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Pharmaceutical PCR & Gene Amplification Glossary & taxonomy
Evolving terminologies for evolving technologies
Questions? Revisions? Comments? Mary Chitty
Last revised January 12, 2015
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What are the odds we'll be able to amplify dinosaur DNA? See Jurassic Park and PCR

Technologies  term index   Finding guide to terms in these glossaries   Site Map 
Related glossaries include
 Technologies Combinatorial libraries & synthesis, Labels, Signaling & Detection, Microarrays, PCR is a key technology for, and an important tool for molecular diagnostics and Sequencing
Biology Gene definitions, DNA, Sequences, DNA & beyond 

Amplified Fragment Length Polymorphism Analysis: The detection of RESTRICTION FRAGMENT LENGTH POLYMORPHISMS by selective PCR amplification of restriction fragments derived from genomic DNA followed by electrophoretic analysis of the amplified restriction fragments.  MeSH 2008

ASO probe (Allele Specific Oligo): The sequence of the oligo is designed in such a way to allow/ inhibit hybridization in the spot where the mutant (resistant) allele differs from the wild type (susceptible) allele. [Schlwindlein]

absolute quantification: To express true value, scientists (including the author) have been using absolute quantification … it does not appear to be a good fit for gene quantification, and thus the use of this terminology should be discouraged … at this low level, a probability rather than an absolute number defines the true copy number value for a given sample.  [Francois. Ferré “Key issues” Gene Quantification Birkhauser 1998]

Attempts to state the number of copies of a specific RNA per cell or unit mass of tissue.  Requires a number of extra conditions and treatments that relative quantification does not. [WM Freeman et al “Quantitative RT-PCR: Pitfalls and Potential” Biotechniques 26: 112-125 Jan 1999] Related term: relative quantification.

allele specific hybridization: SNPs & Genetic variations

amplicon: A cloned, amplified  (by PCR), DNA sequence.  [Glick]

amplification: Narrower terms: gene amplification, kinetic PCR, kinetic RT- PCR, LCR, microamplification, NASBA, nested PCR, nucleic acid amplification, protein amplification, RNA amplification, transcript mediated amplification, target amplification; signal amplification; RNA amplified antisense RNA;   

amplimer: PCR-amplified segment of the genome (including STSs and ESTs) [GDB, ORNL link]

Region of DNA sequence which is amplified during a PCR reaction and which is defined by a pair of PCR primers (these primer pairs are sometimes called amplimers).

anneal: The biochemical process of hybridising or binding two segments of complementary nucleic acid at an optimal temperature of 40-65ºC. [Roche PCR no longer on web] 

branched DNA bDNA: Direct detection of target sequences by hybridization with a branched DNA probe and target specific oligonucleotides. Alkaline phosphatase- conjugated oligonucleotides, complementary to the branched DNA complex, are detected using a chemiluminescent substrate. Whereas PCR amplifies target sequences, the bDNA assay amplifies signal. Urdea, M.S. et al. Clinical Chemistry 35, 1571, 1989 Promega

branched DNA signal amplification assay: A molecular probe technique that utilizes branched DNA (bDNA) as a means to amplify the hybridization signal. One end of the bDNA molecule is designed to bind a specific target, while the other end of the bDNA molecule contains many branches of DNA that are designed to bind a probe used for signal detection. MeSH, 2001

capture probe: Phage or antibody probes that bind proteins in a sample such that their relative expression levels can be detected. Broader term: probe; Related term: reporter probe

comparative genomic hybridization CGH: Cell & tissue technologies

competitive hybridization: Another critical feature of most microarrays is that different samples (often two - control and sample) can be hybridized, competitively, to the same array. This competitive hybridization is possible because fluorescent dyes with different emission spectra can be used on different samples, allowing samples tagged with the various dyes to be discriminated in imaging.  Related term: competitive PCR. 

competitive PCR cPCR: During the last few years, many efforts have been made to provide suitable controls to convert PCR to a quantitative method ... One group [of methods] relies on external calibration ... among [these] the competitive PCR methods (cPCR) are the most robust and reliable.  They are based on a co- amplification of the target DNA (sample) with a homologous or heterologous DNA standard (competitor) which competes with the sample template DNA for the same set of PCR primers. [S Rupf, K. Eschrich,  "Quantification of bacteria by competitive polymerase chain reaction" American Laboratory: 44-  46, July 2000]

