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Related glossaries include Applications Sequencing Technology Assays, Labels, Signaling & Detection; Microarrays. Biology Gene definitions, Sequences, DNA & beyond Additional definitions appear in the In-depth glossary, after the Bibliography.

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. Ferre “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: Genetic variations glossary

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

amplification: Narrower terms gene amplification, nucleic acid amplification, protein amplification, RNA amplification target amplification; signal amplification Assays, Labels, Signaling & Detection  

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

cPCR: See competitive PCR

capture probe: Phage or antibody probes that bind proteins in a sample such that their relative expression levels can be detected. [CHI Microarrays] Broader term probe

comparative genomic hybridization CGH: a molecular Cytogenetic method of screening a tumor for genetic changes. The alterations are classified as DNA gains and losses and reveal a characteristic pattern that includes mutations at chromosomal and subchromosomal levels. [Protocols Online "Comparative Genomic Hybridization] http://www.protocol-online.net/cellbio/cytogenetics/cgh.htm   Related term in situ hybridization

competitive hybridization: See competitive PCR. 

competitive PCR: 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. Ferre "Key issues" in Gene Quantification Birkhauser 1998]

Related terms competitive RT-PCR; competitive immunoassay Assays, labels, signaling & detection glossary

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] http://www.the-scientist.com/yr1998/nov/profile2_981123.html Compare non-competitive RT-PCR

detection: Assays, labels, signaling & detection glossary

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] Broader term in situ hybridization ISH.

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]  Broader term nucleic acid amplification Narrower term PCR. Related terms branched DNA, LCR, NASBA, nested PCR, OLA, PNA, target amplification.

gene quantification: We remain poised at the threshold between relative and absolute quantification. With the resurgence in proteomic activity, the true biological value of low abundance molecules has become alarmingly obvious. An immediate premium is thereby placed on accuracy, sensitivity and reproducibility. This meeting will examine all known approaches to quantifying genes, as well as beginning to consider how quantification of  proteins might clarify or confuse our current picture of biology. Secondarily; that biology has become one of the computer industries fastest growing customer's, points to the problems faced in evaluating huge storehouses of data. Gene Quantification: Introspection and RNA Meditations February 11-12, 2002, San Diego, CA

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]

Narrower terms active hybridization,  competitive hybridization, in situ hybridization ISH, passive hybridization. Related term stringency; Microarrays glossary 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: Use of a DNA or RNA probe to detect the presence of the complementary DNA sequence in cloned bacterial or cultured eukaryotic cells. [DOE]  

Using labeled (radioactive or fluorescent) nucleic acid probes - allows researchers to quantify levels of specific mRNAs within a cell. [CHI Target Validation]

From the Latin "in place". Narrower term FISH, Related term comparative genomic hybridization. Broader term hybridization

In Situ Hybridization Link
Hybridization Histochemistry, WS Young, NIMH and Eva Mezey, NINDS, US http://intramural.nimh.nih.gov/lcmr/snge/Protocols/ISHH/ISHH.html#5

isothermal: Assays, labels, signaling & detection glossary

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]  http://www.ncbi.nlm.nih.gov/Web/Newsltr/Spring99/spring99.htm#DNA 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]

methylation specific PCR: 

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

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. [CHI SNPs Update]

Simultaneous amplification of multiple gene products within the same reaction. Chamberlain, J.S. et al. (1988) Nucl. Acids Res. 16, 11141 [Promega Amplification]

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] http://www.its.bldrdoc.gov/fs-1037/

Originally a math term meaning multiple, later a 19^th century telecommunications term, dating from the telegraph.  [OED]

nucleic acid amplification and detection: Nucleic acid amplification and detection have become the most widely used technique for conducting biological research. Utilization is applied to an increasing range of applications including diagnostics in bench- top research to the clinical arena, genomic screening for drug discovery to toxicology, screening for contamination, and identification of unknown organisms to even discovery of new flora and fauna. Microarray development continues to drive these discoveries.

Nucleic-Acid Based Technologies: Profiling PCR  June 24-26, 2002 • Washington, DC  Related terms gene amplification, PCR, RNA amplification.

