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Evidence classes, types, and subtypes

Definition:

Each annotation in ORegAnno is associated with an evidence class, type, and subtype. Evidence information describes the original biological experiment performed to validate an annotation.

Evidence information is defined as:

  • Evidence class: Information describing the target of a biological assay. This is not the experiment, but whether the experiment confirms binding, a particular transcription factor, or elucidates the regulatory sequence.
  • Evidence type: Information desribing a type of biological assay. This is a generic type of experiment that may have several subtypes of experimentation associated with it.
  • Evidence subtype: Information desribing a subtype of biological assay. This is the specific biological assay that was used in the associated literature.


Evidence:

Currently-defined evidence information: (Click for XML representation)

Evidence Classes

OREGEC00000 UNKNOWN UNKNOWN
OREGEC00001 Transcription regulator site Describes evidence for the identity of a sequence that regulates transcription (e.g. transcription factor binding site).
OREGEC00002 Transcription regulator Describes evidence for the identity of the protein that binds a transcription regulator sequence (e.g. transcription factor).
OREGEC00003 Translation regulator site Describes evidence for the identity of a sequence (usually 3') that regulates protein translation.
OREGEC00011 Transcript stability regulator site Describes evidence for the identity of a sequence that regulates transcript stability.
OREGEC00012 Transcript stability regulator Describes evidence for the identity of the protein that binds a transcript stability regulator site.
OREGEC00013 Translation regulator Describes evidence for the identity of the protein that binds a translation regulator site.

Evidence Types and Subtypes

OREGET00000 UNKNOWN UNKNOWN
Subtype
OREGES00000 UNKNOWN UNKNOWN
OREGET00001 Electrophoretic mobility shift assay (EMSA) Used to detect the interaction of DNA binding proteins with their DNA recognition sequences. Purified proteins or crude cell extracts are incubated with a radiolabelled DNA probe. The complexes are separated from the free probe by migration through a nondenaturing polyacrylamide gel, with the complexes migrating more slowly.
Subtype
OREGES00001 Direct gel shift Gel electrophoresis of DNA in the presence of DNA binding proteins changes the migration relative to homologous protein-free DNA, and can be used to detect the presence and specificity of the proteins.
Subtype
OREGES00002 Supershift Same as direct gel shift except for the addition of an antibody (against the protein under study) which results in a further retardation, a super-shift. The super-shifting allows experiments to be done with crude extracts in place of purified proteins.
Subtype
OREGES00003 Gel shift competition Addition of non-labelled DNA containing the TFBS inhibits the shift induced by TF addition. Addition of other non-labelled DNA competitors are less effective in inhibiting the shift.
Subtype
OREGES00013 UNKNOWN Unknown/unannotated EMSA experiment performed.
Subtype
OREGES00060 Direct gel shift with Western blot detection Gel electrophoresis of DNA in the presence of DNA binding proteins changes the migration relative to homologous protein-free DNA, and can be used to detect the presence and specificity of the proteins. The proteins-DNA complex is then excised, separated with SDS-PAGE and subjected to Western blot analysis.
OREGET00002 Reporter gene assay Used to measure activity of a promoter under different conditions, such as to define elements of a promoter or to study signals that activate an intact enhancer/promoter.
Subtype
OREGES00004 Transient transfection luciferase assay The gene coding for luciferase is linked onto a promoter (or other transcription control region) from another gene, and the construct is "transfected" into cultured cells. The amount of luciferase enzyme produced is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel). A common method for testing the effects of sequence changes on promoter function.
Subtype
OREGES00019 Chloramphenicol acetyltransferase (CAT) Assay Chloramphenicol acetyl transferase (CAT), is a bacterial enzyme which inactivates chloramphenicol by acetylating it. CAT assays are often performed to test the function of a promoter. The gene coding for CAT is linked onto a promoter (transcription control region) from another gene, and the construct is "transfected" into cultured cells. The amount of CAT enzyme produced is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel).