An internal control, close in composition to the target nucleic acid, competes with the latter for reagents (such as common primers) in the same reaction tube … represents the prototype for the so- called end- point quantitative methods, in which quantification is based on the amount of amplified material (amplicon) obtained at the last amplification cycle. [F. Ferré "Key issues" in Gene Quantification Birkhauser 1998]  Related terms competitive RT-PCR; competitive immunoassay Labels, signaling & detection

competitive RT-PCR: Should be that - a competition between a known amount of a template and an unknown target. This method avoids difficulties created by differences in the efficiency of the PCR reaction itself with different template/ primer sets. A competitive template binds the same primers but has been altered in some way (small deletions, point mutation) to provide a product that is distinguishable from the target itself. [Laura De Francesco, "Taking the Measure of the Message" The Scientist 12[23]: 20, Nov. 23, 1998]   Compare non-competitive RT-PCR

cross hybridization: Microarrays categories 

detection, direct detection: Labels, signaling & detection

digital PCR dPCR: A new application in research and development allowing for higher sensitivity and specificity than real-time PCR. This allows researchers to closely examine rare genetic mutations (including single nucleotide polymorphisms), copy number variations, viruses, prenatal defects, and much more. National measurement institutions have also been looking into digital PCR as a reference standard as well as using its precision and accuracy to create reference standards for diagnostic labs.  Digital PCR Technology Report: An Insight to Vendors, Costs & the End User Community, Insight Pharma Reports, 2013 

Digital PCR, at its core, is simply a single molecule counting method that quantitatively measures absolute DNA and eliminates the need for standard curves. However, it has already shown potential to be a disruptive technology in many areas of diagnostics.  Digital PCR Technologies and Tools for Precision Diagnostics  October 6-8, 2014 • La Jolla, CA  Program | Register | Download Brochure   Digital PCR Applications and Advances  

dMIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines.

DNA amplification: See gene amplification, PCR  

DNA probes: Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure DNA- DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA :RNA hybrid- specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections. MeSH, 1989

end-point: The traditional measurements of product … analyze the reaction after it is completed can be accomplished through the use of fluorescent intercalating dyes or through measurement of  incorporated radioactivity by autoradiography or phosphor imaging … Southern blots or fluorescence detection are also used. A third type uses solid- state approaches in which a bound enzyme produces fluorescence or luminescence. Finally, several laboratories measure the production of amplification products following resolution by HPLC or capillary electrophoresis. [WM Freeman et al. "Quantitative RT-PCR: Pitfalls and Potential" Biotechniques 26: 112-125 Jan 1999] Compare real time.

FISH Fluorescence In Situ Hybridization: A type of IN SITU HYBRIDIZATION in which target sequences are stained with fluorescent dye so their location and size can be determined using fluorescence microscopy. This staining is sufficiently distinct that the hybridization signal can be seen both in metaphase spreads and in interphase nuclei. MeSH, 1993  Broader terms: hybridization, in situ hybridization ISH. Narrower term: chromosome painting  Related term: Labels, signaling & detection  fluorescence  

gene amplification: An increase in the number of copies of a specific gene in an organism. This can lead to the production of a corresponding protein at elevated levels. [IUPAC Compendium]

A selective increase in the number of copies of a gene coding for a specific protein without a proportional increase in other genes. It occurs naturally via the excision of a copy of the repeating sequence from the chromosome and its extrachromosomal replication in a plasmid, or via the production of an RNA transcript of the entire repeating sequence of ribosomal RNA followed by the reverse transcription of the molecule to produce an additional copy of the original DNA sequence. Laboratory techniques have been introduced for inducing disproportional replication by unequal crossing over, uptake of DNA from lysed cells, or generation of extrachromosomal sequences from rolling circle replication. MeSH, 1980  Broader term: nucleic acid amplification Narrower term: PCR Related terms branched DNA, LCR, NASBA, nested PCR, OLA, PNA, target amplification  Narrower terms: kinetic PCR, real- time PCR; Related term: Assays & screening quantitative assays
Gene Quantification,
Technical Univ. Munich, Germany

heteroduplex analysis:  A method of detecting gene mutation by mixing PCR- amplified mutant and wild- type DNA followed by denaturation and reannealing. The resultant products are resolved by gel electrophoresis, with single base substitutions detectable under optimal electrophoretic conditions and gel formulations. Large base pair mismatches may also be analyzed by using electron microscopy to visualize heteroduplex regions. MeSH, 1999

hot-start: PCR reaction in which a necessary component for polymerization (polymerase, magnesium, nucleotides, etc.) is withheld from the reaction until all other components achieve a temperature exceeding the annealing temperature of the primers.  This process minimizes amplification artifacts associated with low temperature extension of misprimed oligonucleotides. C.R. Newton et. al. Nucleic Acids Research 17: 2503, 1989. [Promega]

hybridization: 1. The formation of stable duplexes of two DNA and/ or RNA (complementary) strands via Watson- Crick base pairing used for locating or identifying nucleotide sequences and to establish the effective transfer of nucleic acid material to a new host. 2. The formation of a novel diploid organism either by sexual processes or by protoplast fusion. [IUPAC Biotech]

