Solid Phase Sequencing
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Solid Phase Sequencing
The principle of solid phase DNA sequencing was described in 1989 based on binding of biotinylated DNA to streptavidin-coated magnetic beads and elution of single DNA strands selectively using alkali. The method allowed robotic applications suitable for clinical sequencing, but the magnetic handling has also found frequent use in many molecular applications, including sample handling for DNA diagnostics. The use of solid phase methods for DNA handling is now frequently used as an integrated part of many of the next generation DNA sequencing methods, as well as numerous molecular diagnostics Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. In medicine the technique is ... applications. References {{reflist Biotechnology ...
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Biotin
Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', borrowed from the German , derives from the Ancient Greek word (; 'life') and the suffix "-in" (a suffix used in chemistry usually to indicate 'forming'). Chemical description Biotin is classified as a heterocyclic compound, with a sulfur-containing ring fused ureido and tetrahydrothiophene group. A C5-carboxylic acid side chain is appended to one of the rings. The ureido ring, containing the −N−CO−N− group, serves as the carbon dioxide carrier in carboxylation reactions. Biotin is a coenzyme for five carboxylase enzymes, which are involved in the catabolism of amino acids and fatty acids, synthesis of fatty acids, and gluconeogenesis. Biotinylation of histone proteins in nuclear chromatin plays a role in chromatin stability and g ...
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Streptavidin
Streptavidin is a 66.0 (tetramer) kDa protein purified from the bacterium '' Streptomyces avidinii''. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation constant (Kd) on the order of ≈10−14 mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride), detergents (e.g. SDS, Triton X-100), proteolytic enzymes, and extremes of temperature and pH. Structure The crystal structure of streptavidin with biotin bound was reported by two groups in 1989. The structure was solved using multi wavelength anomalous diffraction by Hendrickson et al. at Columbia University and using multiple isomorphous replacement by Weber et al. at E. I. DuPont Central Research ...
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Elution
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions. In a liquid chromatography experiment, for example, an analyte is generally adsorbed, or "bound to", an adsorbent in a liquid chromatography column. The adsorbent, a solid phase (stationary phase), is a powder which is coated onto a solid support. Based on an adsorbent's composition, it can have varying affinities to "hold" onto other molecules—forming a thin film on the surface of its particles. Elution then is the process of removing analytes from the adsorbent by running a solvent, called an "eluent", past the adsorbent/analyte complex. As the solvent molecules "elute", or travel down through the chromatography column, they can either pass by the adsorbent/analyte complex or they can displace the analyte by binding to the adsorbent in its place. After the solvent molecules displa ...
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Alkali
In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a basic, ionic salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as a base that dissolves in water. A solution of a soluble base has a pH greater than 7.0. The adjective alkaline, and less often, alkalescent, is commonly used in English as a synonym for basic, especially for bases soluble in water. This broad use of the term is likely to have come about because alkalis were the first bases known to obey the Arrhenius definition of a base, and they are still among the most common bases. Etymology The word "alkali" is derived from Arabic ''al qalīy'' (or ''alkali''), meaning ''the calcined ashes'' (see calcination), referring to the original source of alkaline substances. A water-extract of burned plant ashes, called potash and composed mostly of potassium carbonate, was mildly basic. After heating this substance with calcium hydroxide (''slaked lime ...
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DNA Sequencing
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers, characterize antibody repertoire, and can be used to guide patient treatment. Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged. The rapid speed of sequencing attained with modern D ...
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Molecular Diagnostics
Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. In medicine the technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients, and in agricultural biosecurity similarly to monitor crop- and livestock disease, estimate risk, and decide what quarantine measures must be taken. By analysing the specifics of the patient and their disease, molecular diagnostics offers the prospect of personalised medicine. These tests are useful in a range of medical specialties, including infectious disease, oncology, human leucocyte antigen typing (which investigates and predicts immune function), coagulation, and pharmacogenomicsthe genetic prediction of which drugs will work best. They overlap with clinical chemistry (medical tests on bodily fluids). History ...
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