Related Research Articles
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 sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.
A primer is a short, single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. A synthetic primer may also be referred to as an oligo, short for oligonucleotide. 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. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. 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. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.
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. Viral load is measured using body fluids sputum and blood plasma. 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.
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
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. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.
Taq polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq or Taq pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.
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.
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.
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 for diagnostic purposes and a low-cost alternative to detect certain diseases. LAMP is an isothermal nucleic acid amplification technique. In contrast to the polymerase chain reaction (PCR) technology, in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler. LAMP was invented in 1998 by Eiken Chemical Company in Tokyo. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA.
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 modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.
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. Many variations and modifications of the PCR procedure have been developed in order to achieve 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 utilizes 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.
Recombinase polymerase amplification (RPA) is a single tube, isothermal alternative to the polymerase chain reaction (PCR). By adding a reverse transcriptase enzyme to an RPA reaction it can detect RNA as well as DNA, without the need for a separate step to produce cDNA,. Because it is isothermal, RPA can use much simpler equipment than PCR, which requires a thermal cycler. Operating best at temperatures of 37–42 °C and still working, albeit more slowly, at room temperature means RPA reactions can in theory be run quickly by simply holding a tube in the hand. This makes RPA an excellent candidate for developing low-cost, rapid, point-of-care molecular tests. An international quality assessment of molecular detection of Rift Valley fever virus performed as well as the best RT-PCR tests, detecting less concentrated samples missed by some PCR tests and an RT-LAMP test. RPA was developed and launched by TwistDx Ltd., a biotechnology company based in Cambridge, UK.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one step nucleic acid amplification method to multiply specific sequences of RNA. It is used to diagnose infectious disease caused by RNA viruses.
Thermostable DNA polymerases are DNA polymerases that originate from thermophiles, usually bacterial or archaeal species, and are therefore thermostable. They are used for the polymerase chain reaction and related methods for the amplification and modification of DNA.
References
- ↑ Wilson, Ian (October 1997). "Inhibition and facilitation of nucleic acid amplification". Applied and Environmental Microbiology. 63 (10): 3741–3751. Bibcode:1997ApEnM..63.3741W. doi:10.1128/aem.63.10.3741-3751.1997. 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. PMID 21917539.
- 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. doi:10.1128/JCM.38.2.881-882.2000. PMC 86233 . PMID 10655406.