A chemical reaction in which single-stranded DNA or RNA molecules combine to form double-stranded complexes, including, for example, the famous DNA double helix. The reaction obeys the usual base-pairing rules, sometimes called Watson- Crick base pairing, in which adenine (A) binds to thymine (T) (or uracil [U], in the case of RNA), and cytosine (C) binds to guanine (G). Binding occurs through the formation of hydrogen bonds between the paired bases, which are much weaker than the covalent bonds that bind the elements of each strand. 
Narrower terms: active hybridization,  competitive hybridization, in situ hybridization ISH, passive hybridization. Related terms: anneal, stringency; Microarrays blotting

hybridization stringency: The percentage of nucleotides which must match on two unrelated single- stranded nucleic acid molecules before they will base pair with each other to form a duplex, given a certain set of physical and chemical conditions. The hybridization stringency is used to determine when a hybridization probe and a target nucleic acid will come together, and can be set by the researcher by varying the conditions. In general, if the percentage of matching nucleotides is lower than 70 percent, the two single- stranded nucleic acid molecules are considered nonhomologous and any hybridization is considered nonstringent. [Life Sciences Dictionary]   Broader term: stringency

in situ hybridization ISH: A technique that localizes specific nucleic acid sequences within intact chromosomes, eukaryotic cells, or bacterial cells through the use of specific nucleic acid-labeled probes. MeSH, 1993

Using labeled (radioactive or fluorescent) nucleic acid probes - allows researchers to quantify levels of specific mRNAs within a cell.  From the Latin "in place".  Narrower terms: FISH, primed in situ labeling; Related term comparative genomic hybridization;  Broader term: hybridization  
, WS Young, NIMH and Eva Mezey, NINDS, US

isothermal: Labels, signaling & detection

Jurassic Park and PCR: Although GenBank lacks dinosaur DNA, fragments of genomes past can be found here. A practical limit of about 100,000 years currently applies to the age of recoverable DNA samples. Beyond this limit, hydrolysis of the phosphate backbone of the DNA and oxidative damage to the bases that make up the DNA sequence become too great to allow for efficient PCR amplification. This is why deposition of significant amounts of dinosaur sequence (age > 65 million years) in GenBank is unlikely to occur in the near future. However, many DNA sequences arising from extinct organisms and ancient genomes [including from a Neanderthal, the late Neolithic "Iceman", Egyptian mummies, woolly mammoths, a quagga, a moa and medieval French rabbits] are in the database today, and the number is expected to grow as technology for the extraction and amplification of aged DNA progresses.  "DNA Sequences from Times Past in GenBank" NCBI News, Spring 1999 Sequences

kinetic PCR: See real time PCR.

kinetic RT-PCR: Direct use of amplification kinetics to quantify RNA without the use of a standard. Started as an attempt to avoid the long development times of standard construction and the problems of designing, storing and accurately quantifying the standard itself. ... never been widely used, recent advances in detection methods suggest that this concept has the greatest potential for future quantitative RT-PCR development. [WM Freeman et al “Quantitative RT- PCR: Pitfalls and Potential” Biotechniques 26: 112- 125 Jan 1999]

LCR Ligase Chain Reaction: A DNA amplification technique based upon the ligation of OLIGONUCLEOTIDE PROBES. The probes are designed to exactly match two adjacent sequences of a specific target DNA. The chain reaction is repeated in three steps in the presence of excess probe: (1) heat denaturation of double- .stranded DNA, (2) annealing of probes to target DNA, and (3) joining of the probes by thermostable DNA ligase. After the reaction is repeated for 20- 30 cycles the production of ligated probe is measured.  [MeSH, 2001]

LAR using a thermal stable DNA ligase. F. Barany Proceedings of the National Academy of Sciences PNAS USA. 88, 189, 1991. [Promega]

linear RCA: See under Rolling Circle Amplification RCA

microamplification: A novel alternative to microcloning for the production of region specific chromosomal DNA is described. In this method, 'microamplification', single bands are dissected from polytene chromosomes and digested with Sau3A. Oligonucleotide adaptors are ligated to these fragments to provide convenient priming sites for polymerase chain reaction amplification. In this way, as much as 1 microgram of DNA can be amplified from a single band. Probes made from PCR amplified DNA from two such dissections have been used to probe cloned DNA form a 100 kb chromosome walk. Whereas conventional microcloning has generated cloned EcoRI fragments corresponding to 3-4 kb of the walk, the PCR probes cover greater than 90% of this chromosomal region. Thus microamplification is significantly more effective than microcloning in providing probes for establishing chromosomal walks. [RD Saunders et. al., PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning, Nucleic Acids Research 17(22): 9027- 9037, Nov. 25, 1989

