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Applications Drug & disease targets allosteric ribozymes (allozymes):
RNA and DNA molecules can be engineered to function as molecular switches
that trigger catalytic events when a specific target molecule becomes bound.
Recent studies on the underlying biochemical properties of these constructs
indicate that a significant untapped potential exists for the practical
application of allosteric nucleic acids. Engineered molecular switches can be
used to report the presence of specific analytes in complex mixtures, making
possible the creation of new types of biosensor devices and genetic control
elements.
Engineered
allosteric ribozymes as biosensor components.
Breaker RR. Curr Opin Biotechnol. 2002
Feb;13(1):31-9. aptamers: Technologies overview Bacterial artificial chromosome BAC: A vector used to clone DNA fragments (100- to 300-kb insert size; average, 150 kb) in Escherichia coli cells. Based on naturally occurring F-factor plasmid found in the bacterium E. coli. Compare cloning vector. DOE DNA constructs that are composed of, at least, a REPLICATION ORIGIN, for successful replication, propagation to and maintenance as an extra chromosome in bacteria. In addition, they can carry large amounts (about 200 kilobases) of other sequence for a variety of bioengineering purposes. MeSH, 2002 Related term: BAC maps. Maps, genetic & genomic chromatin immunoprecipitation: A technique for identifying specific DNA sequences that are bound, in vivo, to proteins of interest. It involves formaldehyde fixation of CHROMATIN to crosslink the DNA-BINDING PROTEINS to the DNA. After shearing the DNA into small fragments, specific DNA-protein complexes are isolated by immunoprecipitation with protein-specific ANTIBODIES. Then, the DNA isolated from the complex can be identified by PCR amplification and sequencing. MeSH 2005 clone: A population of genetically identical cells produced from a common ancestor. Sometimes also used to refer to a number of recombinant DNA molecules all carrying the same inserted sequence. IUPAC Medicinal Chemistry, IUPAC Compendium Clone was coined by Herbert J. Webber in 1903 for "a colony of organisms
derived asexually from a single progenitor" and was quickly adopted by
botanists and cell biologists. But the popular perception of cloning can be
traced to Alvin Toffler's Future Shock (1970) and was quickly popularized
(and extended to items such as computers). But Lee Silver, Professor of
Molecular Biology and Public Affairs, Princeton Univ. concludes that "the
scientific community has lost control over Webber's pleasant sounding little
word. Cloning has a popular connotation that is impossible to dislodge. We must
accept that democratic debate on cloning is bereft of any meaning. Science and
Scientists would be better served by choosing other words to explain advances in
developmental biotechnology to the public". L. Silver "What are
clones? They're not what you think they are" Nature 412 (6842): 21, 5 July
2001 clone bank:
Genomic library, a collection of clones made from
a set of randomly generated overlapping DNA fragments representing the
entire genome of an organism. [Schlindwein] cloning: Using specialized DNA technology (see cloning vector) to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study. This process is used by researchers in the Human Genome Project, and is referred to as cloning DNA. The resulting cloned (copied) collections of DNA molecules are called clone libraries. A second type of cloning exploits the natural process of cell division to make many copies of an entire cell. The genetic makeup of these cloned cells, called a cell line, is identical to the original cell. A third type of cloning produces complete, genetically identical animals such as the famous Scottish sheep, Dolly. DOE The process of making copies of a specific piece of DNA, usually a gene. When geneticists speak of cloning, they do not mean the process of making genetically identical copies of an entire organism. NHGRI Human Cloning
FAQ
, US President's Council on Bioethics http://bioethics.georgetown.edu/pcbe/topics/cloning_faq.html Of course many plants can be cloned (cuttings). And identical twins are (in a
technical sense) clones, who can be organ donors for each other without