Subtype
OREGES00021 In-vivo GFP Expression Assay The gene coding for Green Fluorescent Protein (GFP) is linked to a promoter of another gene, and the construct is transfected into gonadal or embryonic cells. The resulting organisms are observed throughout development and their expression of GFP recorded with respect to time and tissue-type. Putative regulatory elements can be deleted or mutated and the the assay re-done to confirm function.
Subtype
OREGES00026 Dual luciferase reporter gene assay The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter for normalizing the assay. The firefly luciferase gene is fused to the test promoter. Both constructs are integrated or transfectd into the experimental system. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity.
Subtype
OREGES00030 In-vivo LacZ Expression Assay This assay is the same as the GFP Expression assay, except that the LacZ gene (Beta Galactosidase) is used as the reporting gene.
Subtype
OREGES00025 UNKNOWN Unknown reporter contruct assay
Subtype
OREGES00038 Temperature-sensitive mutation in transcription factor and GFP reporter construct First, a strain of the animal is generated that has a temperature-sensitive mutation in the gene of the transcription factor in question. This allows the researcher to control the presence of the transcription factor by growing the animal at either the permissive or non-permissive temperature. The strain is then injected with a GFP reporter gene fused to the regulatory region in question. The researcher can then observe the impact of the presence of the transcription factor on the expression of the reporter gene.
Subtype
OREGES00040 Rabbit Beta-Globin expression assay The gene coding for rabbit beta-globin is linked onto a promoter (or other transcription control region) from another gene, and the construct is "transfected" into cultured cells. Total cell RNA from transfected cells is then isolated, treated by DNaseI, and analyzed by Northern analysis. Next, the RNA undergoes primer extension analysis, and finally is autoradiographed for quantification. The amount of rabbit beta-globin RNA produced is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel)
Subtype
OREGES00043 Stable transfection luciferase assay The gene coding for luciferase is linked onto a promoter (or other transcription control region) from another gene, and the construct is "transfected" into cultured cells. The amount of luciferase enzyme produced is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel). A common method for testing the effects of sequence changes on promoter function. However, in 'stable transfections' (as opposed to 'transient transfections') the transfected DNA is actually incorporated into the cellular genome. Usually this is done by linking the desired gene with a "selectable" gene, i.e. a gene which confers resistance to a toxin (like G418, aka Geneticin).
Subtype
OREGES00037 Secreted alkaline phosphatase (SEAP) assay Secreted alkaline phosphatase (SEAP) is a human placental enzyme which modifies chemiluminescent or fluorescent substrates (CSPD or MUP) leading to changes in optical density. SEAP assays are often performed to test the function of a promoter. The gene coding for SEAP is linked onto a promoter (or transcription control region) from another gene, and the construct is "transfected" into cultured cells. The amount of SEAP enzyme produced is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel).
Subtype
OREGES00045 Transient transfection gene expression assay The promoter (or other transcription control region) of a gene is linked to the transcribed region of either its own gene or a heterologous gene, and the recombinant vector is "transfected" into cultured cells. The amount of mRNA produced is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel).
Subtype
OREGES00047 Transient transfection gene expression assay with ELISA detection The promoter (or other transcription control region) of a gene is linked to a reporter gene, and the recombinant vector is "transfected" into cultured cells. The amount of reporter protein produced is detected by Enzyme-Linked ImmunoSorbent Assay (ELISA).
Subtype
OREGES00048 Transient transfection GFP assay The gene coding for Green Fluorescent Protein (GFP) is linked onto a promoter (or other transcription control region) from another gene, and the construct is "transfected" into cultured cells. The amount of GFP produced is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel). A common method for testing the effects of sequence changes on promoter function.
Subtype
OREGES00049 Targeted in-vivo LacZ expression assay by viral injection The LacZ gene (Beta Galactosidase) is linked to a promoter of another gene, and inserted into a viral DNA construct. The resulting viruses are injected directly into an animal at a targeted site. The targeted tissues are observed for their expression of Beta Galactosidase. Putative regulatory elements can be deleted or mutated and the the assay re-done to confirm function.