miniaturization, PCR: We will create a single, integrated platform where PCR primers are synthesized in tandem, and PCR amplification is initiated by the release of the PCR primers, and progress of the PCR reactions is monitored in Real-Time. Furthermore, such reactions will be multiplexed in a high-throughput device with sub-microliter reaction volumes, and will allow at least 1536 PCR reactions to be performed and monitored in parallel. PCR Miniaturization, Genome Technology Center, Stanford Univ School of Medicine, 2007 

molecular beacons: Oligonucleotide probes that can report the presence of specific nucleic acids in homogeneous solutions (Tyagi and Kramer 1996). They are useful in situations where it is either not possible or desirable to isolate the probe- target hybrids from an excess of the hybridization probes, such as in real- time monitoring of polymerase chain reactions in sealed tubes or in detection of RNAs within living cells. Molecular beacons are hairpin- shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid.  [F Kramer et al "Molecular Beacons" 2000]

multiplex: A sequencing approach that uses several pooled samples, greatly increasing sequencing speed. [DOE]

Simultaneous amplification of multiple gene products within the same reaction. Chamberlain, J.S. et al. Nucleic Acids Research 16, 11141, 1988 

The combining of two or more information channels onto a common transmission medium. Note: In electrical communications, the two basic forms of multiplexing are time- division multiplexing (TDM) and frequency- division multiplexing (FDM). In optical communications, the analog of FDM is referred to as wavelength- division multiplexing (WDM). [Glossary of Telecommunications Terms, National Telecommunications System, Technology and Standards Division, 1996]

In general, primer- extension technologies are amenable to high- throughput applications and automation, yet only very low levels of multiplexing are possible. Higher multiplexing can be accomplished by combining primer- extension technology with microarray technology.  

Originally a math term meaning multiple, later a 19th century telecommunications term, dating from the telegraph. Oxford English Dictionary  Related terms: Sequencing

NASBA Nucleic Acid Sequence Based Amplification: Use Self Sustained Sequence Based Amplification MeSH entry term 2001

non-competitive RT-PCR: The native signal is unaltered by the standard. An increasing series of standard amounts is co- amplified with equal amounts of total experimental RNA; however, this occurs under conditions in which there is no competition for the components in the PCR. The quantification is therefore estimated on a linear scaled graph. The amount of standard signal is plotted against the native signal. When the lines intersect, they reach the equivalence point, and quantification is achieved. .. Generally some estimate of the amount of native signal must be made before deciding on the standard amounts, because they are designed to differ by only one log above and below the native. [WM Freeman et al “Quantitative RT- PCR: Pitfalls and Potential” Biotechniques 26: 112-125 Jan 1999]  Compare competitive RT- PCR.

Nucleic Acid-Based Technologies: Nucleic acid isolation and purification is one of the most technically challenging and labor-intensive procedures performed in any laboratory whether it be for biodefense, drug discovery, or diagnostics. Standardization of sample preparation is an ongoing issue in the field. Whatever technology is selected, success depends on a balanced combination of good experimental design, sample preparation, primer/probe design, amplification, detection, and analysis as well as the selection of equipment and reagents. 

nucleic acid amplification: Narrower terms:  DNA amplification, gene amplification, PCR, RNA amplification.
Nucleic acid amplification protocols,
Promega guide

nucleic acid amplification techniques: Laboratory techniques that involve the in-vitro synthesis of many copies of DNA or RNA from one original template MeSH, 2001

nucleic acid probes: Nucleic acid which complements a specific mRNA or DNA molecule, or fragment thereof; used for hybridization studies in order to identify microorganisms and for genetic studies. MeSH, 1989

Nucleic Acid Testing NAT:  We, FDA, are issuing this guidance to provide you, manufacturers of plasma-derived products, with recommendations for performing nucleic acid testing (NAT) for human provirus B19 as an in-process test for Source Plasma and recovered plasma used in the further manufacturing of plasma-derived products.  Such testing will identify and help to prevent the use of plasma units containing high levels of parvovirus B19.  This guidance also recommends how to report to FDA implementation of parvovirus B19 NAT. We recognize that in the current business practice for parvovirus B19 NAT in-process testing, several weeks can elapse between collection of the units of Source Plasma or recovered plasma and identification of B19 NAT-positive pools or units.  We encourage manufacturers of plasma-derived products to employ practices that will reduce the time between product collection and in-process testing to allow for the meaningful notification of blood and plasma collection establishments of positive test results within the dating period of any blood components intended for use in transfusion. Guidance for Industry Nucleic Acid Testing (NAT) to Reduce the Possible Risk of Parvovirus B19 Transmission by Plasma-Derived Products, FDA 2009 

