DNA Spiking
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DNA Spiking
DNA spiking, also known as custom spiking, is the differing ratio of bases at a single degenerate position when synthesizing oligonucleotides. DNA spiking is a unequal proportions of bases at a given position (for example, 10% Adenine, 75% Guanine, 5% Cytosine & 10% Thymine). As an example, with the degenerate code R = A + G, 50% of the time that R position is adenine and the other 50% of the time it is guanine. However, with DNA Spiking, the R position could be adenine 70% of the time and guanine 30% of the time. The proportions do not need to be 70:30, the ratios can be anything else such as 12:82 and 64:36. DNA spiking can also refer to a spike control in PCR, which is when DNA is added to a sample that will provide some signal (e.g. a plasmid or some synthetic DNA with a specific known sequence) to a reaction, and seeing if the reaction will amplify. This method is used to discover if the PCR method is working correctly, as in a PCR machine it may not amplify DNA properly, so ...
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Nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic building blocks of nucleic acids. The ability of nucleobases to form base pairs and to stack one upon another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are called ''primary'' or ''canonical''. They function as the fundamental units of the genetic code, with the bases A, G, C, and T being found in DNA while A, G, C, and U are found in RNA. Thymine and uracil are distinguished by merely the presence or absence of a methyl group on the fifth carbon (C5) of these heterocyclic six-membered rings. In addition, some viruses have aminoadenine (Z) instead of adenine. It differs in having an ...
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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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Guanine
Guanine () ( symbol G or Gua) is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine (uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is called guanosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with conjugated double bonds. This unsaturated arrangement means the bicyclic molecule is planar. Properties Guanine, along with adenine and cytosine, is present in both DNA and RNA, whereas thymine is usually seen only in DNA, and uracil only in RNA. Guanine has two tautomeric forms, the major keto form (see figures) and rare enol form. It binds to cytosine through three hydrogen bonds. In cytosine, the amino group acts as the hydrogen bond donor and the C-2 carbonyl and the N-3 amine as the hydrogen-bond acceptors. Guanine has the C-6 carbonyl group that acts as the hydrogen bond acceptor, while a group at N ...
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Cytosine
Cytosine () ( symbol C or Cyt) is one of the four nucleobases found in DNA and RNA, along with adenine, guanine, and thymine (uracil in RNA). It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached (an amine group at position 4 and a keto group at position 2). The nucleoside of cytosine is cytidine. In Watson-Crick base pairing, it forms three hydrogen bonds with guanine. History Cytosine was discovered and named by Albrecht Kossel and Albert Neumann in 1894 when it was hydrolyzed from calf thymus tissues. A structure was proposed in 1903, and was synthesized (and thus confirmed) in the laboratory in the same year. In 1998, cytosine was used in an early demonstration of quantum information processing when Oxford University researchers implemented the Deutsch-Jozsa algorithm on a two qubit nuclear magnetic resonance quantum computer (NMRQC). In March 2015, NASA scientists reported the formation of cytosine, along with uracil and thym ...
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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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Nucleic Acid Notation
The nucleic acid notation currently in use was first formalized by the International Union of Pure and Applied Chemistry (IUPAC) in 1970. This universally accepted notation uses the Roman characters G, C, A, and T, to represent the four nucleotides commonly found in deoxyribonucleic acids (DNA). Given the rapidly expanding role for genetic sequencing, synthesis, and analysis in biology, some researchers have developed alternate notations to further support the analysis and manipulation of genetic data. These notations generally exploit size, shape, and symmetry to accomplish these objectives. IUPAC notation Degenerate base symbols in biochemistry are an IUPAC representation for a position on a DNA sequence that can have multiple possible alternatives. These should not be confused with non-canonical bases because each particular sequence will have in fact one of the regular bases. These are used to encode the consensus sequence of a population of aligned sequences and are used f ...
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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 (chemist), 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 DNA, 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 ...
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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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RNA Spike-in
An RNA spike-in is an Messenger RNA, RNA transcript of known Nucleic acid sequence, sequence and Concentration, quantity used to Calibration, calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, Real-time polymerase chain reaction, RT-qPCR, and RNA-Seq. A spike-in is designed to bind to a DNA molecule with a Complementary DNA, matching sequence, known as a Scientific control, control Hybridization probe, probe. This process of specific binding is called Nucleic acid hybridization, hybridization. A known quantity of RNA spike-in is mixed with the experiment sample during preparation. The degree of hybridization between the spike-ins and the control probes is used to Normalization (statistics), normalize the hybridization measurements of the sample RNA. History Nucleic acid hybridization assays have been used for decades to detect specific sequences of DNA or RNA, with a DNA microarray precursor used as early as 1965. In such assays, positive cont ...
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