PCR Polymerase Chain Reaction: 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]

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. [CHI Microarray]

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]

Broader term gene amplification Related terms nucleic acid amplification and detection, polymerase, Q-PCR, RT-PCR

PCR Links
BioGuide-PCR, Weizmann Institute of Science, Israel http://bioinformatics.weizmann.ac.il/mb/bioguide/pcr/contents.html

PCR and multiplex PCR: Guide and troubleshooting, Octavian Henegariu, Yale Univ., US http://info.med.yale.edu/genetics/ward/tavi/PCR.html

Polymerase Chain Reaction (PCR) JumpStation, Horizon Press, UK http://www.horizonpress.com/pcr/

Polymerase Chain Reaction Xeroxing DNA from MIT's Biology Hypertextbook (via Access Excellence) http://esg-www.mit.edu:8001/esgbio/rdna/pcr.html

polymerase: Any of several enzymes that catalyze the formation of DNA or RNA from precursor substances in the presence of preexisting DNA or RNA acting as templates (i.e., patterns). Glossary of HIV/AIDS - Related Terms, HIV/AIDS Treatment Information Service, June 1999]  http://www.hivatis.org/glossary/pglosary.html

Any enzyme that catalyzes the formation of DNA or RNA from deoxyribonucleotides or ribonucleotides. [ORD]

primer: 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]

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 design, EMBL  http://www.embl-heidelberg.de/~toldo/JaMBW/5/2/   http://www.embl-heidelberg.de/ExternalInfo/geerlof/draft_frames/flowchart/clo_pcr_strategy/primer_design.html

primer extension: Uses primers designed to hybridize with a target, ending one base shy of the SNP position. Single-base primer- extension technology acts on primer- target hybrids to add a single chain-ending nucleotide, often a dideoxynucleotide. The only one of four nucleotides that will extend the primer is the one that is complementary. The identity of the added nucleotide is determined in a variety of ways. A method of SNP detection. [CHI SNP Update]

Primer extension analysis is utilized to quantitate mRNA levels, and to detect low abundance mRNA species. In addition, primer extension analysis can also be utilized to map the 5'- end of transcripts to determine the exact start site(s) for transcription.  [Promega FAQ]   http://www.promega.com/faq/primext.html

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"]

Devices which use detector molecules to detect, investigate, or analyze other molecules, macromolecules, molecular aggregates, or organisms. [MeSH "molecular probe techniques"]

Also called DNA probes, hybridization probes. Related terms oligonucleotide primers, target. Narrower terms capture probes; In-depth ASO probes, molecular beacons, PNA probes. 

Probe Databases see Databases & software directory

See also probes Microarrays glossary.

protein amplification: Potentially, the most revolutionary new technologies are those that impart the equivalent of PCR amplification on proteins. Two of these technologies, discussed in this report, are phage display and Profusion technology. In particular, Profusion has the capability of taking any pool of mRNA, translating it into protein/ mRNA fusion products, and following up any kind of selection protocol on the protein with further amplification. The company that is commercializing this technology, Phylos, is seeking to combine the method with other powerful screening techniques such as microarrays. [CHI Summit Proteomics] Related term Rolling Circle Amplification RCA.

quantitative gene amplification: The quantification of genes has grown to the stature of a mature science. We stand on the threshold between relative and absolute quantification. This will cause a ripple effect throughout the entire process — from the concomitant need for more precise sample preparation to a more complete understanding of what it is that these absolute numbers mean in the biological sense. Can we get around sample contamination measurement interfering with sensitivity by simply setting a cutoff point before the negative controls are tripped? This is an exciting time, as traditional research biology evolves and reaches out to impact, with force, medicine and pharmaceutics and healthcare practice. Gene Quantification: Feb. 11-12, 2002 San Diego, CA

quantitative PCR: 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]  http://pubs.acs.org/hotartcl/ac/99/mar/pcr.html

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. Ferre Gene Quantification Birkhauser 1998] Broader term quantitative gene amplification. Related terms absolute 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]  http://www.nature.com/nbt/web_extras/supp_info/nbt0799_720/

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 glossary 

RNA amplification: Amplification of  messenger RNA (mRNA). Related terms In depth amplified antisense RNA; Expression, genes & beyond glossary  

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]

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] http://www.the-scientist.com/yr2000/sep/profile1_000904.html

Narrower term competitive RT- PCR

real time: [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.