OLA Oligonucleotide Ligation Assay: Mutation detection based upon the observation that two oligonucleotides annealing adjacent to each other are a suitable substrate for T4DNA Ligase only if there are no base mismatches. [Landegren, U. et al. Science 241: 1077, 1998] See Ligase- Mediated Gene Detection. [Promega]

oligos, oligonucleotides: Biomolecules

PCR See also Polymerase Chain Reaction
PCR for Molecular Medicine February 16-18, 2015 • San Francisco, CA Program | Register | Download Brochure
PCR for Molecular Medicine

PNA Peptide Nucleic Acid: An analogue of DNA in which the backbone is a pseudopeptide rather than a sugar. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. Peptide Nucleic Acids: Protocols and Applications, Horizon Press, 1999 

DNA analogs containing neutral amide backbone linkages composed of aminoethyl glycine units instead of the usual phosphodiester linkage of deoxyribose groups. Peptide nucleic acids have high biological stability and higher affinity for complementary DNA or RNA sequences than analogous DNA oligomers. MeSH, 1999

PNA probes (peptide nucleic acid) : Made of PNA rather than DNA, attempts to address the intrastrand hybridization problem. [A Marshall & J Hodgson “DNA chips: an array of possibilities” Nature Biotechnology 16 (1): 27- 31 Jan 1998]

padlock probes: Target detection protocol using an oligonucleotide probe that contains two target -complementary sequences (20bp) connected by a 50bp linker. If the target- complementary regions anneal adjacent to each other on the template, they are substrates for DNA ligase (see Ligase Mediated Gene Detection). Ligation concatenates the probe (similar to two intertwined links on a chain) to the target. The concatenated complex can be resolved by denaturing gel electrophoresis.  [Nilsson, M. et al. Science 265: 2085, 1994 [Promega] 
Related term protein amplification

Polymerase Chain Reaction PCR: A laboratory technique to rapidly amplify pre- determined regions of double- stranded DNA. Generally involves the use of a heat stranded DNA polymerase. IUPAC Bioinorganic

The polymerase chain reaction is a technique in molecular biology which is used in genetic engineering and diagnosis. Special techniques are used to identify genes or gene sections (e.g. of viruses or on the chromosomes of cancer cells). These are amplified with the help of an enzyme (polymerase), so that they can be measured or used for other purposes in genetic engineering. 
Applications: Diagnosis of hereditary diseases, even before birth. Diagnosis of certain infectious diseases and types of cancer. The polymerise chain reaction makes it possible to prepare thousands of millions of copies of genetic material within a few hours. This permits the detection of and RNA even before antibodies are formed. The polymerase chain reaction allows not only the diagnosis of a disease at a very early stage, but makes it possible to follow the development of the disease and its response to therapy in a highly precise manner. Roche Glossary, . F Hoffmann-La Roche Ltd, 2011 [no longer on web]

In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double- stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult to isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. MeSH, 1991

Nucleic acid isolation and purification is one of the most technically challenging and labor-intensive procedures performed in any laboratory whether it be for biodefense, drug discovery, or diagnostics. Standardization of sample preparation is an ongoing issue in the field. Whatever technology is selected, success depends on a balanced combination of good experimental design, sample preparation, primer/probe design, amplification, detection, and analysis as well as the selection of equipment and reagents.

Originally described in 1984 by Kary B. Mullis, who shared the Nobel Prize for Chemistry for this invention in 1993, PCR enables the amplification of specific nucleotide sequences through the use of a DNA polymerase. The sequence to be amplified is identified through the use of synthetic oligonucleotides that are complementary to the two terminal regions of the targeted sequence. 
Broader terms: gene amplification, nucleic acid amplification and detection; Related terms: polymerase, primers, RT-PCR; Narrower term: Q-PCR 

PCR and multiplex PCR: Guide and troubleshooting, Octavian Henegariu, Yale Univ., US  
Polymerase Chain Reaction (PCR) JumpStation
, Horizon Press, UK

polymerase DNA or RNA: Enzymes that catalyze the synthesis of nucleic acids on preexisting nucleic acid templates, assembling RNA from ribonucleotides or DNA from deoxyribonucleotides.  DOE Human Genome Program Report, Glossary  

