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Nick Translation
Nick translation (or head translation), developed in 1977 by Peter Rigby and Paul Berg, is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in fluorescent in situ hybridization (FISH) or blotting techniques. It can also be used for radiolabeling. This process is called nick translation because the DNA to be processed is treated with DNAase to produce single-stranded "nicks". This is followed by replacement in nicked sites by DNA polymerase I, which elongates the 3' hydroxyl terminus, removing nucleotides by 5'-3' exonuclease activity, replacing them with dNTPs. To radioactively label a DNA fragment for use as a probe in blotting procedures, one of the incorporated nucleotides provided in the reaction is radiolabeled in the ''alpha'' phosphate position. Similarly, a fluorophore can be attached instead for fluore ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Fluorescent Tag
In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target. History The development of methods to detect and identify biomolecules has been motivated by the ability to improve the stu ... [...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 Ro ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
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 ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Fluorescent In Situ Hybridization
Fluorescence ''in situ'' hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues. Probes – RNA and DNA In biology, a probe is a single strand of DNA or RNA that is complementary to a nucleotide sequence ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Blot (biology)
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA onto a carrier (for example, a nitrocellulose, polyvinylidene fluoride or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the Blotting matrix, blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining (for example, silver staining of proteins), autoradiographic visualization of radioactive tracer, radiolabelled molecules (performed before the blot), or specific labelling of some proteins or nucleic acids. The latter is done with antibody, antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity (and so, the molecules we search in the blot) is visualized by incubation with proper reactive, renderin ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Radiolabeling
A radioactive tracer, radiotracer, or radioactive label is a chemical compound in which one or more atoms have been replaced by a radionuclide so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by tracing the path that the radioisotope follows from reactants to products. Radiolabeling or radiotracing is thus the radioactive form of isotopic labeling. In biological contexts, use of radioisotope tracers are sometimes called radioisotope feeding experiments. Radioisotopes of hydrogen, carbon, phosphorus, sulfur, and iodine have been used extensively to trace the path of biochemical reactions. A radioactive tracer can also be used to track the distribution of a substance within a natural system such as a cell or tissue, or as a flow tracer to track fluid flow. Radioactive tracers are also used to determine the location of fractures created by hydraulic fracturing in natural gas production.Reis, John C. (1976). ''Environmental Control in ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
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 ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Directionality (molecular Biology)
Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-ring means that there will be a 5′ end (usually pronounced "five-prime end"), which frequently contains a phosphate group attached to the 5′ carbon of the ribose ring, and a 3′ end (usually pronounced "three-prime end"), which typically is unmodified from the ribose -OH substituent. In a DNA double helix, the strands run in opposite directions to permit base pairing between them, which is essential for replication or transcription of the encoded information. Nucleic acids can only be synthesized in vivo in the 5′-to-3′ direction, as the polymerases that assemble various types of new strands generally rely on the energy produced by breaking nucleoside triphosphate bonds to attach new nucleoside monophosphates to the 3′- ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Deoxyribonucleotide
A deoxyribonucleotide is a nucleotide that contains deoxyribose. They are the monomeric units of the informational biopolymer, deoxyribonucleic acid ( DNA). Each deoxyribonucleotide comprises three parts: a deoxyribose sugar (monosaccharide), a nitrogenous base, and one phosphoryl group. The nitrogenous bases are either purines or pyrimidines, heterocycles whose structures support the specific base-pairing interactions that allow nucleic acids to carry information. The base is always bonded to the 1'-carbon of the deoxyribose, an analog of ribose in which the hydroxyl group of the 2'-carbon is replaced with a hydrogen atom. The third component, the phosphoryl group, attaches to the deoxyribose monomer via the hydroxyl group on the 5'-carbon of the sugar. When deoxyribonucleotides polymerize to form DNA, the phosphate group from one nucleotide will bond to the 3' carbon on another nucleotide, forming a phosphodiester bond via dehydration synthesis. New nucleotides are always adde ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Processivity
In molecular biology and biochemistry, processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate". For example, processivity is the average number of nucleotides added by a polymerase enzyme, such as DNA polymerase, per association event with the template strand. Because the binding of the polymerase to the template is the rate-limiting step in DNA synthesis, the overall rate of DNA replication during S phase of the cell cycle is dependent on the processivity of the DNA polymerases performing the replication. DNA clamp proteins are integral components of the DNA replication machinery and serve to increase the processivity of their associated polymerases. Some polymerases add over 50,000 nucleotides to a growing DNA strand before dissociating from the template strand, giving a replication rate of up to 1,000 nucleotides per second. DNA binding interactions Polymerases interact with the phosphate backbone and the minor groove of the DNA ... [...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 phos ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Biochemistry Detection Methods
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and metabolism. Over the last decades of the 20th century, biochemistry has become successful at explaining living processes through these three disciplines. Almost all areas of the life sciences are being uncovered and developed through biochemical methodology and research. Voet (2005), p. 3. Biochemistry focuses on understanding the chemical basis which allows biological molecules to give rise to the processes that occur within living cells and between cells,Karp (2009), p. 2. in turn relating greatly to the understanding of tissues and organs, as well as organism structure and function.Miller (2012). p. 62. Biochemistry is closely related to molecular biology, which is the study of the molecular mechanisms of biological phenomena.Astb ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