immunosuppressants cloning vector: DNA molecule originating from a virus, a plasmid, or the cell of a higher organism into which another DNA fragment of appropriate size can be integrated without loss of the vectors capacity for self replication; vectors introduce foreign DNA into host cells, where it can be reproduced in large quantities. Examples are plasmids, cosmids, and yeast artificial chromosomes [YACs]; vectors are often recombinant molecules containing DNA sequences from several sources. DOE conditional knockout: A method by which a gene can be switched off and on. Cre-lox: Tissue- specific gene deletion. A bacteriophage- derived, site- specific recombinase called Cre is used to selectively introduce a deletion into a particular cellular compartment. The method basically involves introducing loxP target sequences into the gene to be deleted, and engineering expression of the Cre recombinase enzyme under the control of a tissue- specific promoter. Thus, the enzyme is expressed only in the desired tissue, and it deletes the gene of interest via the loxP target sites. DNA library: Combinatorial libraries & synthesis DNA nanotechnology: Ned Seeman, DNA Nanotechnology, New York Univ., US http://seemanlab4.chem.nyu.edu/ DNA shuffling: The use of DNA recombination ( RECOMBINATION, GENETIC) to prepare a large gene library of novel, chimeric genes from a population of randomly fragmented DNA from related gene sequences. MeSH 2003 DNA shuffling is the most powerful molecular evolution technique known to date, and it can be used to evolve proteins, plasmids and viruses in vitro. We are applying this method to improve efficacy or pharmacological properties of cytokines with therapeutic potential.... part of the project is done in collaboration with Maxygen (Santa Clara, CA), a biotechnology institute where the gene shuffling technology was developed by Dr. Willem Stemmer (Stemmer, Nature 370: 389- 391, 1994; Crameri et al. Nature 391: 288, 1998). A method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly. Computer simulations called genetic algorithms have demonstrated the importance of iterative homologous recombination for sequence evolution. WP Stemmer, Rapid evolution of a protein in vitro by DNA shuffling, Nature. 1994 Aug 4;370 (6488): 389- 391. Google = about 4,050 Aug. 20, 2003; about 10,100 Nov. 29, 2004; about 91,800 Nov 13, 2007 Related/equivalent? term: gene shuffling. Related terms: domain shuffling, exon shuffling, protein shuffling Wikipedia http://en.wikipedia.org/wiki/DNA_shuffling embryonic lethal trait: In some cases, knockout of a gene believed to be important in a disease occurring in adult life (such as a cancer) will be lethal to the embryo, resulting in little or no information about the function of the gene in adult cells of interest. Related terms knockdown, synthetic lethal screening exon trapping (exon amplification): http://en.wikipedia.org/wiki/Exon_trapping functional genomics technologies include gene disruption, gene manipulation, gene shuffling, gene targeting, gene trapping, knockdowns, knockins, knockouts, mutagenesis, phage display, positional cloning, Post Translational Gene Silencing PTGS, RNA interference RNAi. Related terms chemical genetics, chemical genomics gene disruption: A key methodology in high- throughput gene functional analysis. Involves developing various methods for randomly disrupting genes throughout the genome of a model organism (resulting in knockouts, or null mutations of these genes) and then determining (1) which genes have been disrupted and (2) the phenotype (if any) of the mutant organism. Broader term: gene manipulation Narrower terms: knockdown, knockin, knockout, PTSG Gene Expression Profiling Presentations DVD November 17, 2009 • gene interference: An effect similar to loss- of function mutations in organisms, as if the gene being studied were inactivated. Both sense and antisense RNA are already known to produce interference with the expression of the genes they correspond to by blocking protein synthesis. Antisense RNA is single- stranded RNA that is complementary to a particular mRNA sequence. Sense RNA, also single- stranded, is a shorter version of a particular mRNA strand. Another mechanism for gene interference using RNA has been developed in the past few years. This process, called RNA interference (RNAi) involves double- stranded RNA (dsRNA), and was first developed for use in invertebrates, later vertebrates, and now after much doubt, has been proved to work for mammals, specifically mice. Dr. Bert Ely, Univ. of South Carolina, US http://www.biol.sc.edu/~elygen/caflisch.html Related terms gene disruption, knockout; Broader term: gene manipulation Narrower terms: PTSG, RNAi gene knockout: Use of particular techniques to "knock out" the function of a gene in a model organism. Studying the effects of the gene knockout can help researchers understand the function of the gene that has been inhibited. Related terms: gene manipulation, knockdown, knockin, knockout gene library: A collection of cloned DNA fragments from a variety of species. IUPAC Biotech gene manipulation: The use of in vitro techniques to produce DNA molecules containing novel combinations of genes or altered sequences, and the insertion of these into vectors that can be used for their incorporation into host organisms or cells in which they are capable of continued propagation of the modified genes. IUPAC Biotech Narrower terms: knockdowns, knockins, knockouts, mutagenesis, biochemical genomics, exon trapping, gene disruption, gene targeting, gene trapping; Proteomics protein knockouts gene shuffling:
recombination
between dissimilar genes to create new recombinant genes (see, for example .