Any enzyme that catalyzes the formation of DNA or RNA from deoxyribonucleotides or ribonucleotides. [ORD]  Narrower term: Taq polymerase

primed in situ labeling: A technique that labels specific sequences in whole chromosomes by in situ DNA chain elongation or PCR (polymerase chain reaction).   MeSH, 1999

primer- DNA: Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. MeSH, 1994

A molecule that initiates the synthesis of a larger molecule. For example, a short synthetic piece of DNA serves as a primer to initiate template- directed DNA synthesis in PCR. [NHLBI] 
Narrower term: primer extension. Related term probes primer dimer: 
Artifacts, non- target amplification products, caused by homologies within primers.

primer extension: is used to map the 5' ends of DNA or RNA fragments. It is done by annealing a specific oligonucleotide primer to a position downstream of that 5' end. The primer is labeled, usually at its 5' end, with 32P. This is extended with reverse transcriptase, which can copy either an RNA or a DNA template, making a fragment that ends at the 5' end of the template molecule. DNA polymerase can also be used with DNA templates. John W Little, University of Arizona 2010 

A method of SNP detection.

primers, real time: Considering the time and cost of designing and optimizing primer sets along with the relatively large number of candidate genes that are identified by microarray studies, the development of a central repository for primer sets, reaction conditions and even the actual oligonucleotides would benefit all investigators involved in genomics and other types of experiments. Therefore, we would like to use this site to serve the needs of researchers who are interested in both contributing to and taking advantage of information about quantitative real time PCR.  Steven W. Johnson,  "Real time PCR primer sets" 

probe set: The Affymetrix design represents each gene by a collection of probes called a probe set. Each probe set contains multiple probe pairs; the current design uses 11 probe pairs per gene, while earlier designs used 20. Each probe pair consists of two probes—one called a perfect match (PM) and the other called a mismatch (MM). The perfect match is an oligonucleotide whose sequence exactly matches the gene of interest; the mismatch differs from the perfect match at one base position in the middle of the sequence. 

probes: A specific DNA or RNA sequence which has been labelled by radioactivity, fluorescence labels or chemiluminescence labels and which is used to detect complementary sequences by hybridization techniques, such as blotting or colony hybridization. [IUPAC Compendium]

Biomolecular probes used "to measure the presence or concentration of biological molecules, biological structures, microorganisms, etc. by translating a biochemical interaction at the probe surface into a quantifiable physical signal. MeSH "biosensing techniques", 1999

Devices which use detector molecules to detect, investigate, or analyze other molecules, macromolecules, molecular aggregates, or organisms. MeSH "molecular probe techniques", 1991 Related terms: oligonucleotide primers, target. Narrower terms: capture probes, ASO probes, molecular beacons, nucleic acid probes, PNA probes, padlock probes, RNA, probes; Narrower [or equivalent?] terms: DNA probes, hybridization probes, Labels, signal & detection See also probes Microarrays  discussing the ambiguities of probe and target designations.

quantitative gene amplification: See gene quantification. Narrower term: quantitative PCR

quantitative PCR: A valuable technology used for diagnostics of diseases such as cancer and infectious diseases, and for the detection of bacterial, fungal and viral pathogens. The introduction of novel technology platforms such as digital PCR, high-throughput platforms and improvements in automation and standardization may provide useful tools for diagnosis, prognosis and therapeutic evaluations for both the pharmaceutical industry and the medical community to move the application of qPCR to the next level.
Quantitative Real-Time PCR for Molecular Diagnostics  

Despite recent attention focused on this technique, quantitation is an old idea- almost as old as PCR itself. "Quantitative PCR has been happening all along," says François Ferré, who heads the gene quantification company Althea Technologies. In the early 1990s, for example, Michael Piatak, Ferré, and others used quantitative PCR to show that HIV viral loads - the degree of infection - in patients' blood were higher than previously thought (3, 4). "Even back in 1989, at meetings focused on PCR, quantitation was a hot topic," Ferré says .. As genes are located, their functions need to be determined, and studies of gene expression become the focus. "The next dimension of research is to figure out what is expressed and how much and when," says Mike Lucero, product marketing manager for PCR at Perkin- Elmer. "That is just as basic as knowing what the DNA sequence is." .. When many researchers say "quantitative PCR", they mean kinetic or real-time PCR. [Analytical Chemistry News & Features, March 1, 1999 191A-195A]

Intended either to determine the number of copies of a given nucleic acid sequence, or more generally to determine the relative abundance of two sequences. [J Peccoud and C Jacob "Statistical Estimations of PCR Amplification Rates" in F. Ferré Gene Quantification Birkhauser 1998] Related terms: absolute quantification, gene quantification, QRT- PCR, relative quantification, relative QRT- PCR

quantitative RT-PCR QRT-PCR: Has evolved into a widely used tool for sensitive detection and quantitation of low- abundance RNA species. As the focus of genomic research is shifting from the location of genes towards functional genomics there is a growing demand for techniques capable of accurately quantitating differences in mRNA levels in different settings. J. Stenman et al. Supplement to: Accurate determination of relative messenger RNA levels by RT-PCR" Nature Biotechnology supp 17: 720- 722 July 1999

