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TOPO Cloning
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3'-end of the PCR products. This characteristic is exploited in "sticky end" TOPO TA cloning. For "blunt end" TOPO cloning, the recipient vector does not have overhangs and blunt-ended DNA fragments can be cloned. Principle The technique utilises the inherent biological activity of DNA topoisomerase I. The biological role of topoisomerase is to cleave and rejoin supercoiled DNA ends to facilitate replication. Vaccinia virus topoisomerase I specifically recognises DNA sequence 5´-(C/T)CCTT-3'. During replication, the enzyme digests DNA specifically at this sequence, unwinds the DNA and re-ligates it again at the 3' phosphate group of the thymidine Thymidine (symbol dT or dThd), also known as deoxythymidine, deoxyrib ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Molecular Biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physical structure of biological macromolecules is known as molecular biology. Molecular biology was first described as an approach focused on the underpinnings of biological phenomena - uncovering the structures of biological molecules as well as their interactions, and how these interactions explain observations of classical biology. In 1945 the term molecular biology was used by physicist William Astbury. In 1953 Francis Crick, James Watson, Rosalind Franklin, and colleagues, working at Medical Research Council unit, Cavendish laboratory, Cambridge (now the MRC Laboratory of Molecular Biology), made a double helix model of DNA which changed the entire research scenario. They proposed the DNA structure based on previous research done by R ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Molecular Cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their DNA replication, replication within Host (biology), host organisms. The use of the word ''cloning'' refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living Host (biology), host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with Vector (molecular biology), vecto ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
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 int ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
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 p ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
DNA 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] |
Supercoil
DNA supercoiling refers to the amount of twist in a particular DNA strand, which determines the amount of strain on it. A given strand may be "positively supercoiled" or "negatively supercoiled" (more or less tightly wound). The amount of a strand’s supercoiling affects a number of biological processes, such as compacting DNA and regulating access to the genetic code (which strongly affects DNA metabolism and possibly gene expression). Certain enzymes, such as topoisomerases, change the amount of DNA supercoiling to facilitate functions such as DNA replication and transcription. The amount of supercoiling in a given strand is described by a mathematical formula that compares it to a reference state known as "relaxed B-form" DNA. Overview In a "relaxed" double-helical segment of B-DNA, the two strands twist around the helical axis once every 10.4–10.5 base pairs of sequence. Adding or subtracting twists, as some enzymes do, imposes strain. If a DNA segment under twist s ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Vaccinia Virus
''Vaccinia virus'' (VACV or VV) is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, which encodes approximately 250 genes. The dimensions of the virion are roughly 360 × 270 × 250 nm, with a mass of approximately 5–10 fg. The vaccinia virus is the source of the modern smallpox vaccine, which the World Health Organisation used to eradicate smallpox in a global vaccination campaign in 1958–1977. Although ''smallpox'' no longer exists in the wild, ''vaccinia'' virus is still studied widely by scientists as a tool for gene therapy and genetic engineering. Smallpox had been an endemic human disease that had a 30% fatality rate. In 1796, the British doctor Edward Jenner proved that an infection with the relatively mild cowpox virus would also confer immunity to the deadly smallpox. Jenner referred to cowpox as ''variolae vaccinae'' (smallpox of the cow). However, the origin ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
DNA Replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritance. This is essential for cell division during growth and repair of damaged tissues, while it also ensures that each of the new cells receives its own copy of the DNA. The cell possesses the distinctive property of division, which makes replication of DNA essential. DNA is made up of a double helix of two complementary strands. The double helix describes the appearance of a double-stranded DNA which is thus composed of two linear strands that run opposite to each other and twist together to form. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. As a result of semi-conservative re ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Thymidine
Thymidine (symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA. In cell biology it is used to synchronize the cells in G1/early S phase. The prefix deoxy- is often left out since there are no precursors of thymine nucleotides involved in RNA synthesis. Before the boom in thymidine use caused by the need for thymidine in the production of the antiretroviral drug azidothymidine (AZT), much of the world's thymidine production came from herring sperm. Thymidine occurs almost exclusively in DNA but it also occurs in the T-loop of tRNA. Structure and properties In its composition, deoxythymidine is a nucleoside composed of deoxyribose (a pentose sugar) joined to the pyrimidine base thymine. Deoxythymidine can be phosphorylated with one, two or three phosphoric acid groups, creating dTMP (deoxythymidin ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Cloning
Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cloning is the process of creating cloned organisms (copies) of cells and of DNA fragments (molecular cloning). Etymology Coined by Herbert J. Webber, the term clone derives from the Ancient Greek word (), ''twig'', which is the process whereby a new plant is created from a twig. In botany, the term ''lusus'' was used. In horticulture, the spelling ''clon'' was used until the early twentieth century; the final ''e'' came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling ''clone'' has been used exclusively. Natural cloning Cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years. ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
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