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Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to many of the procedures used in genetic testing and research, including analysis of 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 criminal forensics.
A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable.
Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the assay. When an assay for measuring the infective virus particle is done, viral titre often refers to the concentration of infectious viral particles, which is different from the total viral particles. Sputum and blood plasma are two bodily fluids from which viral load is measured. As an example of environmental specimens, the viral load of norovirus can be determined from run-off water on garden produce. Norovirus has not only prolonged viral shedding and has the ability to survive in the environment but a minuscule infectious dose is required to produce infection in humans: less than 100 viral particles.
Helicase-dependent amplification (HDA) is a method for in vitro DNA amplification that takes place at a constant temperature.
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."
The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. Currently it is a routine procedure in molecular biology or forensic analyses. For the chemical method, there are many different kits used for extraction, and selecting the correct one will save time on kit optimization and extraction procedures. PCR sensitivity detection is considered to show the variation between the commercial kits.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.
The overlap extension polymerase chain reaction is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension (SOE) PCR. It is used to insert specific mutations at specific points in a sequence or to splice smaller DNA fragments into a larger polynucleotide.
Nucleic acid sequence-based amplification, commonly referred to as NASBA, is a method in molecular biology which is used to produce multiple copies of single stranded RNA. NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.
Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.
The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.
Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA and a low-cost alternative to detect certain diseases. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA.
Webtag is an on-line bioinformatics tool providing oligonucleotide sequences that are absent from a specified genome. These tags can be appended to gene specific primers for reverse transcriptase polymerase chain reaction (RT-PCR) experiments, circumventing genomic DNA contamination.
The history of the polymerase chain reaction (PCR) has variously been described as a classic "Eureka!" moment, or as an example of cooperative teamwork between disparate researchers. Following is a list of events before, during, and after its development:
The versatility of polymerase chain reaction (PCR) has led to a large number of variants of PCR.
Multiple displacement amplification (MDA) is a DNA amplification technique. This method can rapidly amplify minute amounts of DNA samples to a reasonable quantity for genomic analysis. The reaction starts by annealing random hexamer primers to the template: DNA synthesis is carried out by a high fidelity enzyme, preferentially Φ29 DNA polymerase. Compared with conventional PCR amplification techniques, MDA does not employ sequence-specific primers but amplifies all DNA, generates larger-sized products with a lower error frequency, and works at a constant temperature. MDA has been actively used in whole genome amplification (WGA) and is a promising method for application to single cell genome sequencing and sequencing-based genetic studies.
A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.
Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.
Hot start PCR is a modified form of conventional polymerase chain reaction (PCR) that reduces the presence of undesired products and primer dimers due to non-specific DNA amplification at room temperatures. Because the results of PCR are so useful, many variations and modifications of the procedure were developed in order to achieve a higher yields, hot start PCR is one of them. Hot start PCR follows the same principles as the conventional PCR - in that it uses DNA polymerase to synthesise DNA from a single stranded template, however, it utilises additional heating and separation methods, such as inactivating or inhibiting the binding of Taq polymerase and late addition of Taq polymerase, to increase product yield as well as provide a higher specificity and sensitivity. Non-specific binding and priming or formation of primer dimers are minimized by completing the reaction mix after denaturation. Some ways to complete reaction mixes at high temperatures involve modifications that block DNA polymerase activity in low temperatures, use of modified deoxyribonucleotide triphosphates (dNTPs), and the physical addition of one of the essential reagents after denaturation. The results of this procedure has many applications both medically and industrially. For example, applications of PCR including forensics, paternity testing, biodefence, cloning, mutation detection, genetic testing and DNA sequencing.
Transcription-mediated amplification (TMA) is an isothermal, single-tube nucleic acid amplification system utilizing two enzymes, RNA polymerase and reverse transcriptase.
References
- ↑ Wilson, Ian (October 1997). "Inhibition and facilitation of nucleic acid amplification". Applied and Environmental Microbiology. 63 (10): 3741–3751. PMC 168683 . PMID 9327537.
- ↑ Alaeddini, Reza (2012). "Forensic implications of PCR inhibition—A review". Forensic Science International: Genetics. 6 (3): 297–305. doi:10.1016/j.fsigen.2011.08.006.
- 1 2 3 4 5 6 7 8 9 10 "An Introduction to PCR Inhibitors (Promega Corporation)" (PDF). Retrieved 2007-12-15.
- ↑ Gieffers J, Reusche E, Solbach W, Maass M (2000). "Failure to detect Chlamydia pneumoniae in brain sections of Alzheimer's disease patients". J. Clin. Microbiol. 38 (2): 881–2. PMC 86233 . PMID 10655406.