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`EP 1 363 125 A2
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`European Patent Office
`Office européen des brevets
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`(19) a
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`(12)
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`EUROPEAN PATENT APPLICATION
`
`(43) Dale OfPUb'icationi
`19.11.2003 Bulletin 2003/47
`
`(21) Application number: 03010388.1
`
`(22) Date of filing: 08.05.2003
`
`(51) Int CI.7: G01N 33/543, C12Q 1/68,
`801;] 19/00, C12M 1/34
`
`(84) Designated Contracting States:
`AT BE BG CH CY CZ DE DK EE ES FI FR GB GR
`HU IE IT LI LU MC NL PT RO SE SI SK TR
`
`Designated Extension States:
`AL LT LV MK
`
`(30) Priority: 08.05.2002 US 140956
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`(71) Applicant: Gentel Corporation
`Madison, Wisconsin 53719 (US)
`
`
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`(72) Inventor: Nelson, Bryce, P.
`Madison, Wisconsin 53703 (US)
`
`(74) Representative: Glawe, Delfs, Moll & Partner
`Patentanwfilte
`Rothenbaumchaussee 58
`
`20148 Hamburg (DE)
`
`(54)
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`Transcription factor profiling on a solid surface
`
`The present invention relates to nOVeI methods
`(57)
`for the analysis of interactions of transcription factors
`with target nucleic acids. In particular, the present inven-
`tion relates to compositions and methods for the detec-
`tion of transcription factors binding to their target pro-
`
`moter regions. The present invention further provides
`methods of screening compounds fortheir abilityto alter
`such binding interactions.
`
`EP1363125A2
`
`Prlnted by Jouve, 75001 PARIS (FR)
`
`

`

`Description
`
`EP 1 363 125 A2
`
`[0001] This application is acontinuation in part of copending application serial number 10/1 40,956, filed 5/8/02, which
`is herein incorporated by reference in its entirety.
`
`FIELD OF TH E INVENTION
`
`[0002] The present invention relates to novel methods for the analysis of interactions of transcription factors with
`target nucleic acids. In particular, the present invention relates to compositions and methods forthe detection of tran-
`scription factors binding to their target promoter regions. The present invention further provides methods of screening
`compounds for their ability to alter such binding interactions.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Transcription factors are proteins that bind to specific enhancer or promoter DNA sequences to regulate the
`transcription of certain genes. Under certain stimuli, transcription factors are created, or become active. To determine
`whether a given transcription factor is active or present in a sample, an electrophoretic mobility shift assay (EMSA) is
`often performed. In this procedure, a radioactively labeled DNA probe is mixed with a protein extract and the entire
`reaction is run on a nondenaturing polyacrylamide gel. Because the protein-bound probe will migrate more slowly than
`a free probe, the experiment is described as a "gel shift." Unfortunately, the gel shift is a cumbersome way to profile
`the activity of many transcription factors at once, because for every factor tested, a separate labeled probe needs to
`be generated. Moreover, results from such techniques are difficult to quantify.
`[0004]
`Several commercially available replacements for the standard gel—shift assay exist. One method attaches the
`consensus DNA binding sequence to wells of a standard ELISA plate. Transcription factor binding is detected via an
`antibody—peroxidase conjugate, much like a standard ELISA detection. Examples include Active Motif's Paradigm Gel
`Shift and CloneTech's Mercury TransFactor kits. These kits can only detect transcription factors for which there is a
`corresponding antibody available. Panomics produces a transcription factor detection kit where fluorescently labeled
`double-stranded oligonucleotides are exposed to nuclear extracts. Protein-oligo complexes are purified, and the oli-
`gonucleotides that were bound by a transcription factor can be detected on an array. This detection method is indirect,
`can only observe binding to known DNA consensus sequences, and requires expensive, and variable, labeling of the
`DNA fragment.
`[0005] What is needed are simple, cost effective methods for screening large numbers of transcription factor binding
`events.
`
`SUMMARY OF THE INVENTION
`
`[0006] The present invention relates to novel methods for the analysis of interactions of transcription factors with
`target nucleic acids. In particular, the present invention relates to compositions and methods forthe detection of tran-
`scription factors binding to their target promoter regions. The present invention further provides methods of screening
`compounds for their ability to alter such binding interactions.
