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Transcription-mediated Amplification
Transcription-mediated amplification (TMA) is an isothermal (performed at constant temperature), single-tube nucleic acid Gene_duplication#As_amplification, amplification system utilizing two enzymes, RNA polymerase and reverse transcriptase. "Amplification" means creating many more copies of a strand of nucleic acid than was present at first, in order to readily detect it or test it. Rapidly amplifying the target RNA/DNA allows a lab to simultaneously detect multiple pathogenic organisms in a single tube. TMA technology allows a clinical laboratory to perform nucleic acid test (NAT) assays for blood screening with fewer steps, less processing time, and faster results. It is used in molecular biology, forensics, and medicine for the rapid identification and diagnosis of pathogenic organisms. In contrast to similar techniques such as polymerase chain reaction and ligase chain reaction, this method involves RNA transcription (via RNA polymerase) and DNA synthesis (via reverse trans ...
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Nucleic Acid
Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is deoxyribose, a variant of ribose, the polymer is DNA. Nucleic acids are chemical compounds that are found in nature. They carry information in cells and make up genetic material. These acids are very common in all living things, where they create, encode, and store information in every living cell of every outline of life forms, life-form on Earth. In turn, they send and express that information inside and outside the cell nucleus. From the inner workings of the cell to the young of a living thing, they contain and provide information via the nucleic acid sequence. This gives the RNA and DNA their unmistakable 'la ...
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Gene Duplication
Gene duplication (or chromosomal duplication or gene amplification) is a major mechanism through which new genetic material is generated during molecular evolution. It can be defined as any duplication of a region of DNA that contains a gene. Gene duplications can arise as products of several types of errors in DNA replication and repair machinery as well as through fortuitous capture by selfish genetic elements. Common sources of gene duplications include ectopic recombination, retrotransposition event, aneuploidy, polyploidy, and replication slippage. Mechanisms of duplication Ectopic recombination Duplications arise from an event termed unequal crossing-over that occurs during meiosis between misaligned homologous chromosomes. The chance of it happening is a function of the degree of sharing of repetitive elements between two chromosomes. The products of this recombination are a duplication at the site of the exchange and a reciprocal deletion. Ectopic recombina ...
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Enzyme
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecules known as product (chemistry), products. Almost all metabolism, metabolic processes in the cell (biology), cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called ''enzymology'' and the field of pseudoenzyme, pseudoenzyme analysis recognizes that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties. Enzymes are known to catalyze more than 5,000 biochemical reaction types. Other biocatalysts include Ribozyme, catalytic RNA molecules, also called ribozymes. They are sometimes descr ...
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RNA Polymerase
In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the double-stranded DNA so that one strand of the exposed nucleotides can be used as a template for the synthesis of RNA, a process called transcription. A transcription factor and its associated transcription mediator complex must be attached to a DNA binding site called a promoter region before RNAP can initiate the DNA unwinding at that position. RNAP not only initiates RNA transcription, it also guides the nucleotides into position, facilitates attachment and elongation, has intrinsic proofreading and replacement capabilities, and termination recognition capability. In eukaryotes, RNAP can build chains as long as 2.4 million nucleotides. RNAP produces RNA that, functionally, is either for protei ...
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Reverse Transcriptase
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, and by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes. The process does not violate the flows of genetic information as described by the classical central dogma, but rather expands it to include transfers of information from RNA to DNA. Retroviral RT has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H (RNase H), and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then integrate into the host genome, from which new RNA copies can be made via host-cell ...
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Nucleic Acid Test
A nucleic acid test (NAT) is a technique used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissue, urine, etc. NATs differ from other tests in that they detect genetic materials (RNA or DNA) rather than antigens or antibodies. Detection of genetic materials allows an early diagnosis of a disease because the detection of antigens and/or antibodies requires time for them to start appearing in the bloodstream. Since the amount of a certain genetic material is usually very small, many NATs include a step that amplifies the genetic material—that is, makes many copies of it. Such NATs are called nucleic acid amplification tests (NAATs). There are several ways of amplification, including polymerase chain reaction (PCR), strand displacement assay (SDA), transcription mediated assay (TMA), and loop-mediated isothermal amplification (LAMP). ...
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Molecular Biology
Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactions. Though cells and other microscopic structures had been observed in living organisms as early as the 18th century, a detailed understanding of the mechanisms and interactions governing their behavior did not emerge until the 20th century, when technologies used in physics and chemistry had advanced sufficiently to permit their application in the biological sciences. The term 'molecular biology' was first used in 1945 by the English physicist William Astbury, who described it as an approach focused on discerning the underpinnings of biological phenomena—i.e. uncovering the physical and chemical structures and properties of biological molecules, as well as their interactions with other molecules and how these interactions explain observ ...
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Forensics
Forensic science combines principles of law and science to investigate criminal activity. Through crime scene investigations and laboratory analysis, forensic scientists are able to link suspects to evidence. An example is determining the time and cause of death through autopsies. This evidence can then be used for proof towards a crime. Forensic science, often confused with criminalistics, is the application of science principles and methods to support legal decision-making in matters of criminal and civil law. During criminal investigation in particular, it is governed by the legal standards of admissible evidence and criminal procedure. It is a broad field utilizing numerous practices such as the analysis of DNA, fingerprints, bloodstain patterns, firearms, ballistics, toxicology, microscopy, and fire debris analysis. Forensic scientists collect, preserve, and analyze evidence during the course of an investigation. While some forensic scientists travel to the scene of t ...
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Medicine
Medicine is the science and Praxis (process), practice of caring for patients, managing the Medical diagnosis, diagnosis, prognosis, Preventive medicine, prevention, therapy, treatment, Palliative care, palliation of their injury or disease, and Health promotion, promoting their health. Medicine encompasses a variety of health care practices evolved to maintain and restore health by the prevention (medical), prevention and treatment of illness. Contemporary medicine applies biomedical sciences, biomedical research, medical genetics, genetics, and medical technology to diagnosis (medical), diagnose, treat, and prevent injury and disease, typically through pharmaceuticals or surgery, but also through therapies as diverse as psychotherapy, splint (medicine), external splints and traction, medical devices, biologic medical product, biologics, and Radiation (medicine), ionizing radiation, amongst others. Medicine has been practiced since Prehistoric medicine, prehistoric times, and ...
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Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study. PCR was invented in 1983 by 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 including biomedical research and forensic ...
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Ligase Chain Reaction
The ligase chain reaction (LCR) is a method of DNA amplification. The ligase chain reaction (LCR) is an amplification process that differs from polymerase chain reaction (PCR) in that it involves a thermostable ligase to join two probes or other molecules together which can then be amplified by standard PCR cycling. Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR. Thus, LCR requires two completely different enzymes to operate properly: ligase, to join probe molecules together, and a thermostable polymerase (e.g., ''Taq'' polymerase) to amplify those molecules involved in successful ligation. The probes involved in the ligation are designed such that the 5′ end of one probe is directly adjacent to the 3′ end of the other probe, thereby providing the requisite 3′-OH and 5′-PO4 group substrates for the ligase. LCR was originally developed to detect point mutations; a single base mismatch ...
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Amplicon
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication. In this context, ''amplification'' refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As it refers to the product of an amplification reaction, ''amplicon'' is used interchangeably with common laboratory terms, such as "PCR product." Artificial amplification is used in research, forensics, and medicine for purposes that include detection and quantification of infectious agents, identification of human remains, and extracting genotypes from human hair. Natural gene duplication plays a major role in evolution. It is also implicated in several forms of human cancer including primary mediastinal B cell lymphoma and Hodg ...
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