Subtype
OREGES00058 Site-specific stable transfection GFP assay by Cre-Lox recombination A method for the introduction of genetic modifications into specific genes by homologous recombination using Cre, a site-specific bacteriophage P1-derived recombinase. Exchange is mediated by loxP sites flanking the test expression cassete and the marker in the recipient DNA. The Cre enzyme mediates the loxP sites coming into close proximity and a site specific recombination. The CreloxP system allows for spatial control of genetic alteration through incorporation of a tissue-specific promoter that drives Cre activity. Cre expression is placed under the control of a specific promoter sequence, which in turn allows for the localized expression of Cre in certain tissues. For example the hemoglobin beta (HBB) promoter would drive recombination specific to human erythrocytes. The DNA fragment targeted by Cre will remain intact in cells which do not express the HBB gene. The donor plasmid has, upstream of the promoter, a multiple cloning site (MCS) into which the test fragment can be inserted. Downstream of the promoter is the Green Florescent Protein gene (GFP) which serves as a reporter for gene activity after induction. The amount of GFP produced is taken to indicate the transcriptional activity of the promoter. This can be used to test for the function of various putative regulatory sequences.
Subtype
OREGES00061 Transient transfection LacZ assay The gene coding for beta-galactosidase (LacZ) is linked onto a promoter (or other transcription control region) from another gene, and the construct is "transfected" into cultured cells. The activity of beta-galactosidase is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel). A common method for testing the effects of sequence changes on promoter function.
Subtype
OREGES00062 In vivo chloramphenicol acetyltransferase (CAT) assay Chloramphenicol acetyltransferase (CAT), is a bacterial enzyme which inactivates chloramphenicol by acetylating it. The gene coding for CAT is linked to a promoter of another gene, and the construct is transfected into gonadal or embryonic cells. The resulting transgenic organisms are tested throughout development for CAT expression, which is recorded with respect to time and tissue-type. Putative regulatory elements can be deleted or mutated and the assay re-done to confirm function.
Subtype
OREGES00063 In-vivo Luciferase expression assay The gene coding for Luciferase is linked to a promoter of another gene, and the construct is transfected into gonadal or embryonic cells. The resulting transgenic organisms are observed throughout development and their expression of Luciferase recorded with respect to time and tissue-type. Putative regulatory elements can be deleted or mutated and the assay re-done to confirm function.
Subtype
OREGES00064 In vivo gene expression assay The promoter (or other transcription control region) of a gene is linked to the transcribed region of either its own gene or a heterologous gene, and the construct is transfected into gonadal or embryonic cells. The resulting transgenic organisms are observed throughout development and the level of mRNA or protein expression for this gene is recorded with respect to time and tissue-type. Putative regulatory elements can be deleted or mutated and the assay re-done to confirm function.
Subtype
OREGES00065 In vitro virus replication assay Potential regulatory sequences are inserted upstream of an endogenous viral gene in a viral DNA construct. The resulting recombinant viruses are injected into cells and the viral titer is measured. Putative regulatory elements can be deleted or mutated and the assay re-done to confirm the effect that the element is having on the replicative ability of the virus.
Subtype
OREGES00066 Stable transfection LacZ assay The LacZ gene, which codes for beta-galactosidase, is linked onto a promoter (or other transcription control region) from another gene, and the construct is "transfected" into cultured cells. The amount of X-gal substrate digested to produce blue color, a characteristic correlated with the amount of beta-galactosidase produced, is taken to indicate the transcriptional activity of the promoter (relative to other promoters which must be tested in parallel). This is a common method for testing the effects of sequence changes on promoter function. However, in 'stable transfections' (as opposed to 'transient transfections') the transfected DNA is actually incorporated into the cellular genome. Usually this is done by linking the desired gene with a "selectable" gene, i.e. a gene which confers resistance to a toxin (like G418, aka Geneticin).
Subtype
OREGES00067 Stably incorporated GFP transgene assay Primary cells from a transgenic animal carrying a promoter-GFP fusion construct are isolated and grown in culture. The amount of GFP produced is taken to indicate the transcriptional activity of the promoter in culture.