We here choose to call only this kind of recombination gene shuffling,
excluding, for example, duplication of domains within a gene. In such a gene
shuffling event, the parental genes may be either destroyed or preserved
. Gene shuffling is clearly the most potent of the three causes of functional
innovation because it can generate new genes with a structure drastically
different from that of either parental gene. Laboratory evolution studies show
that gene shuffling allows new gene functions to arise at rates of orders of
magnitudes higher than point mutations The
rarity of gene shuffling in conserved genes Gavin C Conant1* and Andreas
Wagner2
Genome Biology
2005, 6:R50 doi:10.1186/gb-2005-6-6-r50
http://genomebiology.com/2005/6/6/R50
Encompasses techniques to speed up genetic evolution to screen for high value proteins. Novel recombinant gene products are screened to identify candidate proteins with desired activities. Google = about 1,860 Aug. 20, 2003; about 5, 250 Nov. 29, 2004 Related terms: DNA shuffling, domain shuffling, exon shuffling, molecular evolution, protein shuffling, directed protein evolution gene manipulation, knockdown, knockin, knockout gene silencing: Interruption or suppression of the expression of a gene at transcriptional or translational levels. MeSH, 2000 gene suppression: shRNA is useful where long-term gene suppression is required, or where the cells are resistant to other delivery methods. Its use, however, has been limited by lack of design and processing methods that provide reliable and reproducible gene silencing. BioIT World Weekly Update Sept 5, 2006 Broader term: gene disruption. Related term: Cancer genomics tumor suppressor gene gene targeting: Drug & disease targets gene titration: Dr. [Oliver] Smithies has skillfully wielded a genetic knife - either removing or adding multiple copies of genes to mouse DNA-- to learn more about the roles these proteins play in controlling how much and how fast blood traverses vessels throughout the body. Such "gene titration" experiments, as he calls them, have helped Dr. Smithies evolve a model to describe essential hypertension, a complex disease with multiple genetic and environmental causes. These prior studies have revealed that while increasing the number of AGT genes in a mouse elevates blood pressure, similar manipulations to the number of ACE genes have no effect whatsoever on a mouse's blood pressure. Plugging such data into his customized computer program allows Dr. Smithies to ask -- and answer -- further questions about the effects of genetic and pharmacologic manipulations. "Computers help decipher blood pressure control" National Institute of General Medical Sciences research brief, Mar. 31, 1999 http://www.nigms.nih.gov/news/releases/smithies.html We are investigating the hypothesis that essential hypertension, one of these diseases, is caused primarily by combinations of quantitative genetic variants that individually have only modest effects. To test this hypothesis we have developed a gene targeting approach which allows the level of expression of chosen genes to be varied systematically in different animals by varying the number of functional copies of the target gene from 1 through 4. We have applied this "gene titration" method to several genes in the renin- angiotensin system and in the natriuretic peptide system and we have shown that genetic changes which affect the level of expression of the genes coding for angiotensinogen (AGT), or for renin, or the type 1a receptor for angiotensin II (Atr1a), or the endothelial form of nitric oxide synthase (eNOS), or the atrial natriuretic factor (ANF) or its receptor (NPRA) all affect blood pressures in the mouse. Oliver Smithies, Univ. of North Carolina- Chapel Hill, Lineberger Comprehensive Cancer Center, 2001 http://cancer.med.unc.edu/researchers/DisplayByList.asp?ID=178 gene trapping: Traditional gene- trapping approaches, in which genes are randomly disrupted with DNA elements inserted throughout the genome, have been used to generate large numbers of mutant organisms for genetic analysis. Recent modifications of gene- trapping methods and their increased use in mammalian systems are likely to result in a wealth of new information on gene function. Durick K, et al. “Hunting with traps” Genome Research 9(11): 1019-1025. Nov. 1999 genetic engineering: Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc. MeSH, 1989 Related term: recombinant DNA technology. IUPAC Compendium genetic recombination: Production of new arrangements of genes by various mechanisms such as assortment and segregation, crossing over, gene conversion, transformation, conjugation, transduction, F-duction, or mixed