Biotechnology

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Biotechnology is the use of living systems and organisms to develop or make products, or "any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use" (UN Convention on Biological Diversity).

Glossary of biotechnology

# - A 

A virus used to construct vectors that introduce genes into cultured cells. Recently, researchers started to use AAV as a convenient vector for expression ofshort hairpin RNA (shRNA)inRNAistudies.

AAV integrates into the host cell genome very inefficiently and appears not to generate immune response, but as a vector it cannot carry large genes. It is potentially useful for "hit and run" gene therapy, where a single burst of therapeutic gene expression is desired.

More about adeno-associated viruses and RNAi inPubMed.

A group of non-enveloped icosahedral viruses. Adenoviruses are popular gene delivery vehicles because they efficiently transduce many different cell types, including hard-to-transfect terminally differentiated cells. Also, adenovirus gene delivery typically results in higher levels of RNA expression. Infection is independent of cell cycle, so adenoviruses can be used to express RNA in both dividing and non-dividing cells. Integration of the adenoviral DNA into the host genome is rare, which means that there is little chance of insertional mutagenesis. Because of this feature and the fact that most recombinant adenoviruses elicit an immune response in animal systems, these viral vectors are appropriate only for transient RNA expression.

More about adenoviruses at theAmbion website.

More about adenoviruses and RNAi inPubMed.

Amplified Fragment Length Polymorphism. A hybrid ofRFLPandRAPDtechniques. Genomic DNA is cut with restriction enzymes, as in RFLP. Typically, two different restriction enzymes are used. The idea is to produce a large number of fragments. Some of the fragments are selectively amplified with PCR using "random" primers, as in RAPD. The primers are not really random, however. Specific oligonucleotide "adapters" (these are complementary to the restriction sites) of 25-30 bp are ligated to the restricted DNA fragments. The primers are complementary to these adapters. However, the primers vary at their 3'-end, such that they will amplify only a subset of the restricted DNA fragments. Typically, a moderate-to-high number of bands will be observed, most of which may be monomorphic. Polymorphic bands are identified as in RAPD analysis. Polymorphic bands can even be excised from the gel and sequenced. This allows for specific PCR primers to be developed, if necessary. AFLPs have typically been used to study variation among individuals of a species, most commonly for producing genetic maps (and in trying to find genes responsible for certain traits). They have received limited attention as tools in systematics, perhaps because the method is relatively labor intensive when compared with other methods. The power of AFLP is based upon the molecular genetic variations that exist between closely related species, varieties, or cultivars. These variations in DNA sequence are exploited by the AFLP technology such that "fingerprints" of particular genotypes can be routinely generated. These "fingerprints" are simply RFLPs visualized by selective PCR amplification of DNA restriction fragments.

More about AFLP technology inPubMed.

The specific DNA product generated by PCR using one pair of PCRprimers.

Genetic analyses suggest that an amplification step might be required for efficient RNA-mediated silencing in several systems. Recent studies inC. eleganshave led to a model in which primarysiRNAsmight prime the synthesis of additional dsRNA, using the targetmRNAas a template, in a reaction catalyzed by a putative RNA-directed RNA polymerase (RdRP). The newly synthesized dsRNA would then be cleaved byDicerto generate secondary siRNAs at a sufficient concentration to achieve efficient target mRNA degradation byRISC.

A nucleotide sequence that is complementary to a sequence ofmessenger RNA (mRNA). When antisense DNA or RNA is added to a cell, it binds to a specific mRNA molecule and inactivates it by physically blocking the ability of ribosomes to move along the messenger RNA or by simply accelerating its degradation.

The antisense oligos are potentially the best tools for studying genes. They are also potentially the fastest, simplest, and most cost effective tools fortarget validation. In addition, antisense oligos offer the promise of safe and effective therapeutics for viral diseases, cancers, and a host of other intractable and devastating diseases.

Although the great potential of antisense is widely recognized, that potential remains largely unfulfilled because most available antisense types suffer from such limitations as poor specificity, instability, unpredictable targeting, and undesirable non-antisense effects.Phosphorothioates (S-DNA),morpholinos, andLNAare examples of antisense oligos.

Small, diffusible, highly structured RNAs that act via sequence complementarity on target RNAs called sense RNAs. In the majority of cases, antisense-RNA action entails posttranscriptional inhibition of target RNA function. Naturally occurring antisense RNAs are between 35 and 150 nucleotides long and contain between one and four stem-loops. Almost all naturally occurring antisense-RNA-regulated systems have been found thus far and are characterized in prokaryotes: only a few systems are known from eukaryotes and one from archaea.

More about antisense RNA inPubMed.

DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected that bind nucleic acid, proteins, small organic compounds, and even entire organisms. These novel molecules have many potential uses in medicine and technology.

More about aptamers inPubMed.

A family of evolutionarily conserved genes. Their protein products are involved in various RNA interference (RNAi) processes because of being part of the RNA interference and silencing complex (RISC).

More about Argonaute proteins inPubMed.

B

Bacterial Artificial Chromosome (BAC) is a DNA construct, based on a fertility plasmid, used for transforming and cloning in bacteria, usually Escherichia coli. Its usual insert size is 150 kbp, with a range from 100 to 300 kbp. BACs are often used to sequence the genetic code of organisms in genome projects, for example the Human Genome Project. A short piece of the organism's DNA is amplified as an insert in BACs and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism.

C

A nematode that is widely used as amodel organismin biology.

Accidentally, the term"RNAi"was originally coined byFire et al., Proc Natl Acad Sci U S A. 1998to describe occurrence of a specific-mRNA interference process in the nematode worm Caenorhabditis elegans mediated by homologousdouble-stranded RNA (dsRNA)that are longer than 25 base pairs.

Cleaved Amplified Polymorphic Sequences. CAPS polymorphisms are differences in restriction fragment lengths caused bySNPsorINDELsthat create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus-specific oligonucleotide primers. CAPS assays are performed by digesting locus-specific PCR amplicons with one or more restriction enzymes and then separating the digested DNA on agarose or polyacrylamide gels. CAPS analysis is versatile and can be combined with single strand conformational polymorphism (SSCP), sequence-characterized amplified region (SCAR), or (RAPD) analysis to increase the chance of finding a DNA polymorphism. More about CAPS inPubMed.

Silencing is caused by the presence oftransgenesin the genome. Repression is usually related to copy number. Tandemly arrayed transgenes are more effective inducers of silencing than dispersed transgenes, withinverted repeatsbeing the most effective. Silencing can occur transcriptionally or post-transcriptionally. This transsuppression requires transcription of the transgenes but is independent of the specific promoter sequence used to drive the transcription.

Cosuppression has been recorded in plants, Drosophila, fungi, and C. elegans but not in higher animals. Characteristically, after stable integration of one or more copies of homologous transgene into the genome, reduction in expression of the endogenous gene and the transgenes occur in reciprocal fashion (thus, cosuppression).

D

Derived Cleaved Amplified Polymorphic Sequence. A modification of theCAPStechnique that allows detection of most single-nucleotide changes by utilizing mismatched PCR primers. Using the method, a restriction enzyme recognition site that includes theSNPis introduced into the PCR product by a primer containing one or more mismatches to template DNA. The PCR product modified in this manner is then subjected to restriction enzyme digestion, and the presence or absence of the SNP is determined by the resulting restriction pattern. Similar to the CAPS technique, this method is simple, relatively inexpensive, and uses the ubiquitous technologies of PCR, restriction digestion, and standard agarose gel electrophoresis. More about dCAPS inPubMed.

DNA directed RNA interference (ddRNAi). This technology relies on RNA polymerase III-based promoters (e.g., U6 or H1) for the expression ofsiRNAtarget sequences that have been transfected in mammalian cells.

Pol III directs the synthesis of small RNA transcripts whose 3' ends are defined by termination within a stretch of four to five thymidines. These characteristics allow for the use of DNA templates to synthesize, in vivo, small RNAduplexesthat are structurally equivalent to active siRNAs synthesized in vitro.

Once transfected and transcribed in vivo, siRNA/RISCduplexes form in the cell and lead to the degradation of the targetmRNA.

siRNA target sequences can be introduced into the cell by the generation of a ddRNAi expression cassette or by being cloned in a siRNA expression vector.

More about ddRNAi inPubMed.

One or a few nucleotides are eliminated from a sequence (opposite toinsertionmutation).

A member of the RNase III family of double-stranded RNA-specific endonucleases that processesdouble-stranded RNAs (dsRNAs)to 21-nucleotidesmall interfering RNAs (siRNAs)duringRNA interferenceand excisesmicroRNAs (miRNAs)from precursor hairpins.

More about Dicer inPubMed.

This is an enzyme that catalyze the synthesis of new DNA strand alongside a template DNA strand. All DNA polymerases synthesize DNA in the 5' to 3' direction and can add a nucleotide only onto preexisting 3'-OH group. For this reason, DNA polymerase needs aprimerat which it can add the first nucleotide. DNA polymerase is widely used inPCRfor various research applications. Also seeTaq DNA Polymerase.

RNase III, the core nuclease that executes the initiation step of miRNA processing in the nucleus.

More about Drosha inPubMed.