OREGET00003 Protein Binding Assay Used to determine whether a given protein binds to or is localized to a specific DNA sequence.
Subtype
OREGES00005 Chromatin immunoprecipitation (ChIP) DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo. Isolate the chromatin. Shear DNA along with bound proteins into small fragments. Bind antibodies specific to the DNA-binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins. Use PCR to amplify specific DNA sequences to see if they were precipitated with the antibody.
Subtype
OREGES00015 DNase Footprinting Assay An assay that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. The method uses restriction enzymes or other deoxyribonucleases to cut the DNA, followed by gel electrophoresis to detect the resulting cleavage pattern. To examine DNA-protein interactions, the cleavage pattern of isolated DNA is compared with the cleavage pattern of DNA in the presence of the putative binding protein. If the protein binds the DNA, the binding site is protected against restriction enzyme cleavage and fewer cleavage sites are detected. From the position of the cleavage sites missing in the assay containing the binding protein, the position and extension of the binding site can be inferred.
Subtype
OREGES00022 Yeast 1-hybrid assay Used for determining which transcription factor binds to a functional regulatory element. The sequence of interest tandemly repeated and fused to the promoter of a reporter gene. This construct is integrated into the yeast genome. These yeast are transformed with a library of candidate cDNAs that have been fused to the activation domain for the reporter gene. Only fusion constructs that contain a DNA-binding domain that binds to the regulatory element will activate the reporter gene.
Subtype
OREGES00033 Methylation Interference Assay This method is also called "contact point analysis". In both DNaseI footprint and DMS protection assays only binding site but not contact points, are mapped. In interference assay, the DNA will be methylated before the binding assay. If the contact points are methylated, the protein binding is interfered. After isolating the protein-DNA complex (use EMSA or affinity purification), the methylation sites are cleaved by chemical method. As a result, only those regions out of the binding sites will be cleaved. The protein binding region is not methylated; hence, this region is not cleaved. Although the pattern looks like a footprint, the blank region means "contact points".
Subtype
OREGES00056 DNA-Protein Precipitation Assay An affinity chromatography-based assay.
Subtype
OREGES00057 Western Blot Assay An assay that detects specific proteins within a protein mixture. Samples are subjected to electrophoresis on a slab gel. A replica of the gel is then made on a membrane by electrophoretic transfer. Specific proteins are then detected on the membrane using antibody staining. (a.k.a immunoblot assay)
Subtype
OREGES00059 Chromatin immunoprecipitation with tag sequencing (ChIP-TS) DNA-binding proteins are crosslinked to DNA (e.g. with formaldehyde). Chromatin is isolated and DNA sheared along with bound proteins into small fragments. Antibodies specific to the DNA-binding protein are used to isolate the complex by precipitation. Next, the cross-linking is reversed to release the DNA and digest the proteins. DNA is typically purified, amplified using PCR reactions, and a library constructed for sequencing. Tags or short reads are sequenced for the pool of fragments to some sampling/sequencing depth (usually determined by cost rather than material or runtime). Sequences are aligned to the genome. Genomic locations with high sequence coverage represent potential binding sites for the DNA-binding protein of interest. ChIP-sequencing methods have been reported that use SAGE-like tags, 36 bp paired-end tags (PETs) and variable length sequence reads. Sequencing technologies utilized include traditional Sanger sequencing as well as multiplex 454 sequencing and the Illumina 1G systems.
Subtype
OREGES00068 In-vivo Footprinting Assay An assay that detects DNA-protein interactions based upon the fact that a protein bound to DNA will often protect that DNA from Dimethyl Sulfide (DMS) cleavage. Cells are exposed to DMS leading to DNA cleavage at unprotected sites. Cells are then lysed, and regions of interest are amplified using PCR. If DNA regions of interest were bound by protein, they will have been protected from DMS cleavage, and PCR reactions will be successful.