infection of viruses. MeSH, 1968 Happens during the cell division (meiosis) that occurs during the formation of sperm and egg cells. In this process, chromosomes pair up and may swap portions of genetic material in a phenomenon known as crossing over. The chromosomes then reassemble and separate, with each containing some material from the other. The chromosomes are then divvied out into individual sex cells. During crossing over, it is more likely that far- apart genes will be separated by a break than those that are close together. The genes that tend to stay together are said to be linked and therefore may serve as markers for one another — a pattern that is of particular interest when, for example, one of the genes is a disease gene. Related terms: recombinant, recombination genetic vector : Any DNA molecule capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from plasmids, bacteriophages or viruses. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain genetic markers to facilitate their selective recognition. MeSH, 1980 genomewide knockdowns: RNAi is already making an impact. Genomewide knockdowns have been carried out in organisms including nematodes. Small interfering RNAs (siRNAs), which silence genes in mammalian cells, are now being designed against as many genes as possible. The RNA Revolution, BioIT World April 2003 http://bioitworld.com/archive/041503/blueprint_sidebar_2305.html genomic library: A collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism. DOE A form of GENE LIBRARY containing the complete DNA sequences present in the genome of a given organism. It contrasts with a cDNA library which contains only sequences utilized in protein coding (lacking introns). MeSH, 1990 Genome Technology Program: Supports research to develop new methods, technologies and instruments that enable rapid, low-cost determination of DNA sequence, SNP genotyping, and functional genomics (broadly defined). http://www.genome.gov/page.cfm?pageID=10000368 genomic technologies and tools: One of the primary reasons for the success of the Human Genome Project has been the development and use of high- throughput strategies for data generation, and the placement of the data immediately in the public domain. Most of the sequence data, the underlying maps and the sequence assemblies were generated through the use of large- scale automated processes. Now, methods such as sequence analysis of whole genomes, DNA microarray technology and mass spectrometry have been or are being developed as high- throughput approaches for additional types of genomic analyses, such as determining the parameters of gene expression or the location of gene products by the thousands at a time instead of individually. High- throughput methods to determine the location of cis- regulatory elements and, to a lesser extent, other sequence elements, are also beginning to be developed. However, at present, there is no single approach or compilation of approaches that can accurately and efficiently identify every sequence feature in genomic DNA. DETERMINATION OF ALL FUNCTIONAL ELEMENTS IN HUMAN DNA RELEASE DATE, NHGRI, February 21, 2003 RFA: HG-03-003 http://grants1.nih.gov/grants/guide/rfa-files/RFA-HG-03-003.html See also chromatography & electrophoresis, Gene Amplification & PCR, Microarrays, Sequencing genomics technologies - integrated: The grand challenge of fully characterizing the genomics/ proteomics of the intact living cell … The information coming from these projects [Human Genome Project, variety of plant genome sequencing initiatives, and the completion of an extensive array of microorganism genomes], massive and complex as it will be, provides only the starting point for understanding how the cell, the basic unit of life, interprets the blueprint contained in its genome … What is not understood is how the cell creates and orchestrates its own physiology using the information contained in its DNA …. it is unlikely that a useful understanding of the cell will be possible until a quantitative appreciation of both rates and equilibria of molecular processes in the living cell is achieved. [National Center for Research Resources "Integrated Genomics Technologies Workshop Report" Jan 1999] http://grants.nih.gov/grants/guide/rfa-files/RFA-RR-99-003.html Related terms: Functional genomics insertional mutagenesis: Mutagenesis where the mutation is caused by the introduction of foreign DNA sequences into a gene. This process may occur spontaneously in vivo or be experimentally induced in vivo or in vitro. MeSH, 1991 Enables researchers to both identify and sequence a gene, as well as get functional information about it. Is this related to gene trapping? Does