast agents in MRI scanning, in manufacturing using homogeneous catalysts, in chemical water treatment to assist in the removal of metals, and in fertilizers. Chelate effect The chelate effect is the greater affinity of chelating ligands for a metal ion than that of similar nonchelating (monodentate) ligands for the same metal. ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Pyrococcus Furiosus
''Pyrococcus furiosus'' is a heterotrophic, strictly anaerobic, extremophilic, model species of archaea. It is classified as a hyperthermophile because it thrives best under extremely high temperatures, and is notable for having an optimum growth temperature of 100 °C (a temperature that would destroy most living organisms). ''P. furiosus'' belongs to the '' Pyrococcus'' genus, most commonly found in extreme environmental conditions of hydrothermal vents. It is one of the few prokaryotic organisms that has enzymes containing tungsten, an element rarely found in biological molecules. ''Pyrococcus furiosus'' has many potential industrial applications, owing to its unique thermostable properties. ''P. furiosus'' is used in the process of DNA amplification by way of polymerase chain reaction (PCR) because of its proofreading activity. Utilizing ''P. furiosus'' in PCR DNA amplification instead of the traditionally used ''Taq'' DNA polymerase allows for a significantly more acc ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