Subtype
OREGES00069 DNA-Protein Precipitation Assay with ELISA Detection An affinity chromatography-based assay in which nuclear extracts are incubated with oligonucleotides encoding a potential binding region of interest coupled to a matrix. Proteins which bind to this probe are precipitated, purified by washing, and then detected by staining with primary and secondary antibodies.
Subtype
OREGES00070 ChIP-on-chip (ChIP-chip) ChIP-on-chip (ChIP-chip), also known as genome-wide location analysis, is a technique that combines chromatin immunoprecipitation (ChIP) with microarray technology (chip). As with standard ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo. Coupled with whole-genome DNA microarrays, ChIP allows one to determine the entire spectrum of in vivo DNA binding sites for any given protein. ChIP is performed as usual; DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo. The chromatin is isolated. DNA along with bound proteins is sheared into small fragments and then antibodies specific to the DNA-binding protein are added to isolate the complex by precipitation. The cross-linking is reversed to release the DNA and the proteins are digested. Finally, to determine where the protein binds across the whole genome, the purified DNA and appropriate controls are fluorescently labeled and applied to microscopic slides for microarray analysis.
Subtype
OREGES00074 Chromatin immunoprecipitation with paired-end diTag sequencing (ChIP-PET) ChIP-PET is a technique that combines chromatin immunoprecipitation (ChIP) with high throughput cloning and sequencing approach for mapping full-length transcripts. This technique increases sequencing efficiency by 30-fold as compared to sequencing the entire transcript insert. DNA-binding proteins are crosslinked to DNA (e.g. with formaldehyde). Chromatin is isolated and DNA sheared along with bound proteins into small fragments. Antibodies specific to the DNA-binding protein are used to isolate the complex by precipitation. Next, the cross-linking is reversed to release the DNA and digest the proteins. To construct a ChIP-PET library, ChIP-enriched DNA is purified, end polished and ligated into a cloning vector. The ligations are then transformed into bacteria in order to form the ChIP DNA library. Plasmids of the ChIP DNA library are then digested with appropriate digestion enzymes in a way that the resulting vectors contain a signature tag from each terminal of the ChIP DNA insert. Their ends are then polished to remove 3'-dinucleotide overhangs. The resulting vector is self-ligated to form single-ditag plasmids and then transformed into bacteria to form the "single-ditag" library. The plasmids from this library are then digested to release the paired end ditags, which are then concatenated into long fragments (1-2Kb) and cloned into a vector as the final ChIP-PET library for sequencing. The PET sequences are extracted from the raw sequence obtained from the ChIP-PET library and are mapped to the genome. The specific mapping criteria are that both the 5' and 3' signatures must be present on the same chromosome, on the same strand, in the correct orientation (5' --> 3').
OREGET00004 RNA Expression Assay Used to determine whether a putative transcription factor or binding site affects transcription by measuring levels of RNA produced.
Subtype
OREGES00006 RNase Protection Assay (RPA) Uses a radiolabelled RNA complementary to the mRNA of interest to protect it from the RNase. Purified and digested dsRNA are run on a gel and the amount of radioactive signal is proportional to the amount of specific target mRNA in the original sample. Useful for measuring relative expression of related mRNA species. Can be used in combination with DNA competition to show function of a binding site.
Subtype
OREGES00007 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 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. Variations such as quantitative real-time RT-PCR can be used to accurately measure mRNA transcript levels, even at relatively low copy number.
Subtype
OREGES00009 Allele-specific Transcript Quantification (ASTQ) A variation of RT-PCR where transcripts are Southern blotted with a radio-labelled probe and then measured for signal intensity
Subtype
OREGES00018 Competitive PCR (cPCR) A method to quantify DNA using real-time PCR. A competitor internal standard is co-amplified with the target DNA. Target is quantified from the melting curves of the target and the competitor. The same principle is applied to real-time RT-PCR to quantify RNA.