not require knowing gene's identity or function. Related terms: embryonic lethal, knockdown knockdown: Altering the function of a gene so that it can be conditionally expressed. This is necessary when complete knockout of the gene would be lethal to the organism. Related terms: embryonic lethal trait, knockin, knockout; Pharmaceutical biology antisense; RNAI RNA Interference knockin: Gain of function through addition/ substitution of genetic material. One example of a knockin is deletion of a coding sequence of a gene in a mouse and then replacing it with human coding sequences. Related terms: knockdown, knockout knockout: Inactivation of specific genes. Knockouts are often created in laboratory organisms such as yeast or mice so that scientists can study the knockout organism as a model for a particular disease. NHGRI Narrower term: conditional knockout, random homozygous knockout Related terms gene knockout, knockdown, knockin, protein knockouts knockout mice: Model & other organisms Knockout-mouse technology is considered an essential and standard technique in functional genomics and target validation. knockout mouse phenotyping: Recognizing the value and utility of a readily accessible, genome-wide collection of knockouts as the lynchpin to determine how mammalian genes function, several international programs were launched in 2006 to build such a resource. Partners in this effort include the NIH funded Knockout Mouse Program (KOMP), the European Conditional Mouse Mutagenesis Program (EuCOMM) funded by the European Commission, and the North American Conditional Mouse Mutagenesis Project (NorCOMM) funded by Genome Canada. Collectively, these programs have created almost 8,000 prototype knockout mice, and they are on track to complete the resource by the end of 2011. This collection is complemented by the Texas A&M Institute for Genomic Medicine’s collection of mouse gene traps — mouse knockouts created using high-throughput approaches to introduce mutations across the mouse genome — resulting in a total of some 14,000 knockouts being available to the scientific community. The new Common Fund KOMP2 program will build upon this resource by expanding the efforts to characterize the mutant strains. Knockout Mouse Phenotyping, NIH Common Fund http://commonfund.nih.gov/KOMP2/overview.aspx library; library, genomic: Cell biology morpholinos: http://www.macalester.edu/~montgomery/Morpholinos.html Broader term: antisense oligonucleotides mutagenesis: The introduction of permanent heritable changes (i.e., mutations) into the DNA of an organism. IUPAC Bioinorganic Mutation and gene disruption are powerful tools for investigating gene function and for identifying novel genes without prior knowledge of homology in other systems. Screening for phenotypes of interest leads to discovery of new genes, some of the mutations becoming models for human genetic diseases. Functional Genomics, UK "Analysis of phenotypic changes resulting from mutagenesis and gene disruption" http://www.functionalgenomics.org.uk/sections/programme/mutagenesis.htm Narrower terms: chemical mutagenesis, insertional mutagenesis, saturation mutagenesis, site- directed mutagenesis. Broader terms: gene disruption, gene manipulation Related terms: knockouts, knockins, knockdowns nanogenomics: The main objective of the group is to develop experimental tools that will accelerate the finding of genomic factors (e.g. genes, polymorphisms, methylation patterns) that contribute to complex traits. In the long term this may lead to predictive medicine based on personalized genomic information. Currently available molecular tools are too slow, cumbersome and expensive to allow comprehensive genomic information to be easily obtained from a large number of individuals in a population (e.g. for case/control studies). A radical re-invention of the way genes and DNA are analyzed is needed. We believe that the analysis of single DNA molecules can overcome many of the present limitations and in addition will reveal information which is inaccessible by conventional methods. To this end, we are also developing methods for sequencing DNA in real time using novel means for observing enzymes during template-directed DNA synthesis. Nanogenomics Group, Wellcome Trust Centre for Human Genetics 2008 http://www.well.ox.ac.uk/nanogenomics/ Google = about 14 Aug. 8, 2002; about 18, Jan. 12, 2004; about 24 June 23, 2004; about 64 May 2, 2005, about 118 Dec 26, 2007 Related terms: Ultrasensitivity Post-Transcriptional Gene Silencing PTGS:
Was initially considered a bizarre phenomenon
limited to petunias and a few other plant species, is now one of the hottest
topics in molecular biology (1).