The RT step is the source of most of the variability in a quantitative RT-PCR experiment. The final step in  QRT- PCR is the detection and quantification of amplification products. Inherently an indirect method of measurement. [WM Freeman et al "Quantitative RT-PCR: Pitfalls and Potential" Biotechniques 26: 112-125 Jan 1999]  Related term: differential display Expression, genes & beyond

Random Amplified Polymorphic DNA Technique RAPD: Technique that utilizes low stringency polymerase chain reaction (PCR) amplification with single primers of arbitrary sequence to generate strain specific arrays of anonymous DNA fragments. RAPD technique may be used to determine taxonomic identity, assess kinship relationships, analyze mixed genome samples, and create specific probes. MeSH, 1996

random multirecombinant PCR: See under combinatorial protein libraries Combinatorial Libraries & synthesis 

real time PCR: [Russell] Higuchi and co- workers [at Roche] developed a system in which PCR products can be detected in real time, meaning that the accumulation of PCR products could be visualized at each cycle using an intercalating dye, such as ethidium bromide, an ultraviolet source, and a CCD camera … also referred to as "kinetic PCR". F. Ferre Gene Quantification Birkhauser 1998

Real-time determinations monitor the reaction in the thermal cycler as it progresses …offers the potential for improved quantification. The errors in sample manipulation for end- point quantification are minimized and a great deal more information about the PCR is obtained from the data points for each cycle. [WM Freeman et al "Quantitative RT- PCR: Pitfalls and Potential" Biotechniques 26: 112-125 Jan 1999] Compare end-point Related terms: kinetic PCR, kinetic RT-PCR

relative quantification: The fact that relative quantification is perceived as poor quantitative information has led to the false impression that it is essential to publish copy numbers to increase the credibility of the results. …methods capable of relative quantitation can provide extremely valuable quantitative information. The quantitative power of such methods will be directly proportional to the extent of their dynamic ranges and to the tightness of their precision.  [F. Ferre “Key issues” in Gene Quantification Birkhauser 1998]

Determines the changes in steady-state expression of a gene. For the purposes of the vast majority of investigators, relative quantification is adequate. Semi- quantitative is sometimes used as a synonym for relative quantification; however, it is not an optimal term because of the confusion it causes and its imprecise nature.  [WM Freeman et al “Quantitative RT-PCR: Pitfalls and Potential” Biotechniques 26: 112-125 Jan 1999] Related term: absolute quantification

relative quantitative RT-PCR: Uses an internal standard to monitor each reaction and allow comparisons between different reactions to be made. To do this, a second set of primers is incorporated into the reaction for an invariant, housekeeping message. The difficulty here is matching the level of the internal message to that of the target so that one reaction doesn't dominate ... Finally, purely exogenous standards (template plus primer) may be added to the reaction, which can give a signal against which the unknown can be compared, providing a relative estimate of the amount of a target species. [Laura De Francesco, "Taking the Measure of the Message" The Scientist 12[23]:20, Nov. 23, 1998] 

reporter probe: Broader term: probe; Related term: capture probe
Reverse Transcriptase PCR: See RT-PCR.

reverse transcriptases: Enzymes found in retroviruses that can synthesize complementary single strands of DNA from an mRNA sequence as template. They are used in genetic engineering to produce specific cDNA molecules from purified preparations of mRNA. [IUPAC Compendium]  Related term: RT-PCR

RNA amplification, RNA polymerase : RNA

Rolling Circle Amplification RCA: An alternative technology to PCR, discovered in 1995 by Paul Lizardi at Yale University, and may prove to have as large an impact as PCR.

RT-PCR Reverse Transcriptase Polymerase Chain Reaction: A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols. MeSH, 1999

A method for assessing gene expression, detecting low copy number mRNA transcripts, and generating complementary DNAs (cDNAs) for cloning. [Aileen Constans "Lab Consumer Reverse Psychology" Scientist 14 (7): 29 Sept 4, 2000]  Narrower term: competitive RT- PCR  Related terms: Expression gene & protein