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] http://www.the-scientist.com/yr1998/nov/profile2_981123.html

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

semi-quantitative: See relative quantification.

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): A molecule (usually a protein gene product, but sometimes a DNA sequence, or, in the case of antisense drugs, an mRNA) that may interact with a drug or drug candidate. [CHI Functional Genomics]

There are currently at least two nomenclature systems for referring to hybridization partners. Both use common terms ‘probes’ and ‘targets’ … With respect to the nucleic acids whose entwining represents the hybridization reaction, the identify of one is defined - it tends to be tethered to the solid phase, making up the microarray itself. The identity of the other is revealed by hybridization. The strategy of the ‘standard’ microarray therefore parallels that of a reverse dot- blot, in which the probe is immobilized. For this reason, authors of articles appearing in this supplement have been encouraged to describe the tethered nucleic acid as ‘probe’ and the free nucleic acid as ‘target’. [Chipping Forecast supplement "A note on nomenclature" Nature Genetics 21 (1s): 1 Jan 1999] Has this been standardized yet?  See also target Drug discovery & development glossary.

target amplification: Increasing the amount of target nucleic acid, providing more template for the label, to achieve improved detection. Useful for low levels of expression or abundance or very small sample sizes.

Target amplification increases the amount of target nucleic acid, providing more template for the label and therefore more signal. This approach helps overcome problems associated with low expression of some genes or small sample sizes. The kinetics of the amplification step, however, must be reproduced exactly in these approaches; otherwise, changes observed on the array could be the result of differential amplification. Target- amplification processes include PCR; In- depth Rolling Circle Amplification RCA, Amplified antisense RNA methods and Strand Displacement Amplification SDA. [CHI Microarray]

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

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

Bibliography

[Promega Amplification] Dictionary  http://www.promega.com/amplification/glossary.html

Alpha glossary index

In-depth gene amplification & PCR glossary

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. [Schwindlein]

active hybridization: 

amplified antisense RNA aRNA: Researchers at Stanford University used such a method to produce amplified heterogeneous populations of RNA from limited quantities of cDNA. [Van Gelder RN, et al. "Amplified RNA synthesized from limited quantities of heterogeneous cDNA." Proceedings of the National Academy of Sciences, USA. 1990;87(5):1663-1667.] Specifically, the investigators started by priming whole cerebellar RNA with a synthetic oligonucleotide containing a T7 RNA polymerase promoter sequence. After second- strand cDNA synthesis, T7 RNA polymerase was used to generate aRNA. (aRNA is RNA that is transcribed from the coding, rather than the template, strand of DNA. It is therefore complementary to mRNA.) The investigators reported that this approach achieved up to 80-fold molar amplification from nanogram quantities of cDNA. They also found that the amplicons were similar in size distribution to the parent cDNA and showed sequence heterogeneity. More recently, another group of researchers reported that they had developed a process for optimizing low-abundance RNA, by combining aRNA amplification with template- switching. They found that one round of amplification produced approximately 10³ fold of the estimated amount of starting mRNA, and two rounds produced an approximately 10⁵ fold increase. [Wang E, et al. "High- fidelity mRNA amplification for gene profiling." Nature Biotechnology. 2000;18:457-459.]

aRNA: Amplified antisense RNA.

anneal: The sustained heating of a material such as steel or glass at a specific high temperature, followed by gradual cooling, this is done to eliminate weakness or to produce other qualities.

The pairing of complementary DNA or RNA sequences, via hydrogen bonding, to form a double-stranded molecule. Most often used to describe the binding of a short primer or probe. [OMD]

annealing: The pairing of a mixture of two complementary single- stranded nucleic acids to form double- stranded (duplex) nucleic acids. [Promega website] http://www.promega.com/glossary/

branched DNA: 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. (1989) Clin. Chem. 35, 1571.  [Promega]

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.

exponential RCA: See under Rolling Circle Amplification RCA

hot-start: A method that generally produces cleaner PCR products. Template DNA and primers are mixed together and held at a temperature above the threshold of non- specific binding of primer to template. All the PCR reaction components are added for the extension reaction except one critical reagent (usually the thermostable polymerase). Just prior to the cycling, the missing component is added to allow the reaction to take place at higher temperature. Due to lack of non- specific hybridization of primers to template, the amplified DNA bands tend to be cleaner; the primers don't have a chance to anneal non-specifically. This method is difficult to do because the tubes must be kept on a 100C heat block as your work surface. There are ways to avoid this however. [edited by Jaime Prilusky, from material by: pnh@ncifcrf.gov (Paul N. Hengen) "BioGuide- PCR 1996]  http://bioinformatics.weizmann.ac.il/mb/bioguide/pcr/PCRHot-start.html