In the last few years, it has become clear that PTGS occurs in both plants and
animals and has roles in viral defense and transposon silencing mechanisms.
Perhaps most exciting, however, is the emerging use of PTGS and, in particular,
RNA interference (RNAi) — PTGS initiated by the introduction of
double-stranded RNA (dsRNA) — as a tool to knock out expression of specific
genes in a variety of organisms (reviewed in 1-3).
Ambion, RNA Interference and Gene Silencing, 2002 http://www.ambion.com/techlib/hottopics/rnai/
restriction endonucleases: Stuart Linn and Werner Arber [52] and Matthew Meselson and Robert Yuan [53] found specific restriction endonucleases in bacteria, which act when the latter defend themselves against the attack of bacteriophages; thus these enzymes restrict the host range of the bacteriophages. Harry Smith and K. W. Wilcox [54] were able to purify these enzymes, and Thomas Kelly and Hamilton Smith [55], Kathleen Danna and Daniel Nathans [56] and Philip Sharp et al. [57], determined their mode of action. These enzymes cut DNA molecules each at a specific site. These observations made it possible to isolate genes, to clone them and analyze their biochemical structure in great detail. Following the action of restriction endonucleases, there often arise so- called cohesive ends in the DNA molecules [58], which tend to join together. By this means it is possible for example to join together DNA from any eukaryotic organism and that from the bacterial plasmids. Such recombinant DNA molecules were first constructed by David Jackson et al. [59], Peter Lobban and Armin Kaiser [60] and Stanley Cohen et al. [61]. Cloned DNA molecules can be physically mapped, using the cutting points of the restriction endonucleases as markers, [62] and sequenced by means of sophisticated biochemical methods [63, 64]. Petter Portin in "The Origin, Development and Present Status of the Concept of the Gene: A Short Historical Account of he Discoveries" Univ. of Turku, Finland, 2000 http://www.bentham.org/cg/sample/cg1-1/Portin.pdf
reverse genomics: Cell & tissue technologies
RNAi RNA interference: A gene silencing phenomenon whereby specific dsRNAs ( RNA, DOUBLE- STRANDED) trigger the degradation of homologous mRNA ( RNA, MESSENGER). The specific dsRNAs are processed into SMALL INTERFERING RNA (siRNA) which serves as a guide for cleavage of the homologous mRNA in the RNA- INDUCED SILENCING COMPLEX (RISC). DNA METHYLATION may also be triggered during this process. MeSH 2003 See also RNA Narrower terms: miRNA, siRNA Broader terms: gene expression regulation, gene silencing RNA
Interference
February 3-5, 2010 • San
Francisco, CA Program
| Order
CD
RNA silencing:
Although initially recognized as a handy tool to reduce gene expression, RNA
silencing, triggered by double- stranded RNA molecules, is now recognized as a
mechanism for cellular protection and cleansing: It defends the genome against
molecular parasites such as viruses and transposons, while removing abundant but
aberrant nonfunctional messenger RNAs. The underlying mechanisms in distinct
gene silencing phenomena in different genetic systems, such as cosuppression in
plants and RNAi in animals, are very similar. There are common RNA
intermediates, and similar genes are required in RNA silencing pathways in
protozoa, plants, fungi, and animals, thus indicating an ancient pathway.
Tijsterman M. et. al., The
genetics of RNA silencing, Annual Reviews Genetics; 36: 489- 519, 2002
second- site mutations:
Are not lethal themselves, but in combination
with the primary defect cause lethality. Related term:
synthetic lethal screening
SELEX Systematic Evolution of Ligands by Exponential Enrichment: Process for identifying aptamers by iterative enrichment of oligonucleotide mixtures with respect to their ability to bind a target. [IUPAC COMBINATORIAL CHEMISTRY spiegelmer: The chiral inversions or mirror images of aptamers. Related term: photoaptamer; Broader term: aptamers systems biology
Bibliography IUPAC definitions are reprinted with the permission of the International Union of Pure and Applied Chemistry. |
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