Double-stranded RNA. RNA with two strands instead of the typical one. dsRNAs longer than 30 nucleotides are normally referred as long dsRNAs. In general, long dsRNA is the precursor of thesiRNAthat can triggerRNAi.

Although there are different methods to generatesiRNAfor gene silencing, the easiest and most efficient way to achieveRNAiis to use synthetic small-interfering RNA (siRNA). siRNAduplexesare typically made of 19 RNA nucleotides followed by a 3' dTdT overhang.

As the field has progressed, researchers have found that the composition of the overhang does not appear to play a critical role in targetmRNArecognition and cleavage. The overhangs may play a structural role for presenting a symmetrical duplex to theRISC. Many investigators select dTdT because early evidence suggested that deoxyribonucleotides might confer nuclease resistance.

Nevertheless, some investigators prefer overhangs that are complementary to the authentic mRNA target. What is most important, however, is the identity of the 19-base core of the duplex toward a unique mRNA target.

A molecular structure consisting of two strands of opposed polarity.

E

A mitotically and/or meiotically heritable trait that is not accompanied by a change in the DNA sequence.

Endoribonuclease-prepared siRNA. Mixture of siRNA oligos resulting from cleavage of longdouble-stranded RNA (dsRNA)with Escherichia coli RNase III. esiRNA can target multiple sites within anmRNAand mediates effectiveRNA interferencewithout apparent nonspecific effect in cultured mammalian cells.

More about esiRNA inPubMed.

Expressed Sequence Tag. A short (200-500 nucleotides) DNA sequence that can be used to identify a gene that is being expressed in a cell at a particular time. Individual ESTs are neither especially informative nor highly accurate; however, they are easy to collect in large numbers. A database of ESTs can be helpful for picking protein-coding sequences from a long stretch of DNA or for providing a larger context for very short sequences, such as the small pieces of protein sequence obtainable by mass spectrometry. ESTs are compiled by theNational Center for Biotechnology InformationinEST database.

F

Fluorescence In Situ Hybridization. This is a variant ofISHwhereriboprobesare fluorescently labeled.

The large-scale exploration of gene function that includes the analysis of regulatory networks, biochemical pathways, protein-protein interactions, the effects of gene knockouts, and the results of functional complementation of knockouts.

G

Gene impedance. This is the coordinated cellular mechanisms for efficient and specific transcriptional inhibition, degradation of themRNA, and inhibition of translation of the transcripts of a target gene in a cell or a population of cells in response to physiological signals. Gene impedance can also be triggered by transient introduction of double-stranded polynucleotides homologous to regions of the target gene. GENEi functions transcriptionally and post-transcriptionally and could operate independently of the level of transcription of thetransgenes.

It was first reported in mammalian cells but applies to various eukaryotic organisms.

More about GENEi inBahramian MB, Zarbl H. J Theor Biol. 2005 Apr 7;233(3):301-14.

Any mechanism that silences a gene, such as various sequence homology-dependent silencing mechanisms (RNAi,quelling,PTGS,TGS,VIGS), gene knockout by homologous recombination, imprinting, DNA methylation, andtransvection.

The therapeutic application of either blocking the unwanted result of a defective gene or repairing a defective gene by inserting a new gene into the chromosome of the cell.

A phenotypically recognizable genetic trait that can be used to identify a genetic locus, a linkage group, or a recombination event. Year introduced: 1989 (MeSH)

The process of analyzing the particular genetic variations (polymorphisms) existing in an individual DNA sample.

H

The genetic constitution of an individual with respect to one member of a pair of allelic genes. A haplotype can refer to only one locus or to an entire genome. A genome-wide haplotype would comprise half of a diploid genome, including one allele from each allelic gene pair (Wikipedia).

More about haplotype inPubMed.

The process of replacing an endogenous gene with an artificial cassette that is flanked by regions that are homologous to sequences bordering the targeted gene. It is used as a technique to inactivate a gene and determine its function in a living animal.

The annealing of two complementary nucleic acid strands to form a double-stranded molecule; a technique for detecting specific nucleic acid sequences.

I

Insertion/Deletion (InDel) markers are an important class of molecular markers for mapping heritable traits in animals and plants because the markers with large insertions or deletions (>10 bp) can be directly scored on agarose gels. The advent of fragment analysis with fluorescence-tagged primers made this class of markers especially useful because a single bp insertion or deletion can be reliably resolved by electrophoresis on acrylamide gels or capillary columns.

One or a few nucleotides are inserted into a sequence; in rare cases, this involves transposition from another locus via an RNA intermediate. Large insertions and duplications are rare, as insertions of transposable elements (transposons).

In Situ Hybridization. A technique that allows for precise localization of a specific segment of nucleic acid.