Subtype
OREGES00020 RLM-RACE (RNA Ligase-mediated Rapid Amplification of cDNA ends) RNA ligase-mediated RACE (RLM-RACE), and involves the use of bacteriophage T4 RNA ligase to covalently attach a single strand RNA anchor molecule to the de-capped 5’ end of mRNA. cDNA synthesis can then be performed using a gene-specific primer to produce a pool of cDNAs encoding the anchored primer sequence.
Subtype
OREGES00023 Whole-mount in situ hybridization A method to semi-quantitatively assess the temporal and/or spatial patterns of gene expression. For example, labelled anti-sense RNA probes for a gene of interest and its transcription factor can be hybridized to whole-mount preparations of mouse embryos at different developmental stages. Coexpression of the gene and its transcription factor at specific stages or tissues can be used to infer regulation.
Subtype
OREGES00039 Northern Blot RNA is extracted from cells and run on a gel. The bands are then transferred to a filter membrane and blotted with a radioactively-labelled cDNA probe for the transcript in question. This technique can be used to identify which RNA band contains the transcript; it can also be used to quantify the amount of transcript present under different conditions.
Subtype
OREGES00050 In-vitro Transcription Assay with detection by primer extension Used to determine whether a putative regulatory element affects the transcription of a gene in-vitro. Purified linear DNA template is incubated with rNTPs and RNA polymerase in a compatible buffer. Amount of transcript produced is quantitated by primer extension.
Subtype
OREGES00044 Primer Extension Analysis with poly(A)+ RNA Mapping technique for detecting TSS (transcription start sites).
OREGET00005 Protein Expression Assay Used to determine whether a putative transcription factor or binding site affects translation.
Subtype
OREGES00008 Western Blot Assay An assay that detects specific proteins within a protein mixture. Samples are subjected to electrophoresis on a slab gel. A replica of the gel is then made on a membrane by electrophoretic transfer. Specific proteins are then detected on the membrane using antibody staining.
Subtype
OREGES00012 Luciferase Expression Assay Protein is quantified using luciferase protein fusion with target gene.
Subtype
OREGES00014 Enzyme-linked immunosorbent assay (ELISA) An assay that detects specific proteins within a sample. There are variations of this test, but the most basic consists of an antibody attached to a solid surface. This antibody has affinity for the protein of interest (e.g. protein A). A mixture of purified protein A linked to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no protein A is present in the test sample, then only the purified protein A with linked enzyme will bind to the solid surface. The more protein A which is present in the test sample, the less enzyme-linked protein A will bind. The substance the enzyme acts on is then added, and the amount of product measured in some way, such as a change in colour of the solution.
Subtype
OREGES00024 Indirect Immunofluorescence A method of using fluorescence microscopy to detect the presence of an antigen indirectly. An antibody is applied to the protein of interest, excess antibody washed away, and then a second labelled antibody that binds the first antibody is added. Labelled antibody is visualized with conventional immunofluorescence microscopy and the amount of target protein estimated.
OREGET00006 Association Study Used to determine if a particular allele is present in a population exhibiting a particular phenotype
Subtype
OREGES00010 Resequencing Controls and target subjects were subjected to resequencing to demonstrate a particular association.
Subtype
OREGES00011 Single-Stranded Conformational Polymorphism (SSCP) Identification of mutations/polymorphisms based on the observation that certain mutations alter the manner in which single-stranded DNA folds upon itself and thus affect its mobility on gel electrophoresis; mutant sequences can be distinguished from wild-type sequences by comparing the electrophoretic pattern
Subtype
OREGES00017 Restriction Fragment Length Polymorphism (RFLP) Analysis Makes use of variation in DNA fragment banding patterns of electrophoresed restriction digests of DNA from different individuals of a species. For example, if a SNP creates or destroys a restriction enzyme site, this will be visualized as a different banding pattern on the gel.
OREGET00007 RNA Stability Assay An assay that measures mRNA turnover rate or mRNA stability
Subtype
OREGES00016 RNA synthesis blocking A blocking agent such as Actinomycin-D (a DNA binding compound that prevents RNA synthesis) is used to block transcription. mRNA levels are measured (usually by RT-PCR) upon addition and then again after a set time period. The change in RNA concentration is a measure of stability. RNA stability can be compared using this method for different regulatory variants or mutants.
