The 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 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.
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). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
Keck Graduate Institute (KGI) is a private graduate school in Claremont, California. Founded by Henry Riggs and David Galas in 1997, it is the seventh and newest member of the Claremont Colleges.
Cycling probe technology (CPT) is a molecular biological technique for detecting specific DNA sequences. CPT operates under isothermal conditions. In some applications, CPT offers an alternative to PCR. However, unlike PCR, CPT does not generate multiple copies of the target DNA itself, and the amplification of the signal is linear, in contrast to the exponential amplification of the target DNA in PCR. CPT uses a sequence specific chimeric probe which hybridizes to a complementary target DNA sequence and becomes a substrate for RNase H. Cleavage occurs at the RNA internucleotide linkages and results in dissociation of the probe from the target, thereby making it available for the next probe molecule. Integrated electrokinetic systems have been developed for use in CPT.
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
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.
Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism.
TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-La Roche for diagnostic assays and by Applied Biosystems for research applications.
New England Biolabs (NEB) produces and supplies recombinant and native enzyme reagents for the life science research, as well as providing products and services supporting genome editing, synthetic biology and next-generation sequencing. NEB also provides free access to research tools such as REBASE, InBASE, and Polbase.
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.
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.
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 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), or transcription mediated assay (TMA).
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.
MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads of 100x100x20µm or smaller size. This technology is used for analysis of nucleic acid hybridization, specific binding of DNA, and low-molecular weight compounds with proteins, and protein-protein interactions.
The Xpert MTB/RIF is a cartridge-based nucleic acid amplification test (NAAT) for simultaneous rapid tuberculosis diagnosis and rapid antibiotic sensitivity test. It is an automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) DNA and resistance to rifampicin (RIF). It was co-developed by the laboratory of Professor David Alland at the University of Medicine and Dentistry of New Jersey (UMDNJ), Cepheid Inc. and Foundation for Innovative New Diagnostics, with additional financial support from the US National Institutes of Health (NIH).
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 simply by holding a tube. 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.
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.
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.
Transcription-mediated amplification (TMA) is an isothermal, single-tube nucleic acid amplification system utilizing two enzymes, RNA polymerase and reverse transcriptase.
Seegene, Inc is a Korean manufacturer of in vitro diagnostic (IVD) products, particularly molecular diagnostics. Its portfolio includes a range of assays and screening products for sepsis, respiratory diseases such as influenza and respiratory syncytial virus, as well as sexually transmitted infections (STIs). It was founded in 2000. In early 2020, it began developing and distributing a range of tests for SARS-CoV-2, the virus that causes COVID-19.
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