Taq DNA Polymerase
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Taq DNA Polymerase
''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to ''Taq'' or ''Taq'' pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA. ''T. aquaticus'' is a bacterium that lives in hot springs and hydrothermal vents, and ''Taq'' polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. Therefore, it replaced the DNA polymerase from '' E. coli'' originally used in PCR. Enzymatic properties ''Taqs optimum temperature for activity is 75–80 °C, with a half-life of greater than 2 hours at 92.5 °C, 40 minutes at 95 °C and 9 minutes at 97.5 °C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72 °C. ...
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DNA Polymerase I
DNA polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and the first known of any kind of polymerase). It was initially characterized in '' E. coli'' and is ubiquitous in prokaryotes. In ''E. coli'' and many other bacteria, the gene that encodes Pol I is known as ''polA''. The ''E. coli'' Pol I enzyme is composed of 928 amino acids, and is an example of a processive enzyme — it can sequentially catalyze multiple polymerisation steps without releasing the single-stranded template. The physiological function of Pol I is mainly to support repair of damaged DNA, but it also contributes to connecting Okazaki fragments by deleting RNA primers and replacing the ribonucleotides with DNA. Discovery In 1956, Arthur Kornberg and colleagues discovered Pol I by using ''Escherichia coli'' (''E. coli'') extracts to develop a DNA synthesis assay. The scientist ...
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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 ...
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Biotechnology
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used by Károly Ereky in 1919, meaning the production of products from raw materials with the aid of living organisms. Definition The concept of biotechnology encompasses a wide range of procedures for modifying living organisms according to human purposes, going back to domestication of animals, cultivation of the plants, and "improvements" to these through breeding programs that employ artificial selection and hybridization. Modern usage also includes genetic engineering as well as cell and tissue culture technologies. The American Chemical Society defines biotechnology as the application of biological organisms, systems, or processes by various industries to learning about the science of life and the improvement of the value of materials ...
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Cetus Corporation
Cetus Corporation was one of the first biotechnology companies. It was established in Berkeley, California, in 1971, but conducted most of its operations in nearby Emeryville. Before merging with Chiron Corporation in 1991 (now a part of Novartis), it developed several significant pharmaceutical drugs as well as a revolutionary DNA amplification technique. History Cetus was founded in 1971 by Ronald E. Cape, Peter Farley, and Nobelist Donald A. Glaser. Its early efforts involved automated methods to select for industrial microorganisms that could produce greater amounts of chemical feedstocks, antibiotics, or vaccine components. By the late 1970s, however, three new revolutionary techniques had been developed: recombinant DNA, monoclonal antibodies, and gene expression, the foundations of the biotechnology industry. In order to enter these new fields, Cetus raised $108 million in an initial public offering (IPO) in 1981, the largest IPO to that date. Its first large deve ...
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Kary Mullis
Kary Banks Mullis (December 28, 1944August 7, 2019) was an American biochemist. In recognition of his role in the invention of the polymerase chain reaction (PCR) technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and was awarded the Japan Prize in the same year. PCR became a central technique in biochemistry and molecular biology, described by ''The New York Times'' as "highly original and significant, virtually dividing biology into the two epochs of before PCR and after PCR." Mullis attracted controversy for downplaying humans' role in climate change and for expressing doubts that HIV is the sole cause of AIDS. Early life Mullis was born in Lenoir, North Carolina, near the Blue Ridge Mountains, on December 28, 1944 to Cecil Banks Mullis and Bernice Barker Mullis. His family had a background in farming in this rural area. As a child, Mullis said, he was interested in observing organisms in the countryside. He and his cousins would often taunt live ...
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DNA Ligase
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as DNA ligase IV) may specifically repair double-strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA. DNA ligase is used in both DNA repair and DNA replication (see '' Mammalian ligases''). In addition, DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments (see '' Research applications''). Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA. Enzymatic mechanism The mechanism of DNA ligase is to form two covalent phos ...
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Thymine
Thymine () ( symbol T or Thy) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. In RNA, thymine is replaced by the nucleobase uracil. Thymine was first isolated in 1893 by Albrecht Kossel and Albert Neumann from calf thymus glands, hence its name. Derivation As its alternate name (5-methyluracil) suggests, thymine may be derived by methylation of uracil at the 5th carbon. In RNA, thymine is replaced with uracil in most cases. In DNA, thymine (T) binds to adenine (A) via two hydrogen bonds, thereby stabilizing the nucleic acid structures. Thymine combined with deoxyribose creates the nucleoside deoxythymidine, which is synonymous with the term thymidine. Thymidine can be phosphorylated with up to three phosphoric acid groups, producing dTMP (deoxythymidine monophosphate), dTDP, or dTTP (for the di- and tr ...
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Plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the inter ...
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Cloning Vector
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined together by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use. There are many types of cloning vectors, but the most commonly used ones are genetically ...
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Adenine
Adenine () ( symbol A or Ade) is a nucleobase (a purine derivative). It is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The three others are guanine, cytosine and thymine. Its derivatives have a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate (ATP) and the cofactors nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) and Coenzyme A. It also has functions in protein synthesis and as a chemical component of DNA and RNA. The shape of adenine is complementary to either thymine in DNA or uracil in RNA. The adjacent image shows pure adenine, as an independent molecule. When connected into DNA, a covalent bond is formed between deoxyribose sugar and the bottom left nitrogen (thereby removing the existing hydrogen atom). The remaining structure is called an ''adenine residue'', as part of a larger molecule. Adenosine is ad ...
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Pfu DNA Polymerase
''Pfu'' DNA polymerase is an enzyme found in the hyperthermophilic archaeon ''Pyrococcus furiosus'', where it functions to copy the organism's DNA during cell division. In the laboratory setting, ''Pfu'' is used to amplify DNA in the polymerase chain reaction (PCR), where the enzyme serves the central function of copying a new strand of DNA during each extension step. It is a family B DNA polymerase. It has an RNase H-like 3'-5' exonuclease domain, typical of B-family polymerase such as DNA polymerase II. Proofreading ability of ''Pfu'' polymerase ''Pfu'' DNA polymerase has superior thermostability and proofreading properties compared to ''Taq'' DNA polymerase. Unlike ''Taq'' DNA polymerase, ''Pfu'' DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that as the DNA is assembled from the 5' end to 3' end, the exonuclease activity immediately removes nucleotides misincorporated at the 3' end of the growing DNA strand. Consequently, ''Pfu'' DNA polyme ...
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