LongdsRNA(<30 bp) introduced into mammalian cells can induce (in a manner similar to the silencing observed during viral infection) the up-regulation of type I interferons (a group of glycoprotein cytokines) and activation of RNAse L and PKR (protein kinase R) pathways. RNAse L leads to nonspecific mRNA cleavage. PKR phosphorylates the translation initiation factor eI2α, which results in global inhibition of mRNA translation and induction of apoptosis.

In the RNAi world, a stretch of DNA encoding for about 800 nucleotides of the targetmRNAin a sense and antisense orientation, often separated by non-homologous sequence. During transcription of the inverted repeat, an RNA molecule is formed that is supposed to fold back into a hairpin-like structure by intramolecularhybridizationresulting in formation ofdsRNA. These 800 nucleotide-long dsRNAs are believed to be processed by Dicer tosiRNAsthat can target endogenous mRNA for cleavage.

J

None

K

Conditional down-regulation of a gene.

More about gene knockdown inPubMed.

An alteration of a gene in a way that results in a gain of function.

An alteration of a gene that results in loss of function; a transgenic organism in which a gene has been inactivated.

More about gene knockout inPubMed.

L

A class of retroviruses used for gene therapy vectors. Lentiviruses, such as the human immunodeficiency virus (HIV), are capable of infecting non-dividing cells, including differentiated neurons of the brain.Short hairpin RNAscan be expressed from lentiviruses, allowing for high efficiency transfection of a variety of cell types. The lentivirus vectors have the advantage overadenovirusof being able to introduce RNAs into blood and bone marrow cells.

More about lentiviral RNAi inPubMed.

Locked Nucleic Acid. A novel class of nucleotide analogue in which the conformational freedom of the ribose moiety is restricted by a methylene 2'-heteroatom, 4'-C linkage. The most commonly used LNA is the β-D-oxy-LNA that has a methylene 2'-O, 4'-C linkage, which restricts the sugar in the C3'-endo (N-type) conformation characteristic of RNA. This feature enables β-D-oxy-LNA to be used in the development of RNA antagonists that exploit both the antisense andsiRNAprinciples.

Long range Polymerase Chain Reaction (PCR) allows the amplification of DNA fragments, which are much larger than those achieved in conventional PCR. Up to 27 kb fragments are possible from good quality genomic DNA. The method uses twoDNA polymerases, usuallyTaq DNA polymerasefor high processivity (i.e. 5’-3’ polymerase activity) and another DNA polymerase with 3’-5’ proofreading abilities (usuallyPwo). This combination of features allows longerprimerextension than can be achieved withTaqalone.

Perlegen Sciencesutilized long range PCR in the Phase IIHapMapgenotyping project.

M

A rapid method to produce 17 base pair sequence tags that are precisely representative of the population of messenger RNAs (mRNAs) in a given tissue. The 17-bp tag is derived from 3' end of a transcript and provides a virtually unique, experimentally derived identifier for each gene.

More about MPSS technology.

microRNA. Naturally expressed small RNAs that interact with components shared by the RNA-induced silencing complex (RISC). In animals, the complex-bound, single-stranded miRNA binds specific mRNAs through sequences that are significantly. Al though not completely, complementary to themRNA. By a mechanism that is not fully characterized but which apparently does not involve mRNA degradation as inRNAithe bound mRNA remains untranslated, resulting in reduced expression of the corresponding gene. It is increasingly believed that microRNAs play a central role in the regulation of gene expression in mammalian cells, and abnormalities in their function may play a role in human disease.

More about miRNA inPubMed.

The RNA template for protein synthesis. mRNA is formed by transcription of the template DNA strand, followed by the excision of introns and the joining of exons to form mature mRNA.

One that is extensively studied to understand particular characteristics inherent to it that can provide insights into important biological phenomena. Most common factors that influence selection of model organisms are size, life span, accessibility, ease of manipulation, genetics, conservation of mechanisms, and potential economic benefits.

Most popular model organisms are Mus musculus (mouse), Rattus norvegicus (rat),Caenorhabditis elegans(nematode), Drosophila melanogaster (fruit fly), and several others.

A single-stranded DNA molecule containing several features enabling target identification, detection, and quantification. Typically it contains two regions complementary to adjacent regions in the target DNA sequence that are allowed to anneal directly to the genomic DNA, followed by enzyme-assisted detection of theSNPalleles in the Molecular Inversion Probe assay.

More about MIP technology.

Morpholino oligos were devised by James Summerton in 1985 and were developed at ANTIVIRALS Inc. (now AVI BioPharma), the pioneerantisensecompany founded by Summerton in 1980.