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]

immuno-RCA: See under Rolling Circle Amplification RCA

LNA: Long nucleic amplification

linear RCA: See under Rolling Circle Amplification RCA

LCR ligase chain reaction: (Ligase Chain Reaction) - LAR using a thermal stable DNA ligase. Barany, F. (1991) Proc. Natl. Acad. Sci. USA. 88, 189. [Promega]

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] http://www.molecular-beacons.org/#cap1

NASBA: (Nucleic Acid Sequence Based Amplification) - See 3SR. Compton, J. (1991) Nature 350, 91  [Promega]

nested PCR:  A second PCR is performed on the product of an earlier PCR using primers which are internal to the originals. This improves sensitivity without impairing specificity. [Glossary, Pathogen Molecular Biology and Biochemistry Unit, London School of Tropical Medicine and Hygiene ] http://www.lshtm.ac.uk/itd/units/pmbbu/mcnerney/glossary.html

Two-stage amplification reaction. Primers that complement regions of the first stage amplification product are used to amplify a portion of the original PCR product. The use of nested primer pairs significantly increases amplification specificity. Porter-Jordan, K. et. al. (1990) J. Med. Virol. 30, 85.  [Promega]

non-competitive PCR: 

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.

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]

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. [PNA Protocols & Applications, Horizon Press, UK]  http://www.horizonpress.com/hsp/pna.html

PNA JumpStation  http://www.horizonpress.com/gateway/pna.html

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. (1994) Science 265, 2085. [Promega]

passive hybridization: 

probe immobilization:

Profusion technology: Enables one to link messenger RNA (mRNA) to the protein that it encodes. The significance of this is the ability to use advanced nucleic acid- based technologies to access protein, or alternatively to use protein selection methods to access the gene. The procedure begins by isolating mRNA and then translating it in rapid reticulocyte lysates - an in vitro method for generating protein. The mRNA has a DNA linker with a puromycin moiety at its three prime end. During translation in the reticulocytes, the ribosome reads through the mRNA, slows down at the DNA, but continues until it reaches the puromycin. As the ribosome reaches the end of the DNA linker, the puromycin enters into the A-site of the ribosome where it is covalently linked to the carboxy- terminal amino acid of the growing peptide chain. The result is a fusion between mRNA and protein. Because the bond between the gene product and the protein product is covalent, it is very robust and can be purified under a wide variety of conditions. [CHI Summit Proteomics] Related term protein amplification.

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]

RNA polymerase: Sequences, DNA & beyond glossary

reverse transcriptases: The enzymes that perform [the copying of an RNA molecule back into its DNA complement]. [J Buhler, Washington Univ.] http://www.cs.washington.edu/homes/jbuhler/research/array/glossary.html

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. Interestingly, it borrows from another organism in its ability to replicate DNA - the virus. In RCA, similar to many viruses, a polymerase enzyme reads off of a single promoter around a circle of DNA - continuously rolling out linear stretches of the circle. At first it creates a copy of itself, and then it continues to create a concatenated string of multiple copies. This linear RCA reaction can run for three days, producing millions of copies of the small circle sequence. The beauty of the method is that each copy in the amplification is covalently attached. In exponential RCA, a second promoter displaces the double strands at each repeat and initiates hyperbranching in the DNA replication, creating as many as 10¹² copies per hour.  ... Immuno- RCA can provide that capability [protein amplification, analogous to DNA or RNA amplification] Perhaps the most distinctive advantage is the physical localization of signal product to the triggering protein, which enables large scale multiplexing - the ability to resolve the presence of hundreds of analytes at once. This characteristic is essential to the development of proteomic microarrays, it enables in situ cellular assays, and direct molecular haplotyping.  [CHI Summit Proteomics] 

SBH Sequencing by Hybridization: 

Strand Displacement Amplification SDA: 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. 2000;18:199-204.]