OREGET00010 Sequence Conservation In some studies, bioinformatic methods are used to identify conserved sequences in a set of orthologous or simultaneously expressed genes. This information narrows down the likely locations of the regulatory elements, some of which are subsequently confirmed using laboratory methods.
Subtype
OREGES00028 Orthologous gene conservation Conservation of lab-verified binding site (sequence and position) in the orthologous gene of a related species. The comment field can be used to indicate which orthologous gene this site is confirmed in.
Subtype
OREGES00029 Co-expressed gene conservation Conservation of lab-verified binding site in a gene expressed in the same tissue and developmental timepoint. The comment field can be used to indicate which gene this site is confirmed in.
OREGET00008 Orthologous gene conservation Conservation of an experimentally-verified regulatory element (sequence and position) in a homologous region of at least one other species.
Subtype
OREGES00027 Conservation found by alignment Conserved regulatory element detected in an alignment of homologous sequences.
Subtype
OREGES00031 Conservation found by scanning with a motif model Regulatory element identified in multiple homologous sequences by scanning with a motif model
OREGET00011 Linkage Analysis The use of several DNA sequence polymorphisms (normal variants) that are near or within a gene of interest to track within a family the inheritance of a disease-causing mutation in that gene.
Subtype
OREGES00036 Resequencing Target subjects were subjected to resequencing to demonstrate a particular inheritance pattern.
OREGET00013 Gene co-expression Identification of a homologous regulatory element in a gene which is expressed in the same tissue or developmental timepoint as a previously-identified gene regulated by an experimentallly-verified regulatory element.
Subtype
OREGES00032 Co-expressed genes determined through expression patterns Co-expressed genes determined from expression pattern data in situ
Subtype
OREGES00034 Co-expressed genes determined through reporter gene experiments Co-expressed genes were found by observing the expression of reporter gene constructs.
Subtype
OREGES00035 Co-expressed genes determined through microarray experiments Co-expressed genes were found using microarray experiments.
OREGET00009 Mutagenesis A mutation is artificially introduced into biological sequence to identify, characterize and/or elucide the mechanisms of action of physical, chemical and biological agents capable of producing genetic change in living organism to thereby analyze the consequences of such changes.
Subtype
OREGES00041 UNKNOWN UNKNOWN
Subtype
OREGES00042 Rescue construct A construct recovers expected activity in a mutant background.
Subtype
OREGES00051 Exonuclease digestion An exonuclease is used to specifically digest DNA from a 5' protruding or blunt end in a progressive unidirectional manner. The rate of digestion is uniform, allowing deletion of predetermined lengths by stopping the reaction at timed intervals.
Subtype
OREGES00052 PCR DNA fragments are generated by PCR using a sense and antisense primer complementary to the flanking regions of the subsequence of interest.
Subtype
OREGES00053 Restriction endonuclease digestion DNA sequence is digested with restriction endonuclease, resulting in a deletion construct.
Subtype
OREGES00054 Site-directed Mutated oligonucleotide primers are used in a PCR to insert specific point mutation(s) into a wildtype sequence.
Subtype
OREGES00055 Oligonucleotide synthesis Chemical DNA synthesis in which specific single nucleotides are linked together into a growing chain by multi-step reactions.
OREGET00016 Expert curated Regulatory sequences which have been identified by recognized experts in the field and can be included in ORegAnno without re-annotation.
Subtype
OREGES00046 Literature derived The publication provides a set of curated regulatory sequences based on a review of other publications by experts in the field. In some cases, information from multiple publications are combined in such a way that the reported sequences do not correspond exactly to any one of the original publications. It may be that any one of the original publications would not be considered strong evidence for an ORegAnno record. But together, the publications make a convincing case for a functional regulatory sequence. This evidence type should only be used in cases where the expert curator is highly trusted and the source publications would not be easily re-curated. Wherever possible, the original publications upon which the review was based should be cited in the evidence comment.
 

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