Morpholino oligos are so named because they are assembled from four different Morpholino subunits, each of which contains one of the four genetic bases (adenine, cytosine, guanine, and thymine) linked to a six-membered morpholine ring. Eighteen to 25 subunits of these four subunit types are joined in a specific order by non-ionic phosphorodiamidate intersubunit linkages to give a Morpholino oligo.

More about morpholino technology.

Any change in a specific DNA sequence.

N

Nonsense-mediatedmRNAdecay. A process that appears to be a quality control mechanism that eliminates nonsense transcripts such as mRNAs with premature termination codons. First discovered in yeast, this surveillance mechanism is ubiquitous among eukaryotes. Coupled to mRNA splicing, this pathway results in the degradation of aberrant mRNAs. There is evidence that NMD is involved in thePTGS-related degradation of the mRNA, because someC. elegansgenes are required for bothRNAiand NMD. The two mechanisms are different, however, because NMD is dependent on translation of the mRNA, whereas the decrease in mRNA observed in RNAi and related PTGS phenomena is not prevented by inhibitors of translation. Also, the mRNA degradation associated with NMD begins with de-capping followed by 5' to 3' exonuclease degradation, whereas the degradation associated with PTGS appears to begin with endonucleotidic cleavage.

O

A key feature ofsiRNAsis that they inhibit genes in a highly specific manner. Indeed, the initial description of these RNAs demonstrated that alternation of a single base pair was sufficient to disruptgene silencing. For many subsequent studies with siRNAs this specificity was shown to hold true. A number of more recent studies have suggested that there are situations where mismatches between the siRNA and target sequence can be tolerated. This observation has raised the concern that siRNAs may have effects on genes that are not considered targets, so called off-target effects. This concern has been addressed by a number of groups that have examined genome-wide changes in gene expression following the introduction of siRNAs. Some of these studies found that a number of genes unrelated to the target are changed in expression, mostly by a factor of twofold. These off-target effects have been correlated with the concentration of siRNAs, as well as similarities between the off-target transcripts and the 5' ends of siRNAs. It seems plausible on the basis of recent work that the decreased off-target mRNA levels are the consequence of siRNAs adopting miRNA-like properties, resulting in slightly decreased levels of mRNAs, possibly through alterations in mRNA stability. The exact mechanism remains to be determined.

More about RNAi off-target effects inPubMed.

Overlapping oligonucleotide. Overgo probes are produced using two oligos that are complementary to each other and anneal to form a double-stranded region. For 36-mer overgo probes, two 22-mers create the initial template for DNA synthesis, overlapping in 8 bp, leading to the production of a labeled probe.

More about overgo probes.

P

P1-derived Artificial Chromosome. DNA constructs that are derived from the DNA of P1 bacteriophage. They can carry large amounts (about 100-300 kilobases) of other sequence for a variety of bioengineering purposes.

Polymerase Chain Reaction. A technique for amplifying DNA sequences in vitro by separating the DNA into two strands and incubating it with oligonucleotideprimersandDNA polymerase. It can amplify a specific sequence of DNA by as many as one billion times. For more information about PCR seeintroduction to PCR. See alsoRT-PCRandqRT-PCR

PCR followed by Sequence-Specific Oligonucleotides (also SSOP, ASO). Fixed amplicons are hybridized under discriminatory conditions with a panel of probes in solution after locus-specific amplification. Complete hybridization or specific binding of probes is more stable than a mismatched hybridization. Therefore, a specifically bound probe is not washed out during stringent washes and can be subsequently detected. First, amplicons are transferred onto membrane, forming a dot or slot. Each membrane is then hybridized with one probe in solution and washed, and the attached probe is detected.

Drabek J. A commented dictionary of techniques for genotyping. Electrophoresis. 2001 Apr;22(6):1024-45.

PCR with Sequence-Specific Primers (also ARMS, ASA, PASA, MAMA, and ASP). Stringent reaction conditions and Taq without proof-reading activity ensure that only primers fully complementary to the template allow for amplification of the target. The specific step is primer hybridization and extension in contrast to sole probe hybridization in PCR-SSO. In primer design, matching the 3'-end of the primer with its template is the most important step. Alleles are distinguished by detection of the amplicon in an agarose gel. If the specific amplicon is absent, the sample is assumed to lack a specific motif. Because an amplification failure can mask the presence of the amplicon and lead to a false-negative result, a pair of primers reacting constitutionally with each sample is added into each tube as inner amplification control.

Drabek J. A commented dictionary of techniques for genotyping. Electrophoresis. 2001 Apr;22(6):1024-45.

The degree to which a genotype is expressed as a phenotype; also used to characterize the likelihood that a gene will result in a disease.

Measurable functional and structural characteristics of an organism as determined by the interaction of the genotype with the environment.