SBH Sequencing by Hybridization: A novel DNA sequencing technique in which an array (SBH chip) of short sequences of nucleotides (probes) is brought in contact with a solution of (replicas of) the target DNA sequence. A biochemical method determines the subset of probes that bind to the target sequence (the spectrum of the sequence), and a combinatorial method is used to reconstruct the DNA sequence from the spectrum. [Franco Preparata, Eli Upfel, Samuel Health, Sequencing by Hybridization, Brown Univ. 1999]

self-sustained sequence replication: An isothermal in-vitro nucleotide amplification process. The process involves the concomitant action of a RNA- DIRECTED DNA POLYMERASE, a ribonuclease (RIBONUCLEASES), and a DNA- DIRECTED RNA POLYMERASE to synthesize large quantities of sequence- specific RNA and DNA molecules. MeSH, 2001

semi-quantitative: See relative quantification.

sexual PCR (molecular diversity):  A form of PCR in which similar, but not identical, DNA sequences are reassembled to obtain novel juxtapositions, simulating the result of genetic recombination. The result is the creation of an array of related genes which may possess improved characteristics. By repeated rounds of recombination, selection and PCR- based amplification vastly improved gene- products, such as enzymes with greater activity, may be generated and selected.  [Bioinformatics glossary, Bioinformatics Research Group, Roswell Park Cancer Institute, US, 2002]   Related terms: gene shuffling, genome shuffling, molecular breeding, molecular diversity 

signal amplification methods:  Increasing the amount of signal per unit of target for better detection. Because of the minute scale of hybridization, and the small amounts of target present, it is desirable to increase the signal from an array. This signal- boosting can be accomplished either by increasing the amount of target (target amplification) or increasing the amount of signal per unit of target (signal amplification). Narrower terms:  branched DNA bDNA, Rolling Circle Amplification, Hybrid Capture® (HC) technology, Tyramide Signal Amplification TSA 

Strand Displacement Amplification SDA: Uses two types of primers and two enzymes (DNA polymerase and restriction endonuclease) to exponentially produce single- stranded amplicons asynchronously. A group of researchers described the use of this method in microelectronic chip experiments. In particular, sets of the amplification primers were electronically anchored to distinct zones on the chip to reduce primer- primer interactions. The researchers reported that this "anchored SDA" approach enabled multiplex DNA or RNA amplification without decreasing amplification efficiency. [Westin L, et al. "Anchored multiplex amplification on a microelectronic chip array." Nature Biotechnology 18: 199- 204, 2000]

stringency: Reaction conditions - notably temperature, salt, and pH - that dictate the annealing of single- stranded DNA/ DNA, DNA/ RNA, and RNA/ RNA hybrids. At high stringency, duplexes form only between strands with perfect one- to- one complementarity; lower stringency allows annealing between strands with some degree of mismatch between bases. Susan A. Hagedorn [Life Sciences Dictionary]  Narrower term: hybridization stringency

Taq polymerase: The thermostable DNA polymerase from Thermus aquaticus (Taq) has been the most extensively used enzyme in PCR. T. aquaticus was first isolated from a hot spring in Yellowstone National Park. CR Newton & A Graham PCR Bios Scientific Publishers 1994

target (hybridization), target amplification: Drug targets 

template: The nucleic acid single strand that is copied during replication or transcription. [IUPAC Biotech]

transcription mediated amplification TMA: Infectious disease diagnostic assays using transcription- mediated amplification (TMA) NAAT have become increasingly popular in many clinical microbiology laboratories. Recent technology developments have improved the performance and simplified the use of the TMA assays. These new technologies have been applied to the development of multiplex TMA tests to improve the testing accuracy for organisms, such as Chlamydia trachomatis and Neisseria gonorrhoeae in clinical microbiology laboratories. TMA tests for HIV-1 and HCV have also led to improvements in blood bank testing which can improve the safety of the public blood supply.  CS Hill, Molecular diagnostic testing for infectious diseases using TMA technology, Expert Rev Mol Diagn 1 (4) :445- 455, Nov 2001

whole genome amplification:  The concept of whole genome amplification is something that has arisen in the past few years as the polymerase chain reaction (PCR) has been adapted to replicate regions of genomes that are of biological interest. The applications are many - forensic science, embryonic disease diagnosis, bioterrorism genome detection, "immortalization" of clinical samples, microbial diversity, and genotyping. [TL Hawkins et. al. "Whole genome amplification: applications and advances" Current Opinion in Biotechnology 13(1): 65- 67, Feb. 2002] 

Digital PCR Technology Report: An Insight to Vendors, Costs & the End User Community, Insight Pharma Reports, 2013 
Advances: Comparative Genomic Hybridization: Current State and Future Directions , 2006  Insight Pharma Reports

454 Glossary, Roche Diagnostics 1996-2011 
Real Time PCR Glossary, M Tevfik Dorak 2010 

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IUPAC definitions are reprinted with the permission of the International Union of Pure and Applied Chemistry.

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