Phosphorothioates are the first generation ofantisense oligonucleotides. They are a variant of normal DNA in which one of the non-bridging oxygens is replaced by a sulfur. The sulfurization of the internucleotide bond dramatically reduces the action of endo- and exonuclease 2, including 5' to 3' and 3' to 5' DNA POL 1 exonuclease, nucleases S1 and P1, RNases, serum nucleases, and snake venom phosphodiesterase. In addition, the potential for crossing the lipid bilayer increases. Because of these important improvements, phosphorothioates have found increasing application in cell regulation studies.

More about phosphorothioates inPubMed.

Chromodomain proteins that transcriptionally repress genes by favoring the formation of closed chromatin structures. These proteins play a role inRNAiinCaenorhabditis elegansand Drosophila. More about polycomb proteins inPubMed.

A short oligonucleotide complementary to target DNA and acts as the leader for DNA extension.

DNA or RNA molecules of specific base sequence, often labeled either radioactively or immunologically, that are used to detect the complementary base sequence byhybridization.

Post-Transcriptional Gene Silencing. This is a general term that applies to all eukaryotic organisms and involves double-stranded (ds)RNA. The transcription of the gene is unaffected; however, gene expression is lost becausemRNAmolecules become unstable. PTGS provides nucleotide sequence-specific protection against a variety of foreign genetic elements.

In higher plants, this system provides protection against viruses, whereas in Caenorhabditis elegans, Drosophila, and Chlamydomonas, the targeted elements aretransposons. Presumably, in PTGS oftransgenes, the cell perceives the foreign DNA or the corresponding RNA as if it were a virus or a transposon.

Q

Quantitative Real Time Polymerase Chain Reaction. Quantitative Real Time PCR was commercialized in 1995. This method capitalizes on the fact that there is a quantitative relationship between the amount of starting target sample and the amount ofPCRproduct at any given PCR cycle number. This chemistry exploits the exonuclease activity ofTaq DNA polymeraseby using a cleavable labeled probe that hybridizes with target sequence in combination with forward and reverse PCRprimers.

This term is specific fortransgene-inducedPTGSin Neurospora crassa.

During the vegetative phase in Neurospora crassa, the transgene can induce reversible gene silencing.

R

Random Amplified Polymorphic DNA. The complexity of eukaryotic nuclear DNA is sufficiently high that randomly generated probes that are 8 or 10 nucleotides long will, by chance, be complementary to some sequences within the genome and will sometimes be close enough to one another for PCR amplification. With some random probes, no sequences are amplified. With others, the same length products are generated from DNAs of different individuals. With still others, different patterns of hybridization will occur in different individuals, leading to different patterns of bands after DNA electrophoresis of the amplified DNA. That is, some individuals will have sequences complementary to the two probes at a particular place in the genome, the probes will hybridize, and PCR will amplify the DNA fragment between the probes. However, other individuals may have sequences at those sites that are somewhat different and therefore not complementary to the probes. The probes will not hybridize, and no amplification will take place. These variable bands are commonly called random amplified polymorphic DNA (RAPD) bands. RAPDs exhibit polymorphism and thus can be used as genetic markers. RAPDs are dominant in the sense that the presence of a RAPD band does not allow distinction between heterozygous and homozygous states. More about the RAPD technology inWilliams JG, Kubelik AR et al. Nucleic Acids Res. 1990 Nov 25;18(22):6531-5.

Repeat-associated RNA. rasiRNAs are presumably derived from longdsRNAsand match to repetitive sequence elements in sense and antisense orientation. Repetitive sequences are often associated with regions of heterochromatin. Transposable elements (TEs ortransposons), for example, may producedsRNAupon random integration in nearby transcriptionally active sequences. The high density of transposons, in particular genomic regions, makes it more likely that dsRNA is generated by transcription from adjacent opposing promoters of transposons rather than cellular genes. rasiRNAs are likely to function as guide RNAs during the establishment and/or maintenance of heterochromatin in plants, Trypanosoma brucei, Drosophila melanogaster, and fission yeast. Small RNAs and proteins related to RNA silencing have also been identified in Tetrahymena thermophila and are thought to participate in guiding programmed DNA elimination of dispersed sequence elements to form the transcriptionally active macronucleus after sexual conjugation (Aravin AA, Lagos-Quintana M et al.).

Restriction endonucleases (REs) are enzymes that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Enzyme recognition sites are usually 4 to 6 base pairs in length. Generally, the shorter the recognition sequence, the greater the number of fragments generated. Restriction enzymes are isolated from a wide variety of bacterial genera. These enzymes are named by using the first letter of the genus, the first two letters of the species, and the order of discovery. See about using REs inRFLP,AFLPandCAPSassays.

A virus used to construct vectors that are able to stably integrate any gene cassette into the host cell chromosome. This characteristics made it popular inRNAistudies. Disadvantage of the retroviral delivery system is that retrovirus only infects dividing cells.

More about retroviral RNAi inDevroe E, Silver PA. BMC Biotechnol. 2002 Aug 28;2(1):15.

Restriction Fragment Length Polymorphism. RFLPs are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites. RFLP assays are performed by hybridizing a chemically labelled DNA probe to a Southern blot of DNA digested with a restriction endonuclease.

More about RFLP technology inTodd R, Donoff RB et al. J Oral Maxillofac Surg. 2001 Jun;59(6):660-7.

RNA probe. Riboprobes are reporter molecules that are used inISH.

A ribozyme, or RNA enzyme, is an RNA molecule that can catalyze a chemical reaction. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome.

Some known ribozymes include RNase P, Group I and Group II introns, leadzyme, hairpin ribozyme, hammerhead ribozyme, hepatitis delta virus ribozyme and tetrahymena ribozyme. Artificially produced self-cleaving RNAs have been produced that have good enzymatic activity.

The ribozymes have an advantage overantisense oligonucleotides (ODNs)in that ribozyme genes can be delivered to cells with plasmid or viral vectors, and ribozyme expression can be controlled with promoter-based expression.

More about ribozymes inPubMed.

RNA-induced silencing complex. This enzyme complex contains an endoribonuclease (termed "Slicer") that uses the sequence encoded by the antisensesiRNAsstrand to find and destroymRNAof complementary sequence. The siRNA thus acts as a guide restricting the ribonuclease to cleave only RNAs complementary to one of the two siRNA strands.

More about RISC inPubMed.

RNA-dependent RNA polymerase. RdRP is believed to be involved in the amplification ofdsRNAsin some organisms (C. elegans, Arabidopsis, Neurospora, Dictyostelium) but not in others (Drosophila, humans).

RNA interference. This type ofPTGSis induced directly bydsRNA. It was first defined inC. elegansand seems to be mechanistically related, if not identical, to PTGS in plants.

Reverse Transcription Polymerase Chain Reaction. This is aPCRpreceded with conversion of sample RNA into cDNA suitable for amplification byDNA polymeraseenzyme.


S

Short hairpin RNA. A structure in which adjacent segments of DNA or RNA fold together and are stabilized by base pairing, creating a loop of single-strand DNA or RNA.

Short hairpin RNAs can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines ortransgenicanimals in whichRNAienforces stable and heritablegene silencing.

More about shRNA inPaddison PJ, Caudy AA et al. Genes Dev. 2002 Apr 15;16(8):948-58.

Small interfering RNA. "Short pieces" ofdsRNA, approximately 21-25 base pairs long, which are central toRNAi.

In the majority of cases, siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, are paired in a manner to have a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair. 2'-Deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides but are often cheaper to synthesize and probably more nuclease resistant. More about siRNA inPubMed.

Three or more different siRNAs designed from distinct sequences of one target gene and mixed together are called 'siRNA pool'. This increases efficiency in knockdown of target gene expression.

SeeRISC.

Single Nucleotide Polymorphism. The most common form of DNA variation, alterations to a single base. If the SNP is in a gene, it can disrupt the gene's function. Most SNPs do not occur in genes but can be associated with other types of DNA variation and so are used effectively as markers. Typically, SNPs are biallelic, although very rarely tri- or tetraallelic forms can be found. The average frequency of SNPs in the human genome is approximately one per 1,000 bp.

More about SNPs and their application inPubMed.

SeePCR-SSO.

SeePCR-SSP.

Simple Sequence Repeats or microsatellites are tandemly repeated mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs. SSR length polymorphisms are caused by differences in the number of repeats. SSR loci are "individually amplified by PCR using pairs of oligonucleotide primers specific to unique DNA sequences flanking the SSR sequence". More about SSR inKashi Y, King D, Soller M. Trends Genet. 1997 Feb;13(2):74-8.

Small temporal RNA. stRNAs exert negative post-transcriptional regulation by binding to complementary sequences in the 3' untranslated regions of their target genes. stRNAs are transcribed as longer precursor RNAs that are processed by the RNase Dicer/DCR-1 and members of the RDE-1/AGO1 family of proteins, which are better known for their roles inRNA interference (RNAi). stRNAs may function to control temporal identity during development in C. elegans and other organisms.

More about stRNA inBanerjee D, Slack F. Bioessays. 2002 Feb;24(2):119-29.

Sequence Tagged Site. A short DNA sequence that has been shown to be unique. To qualify as an STS, the exact location and order of the bases of the sequence must be known, and this sequence may occur only once in the chromosome being studied or in the genome as a whole if the DNA fragment set covers the entire genome.

More about STSs inPubMed.

T

Heat resistantDNA polymeraseisolated from Thermus aquaticus. This is one of most popular DNA polymerase used forPCR.

The ability of a chemical, synthetic or of natural origin, to distinguish between highly similar target molecules.

Blocking the activity or preventing the synthesis of a selected protein to predict if developing a drug targeted against that protein would likely produce the desired therapeutic effect. The demand for fast, reliable, and cost effective target validation is rapidly growing because the Human Genome Project, proteomics research, and our increasing understanding of many diseases at the molecular level is leading to identification of a host of new proteins having potential as drug targets.

The sequencing of the human genome revealed vast numbers of potential new targets. Unfortunately, most of these are poorly characterized.

Currently marketed drugs target only between 272 and 482 molecules. The vast majority of these targets fall into a few protein families: G protein-coupled receptors (the most popular targets by far), ion channels, certain types of enzymes (e.g., serine proteases), and nuclear hormone receptors (e.g., steroid receptors).

RNAihas rapidly become a widely used tool for determining gene function and validating potential therapeutic targets both in cell culture and in vivo. With the present rate of progress and most recent animal experiments, RNAi is poised to become a new generation of rationally designed drugs.

More about target validation and RNAi inPubMed.

Transcriptional Gene Silencing. Gene expression is reduced by a blockade at the transcriptional level. Evidence indicates that transcriptional repression might be caused by chromatin modification or DNA methylation.

This is generally observed in plants but has also been seen in animals. Generally in plants, transcriptional inactivation requires homology between promoters.

All the DNA transcripts, mostly messenger RNAs (mRNAs), being produced in a cell.

The gene(s) transferred into another organism using genetic engineering.

An organism in which a foreign DNA gene (a transgene) is incorporated into its genome early in development. The transgene is present in both somatic and germ cells, is expressed in one or more tissues, and is inherited by offspring in a Mendelian fashion.

A phenomenon when a restricted portion of a particularmRNAis targeted byRNAiandsiRNAcorresponding to other portions of the mRNA can also be detected. In C. elegans, these secondary siRNAs are homologous to sequences upstream of those targeted by the primary trigger. This 5'->3' directionality (with respect to the antisense strand) is in agreement with a model where secondary siRNAs are produced byamplification. Similarly, secondary siRNAs can also be observed in plants where they spread in both the 5'->3' and 3'->5' directions.

A segment of DNA that can move around to different positions in the genome of a single cell, thereby causing mutations and changes in genome size. The so-called retro-transposons move by a copy-paste mechanism using an RNA intermediate. Their exact function is still a debate, but it has been estimated that about 40% of our genome consist of retro-transposons. They have been called "junk" DNA and "selfish" DNA. "Selfish" because their only function seems to make more copies of themselves, and "junk" because there is no obvious benefit to their host.

A process by which the activity of a gene is altered when it is paired with a homologue.

U

The UAS/GAL4 binary ectopic expression system is widely used in Drosophila melanogaster for the overexpression oftransgenesand allows for heritable and inducibleRNA interference. This system operates under the assumption that the yeast transcription factor, GAL4, is inactive in D. melanogaster. Thus, GAL4 can be expressed under the control of D. melanogaster-specific promoters with little effect upon the organism. Twotransgenicfly stocks are used for this system. The first one contains GAL4 under control of tissue-specific promoter (GAL4-driver transgene), and the second one contains aninverted repeatunder control of the yeast upstream activating sequences (UAS) which are binding sites for GAL4 (UAS-IR transgene). When the GAL4-driver fly is crossed with the UAS-IR stock, the resulting progeny have both components of the system, and thus the target gene is silenced in those tissues in which GAL4 is expressed.

More about UAS/GAL4 system inGiordano E, Rendina R et al. Genetics. 2002 Feb;160(2):637-48.

V

Silencing that is induced by the presence of viral genomic RNA. Only replication-competent viruses cause silencing, indicating thatdsRNAmolecules might be the inducing agents.

Restriction of virus growth in plants is mediated byPTGS, which can be initiated by production of dsRNA replicative intermediates. This silencing of gene expression is gene specific.

More about VIGS inBaulcombe DC. Curr Opin Plant Biol. 1999 Apr;2(2):109-13.

W

Nonspecific base-pairing of the base at the 5' end of a tRNA anti-codon to alternative bases at the 3' end of a mRNA codon.

X

None

Y

Yeast Artificial Chromosomes are extremely large segments of DNA from another species spliced into DNA of yeast. YACs are used to clone up to one million bases of foreign DNA into a host cell, where the DNA is propagated along with the yeast cell's other chromosomes.

Z


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