`[0007]
`For example, in some embodiments, the present invention provides a composition comprising an arrayed
`solid surface, the solid surface comprising an array of transcription factor binding targets. In some embodiments, the
`array of transcription factor binding targets comprises at least 20, preferably at least 50, even more preferably at least
`100, and still more preferably, at least 1000 distinct target nucleic acid sequences. In some preferred embodiments,
`the solid surface is configured for label free detection. Accordingly, in some embodiments, the solid surface is an SPR
`surface (e.g., an SPR prism). In some embodiments, the solid surface further comprises a plurality of microfluidics
`channels (9.9., one dimensional ortwo dimensional arrays). In other embodiments, the solid surface further comprises
`a plurality of etched microchannels. In some embodiments, the transcription factor targets are double—stranded DNA
`molecules.
`
`[0008] The present invention further provides a composition comprising an arrayed solid surface comprising a plu-
`rality of microfluidics channels, the solid surface comprising an array of transcription factor binding targets.
`[0009] The present invention additionally provides a composition comprising an arrayed solid surface comprising a
`plurality of etched microchannels, the solid surface comprising an array of transcription factor binding targets.
`[0010] The present invention also provides a composition comprising an arrayed solid surface comprising an array
`of transcription factor binding targets in contact with a biological sample containing at least one transcription factor.
`[0011] The present invention further provides a composition comprising an arrayed solid surface comprising an array
`of transcription factor binding targets in contact with a biological sample containing at least one transcription factor,
`wherein the biological sample has been treated with a small molecule.
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`EP 1 363125 A2
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`In still further embodiments, the present invention provides a composition comprising an arrayed solid surface
`[0012]
`comprising an array of transcription factors. In some embodiments, the array of transcription factors comprises at least
`20, preferably at least 50, even more preferably at least 100, and still more preferably at least 1000 distincttranscription
`factors. In some preferred embodiments, the solid surface is configured for label free detection. Accordingly, in some
`embodiments, the solid surface is an SPR surface (9.9., an SPR prism). In some embodiments, the solid surface further
`comprises a plurality of microfluidics channels (9.9., one dimensional ortwo dimensional arrays). In other embodiments,
`the solid surface further comprises a plurality of etched microchannels.
`[0013] The present invention further provides a composition comprising an arrayed solid surface comprising an array
`of transcription factors in contact with a sample comprising at least one transcription factor binding target.
`[0014] The present invention also provides a composition comprising an arrayed solid surface comprising an array
`of transcription factors in contact with a treated biological sample, the treated biological sample used to prepare a cell
`lysate containing at least one test compound.
`[0015] The present invention additionally provides a system comprising a composition comprising an arrayed solid
`surface, the solid surface comprising an array of transcription factor binding targets; at least one transcription factor
`polypeptide; and a detection apparatus in communication with the arrayed solid surface. In some embodiments, the
`array of transcription factor binding targets comprises at least 20, preferably at least 50, even more preferably at least
`100, and still more preferably, at least 1000 distinct target nucleic acid sequences. In some embodiments, the at least
`one transcription factor polypeptide comprises at least 20, preferably at least 50, even more preferably at least 100,
`and still more preferably at least 1000 distincttranscription factors. In some embodiments, the system further comprises
`competitor DNA, wherein the competitor DNA has an identical nucleic acid sequence as the transcription factor binding
`targets. In other embodiments, the system further comprises an antibody that specifically recognizes the at least on
`transcription factor polypeptide. In some embodiments, the antibody is conjugated to a gold particle. In yet other em-
`bodiments, the system further comprises a second transcription factor target sequence, wherein a portion ofthe second
`transcription factortarget sequence is complementaryto the arrayedtranscription factortarget sequences. In still further
`embodiments, the system furthercomprises at least one test compound (9.9., a drug). In some preferred embodiments,
`the solid surface is an SPR surface (9.9., an SPR prism).
`In some embodiments, the solid surface further comprises
`a plurality of microfluidics channels (9.9., one dimensional ortwo dimensional arrays). In other embodiments, the solid
`surface further comprises a plurality of etched microchannels. In some embodiments, the detection apparatus is con-
`figured for label-free detection. In other embodiments, the detection apparatus is configured for detection of a label.
`[0016] The present invention further provides a system comprising a composition comprising an arrayed solid sur-
`face, the solid surface comprising an array of transcription factor polypeptides; at least one transcription factor binding
`target; and a detection apparatus in communication with the arrayed solid surface.
`[0017] The present invention additionally provides a system comprising an arrayed solid surface, the solid surface
`comprising an array of transcription factor binding targets; and a biological sample containing at least one transcription
`factor, the biological sample in communication with the array solid surface.
`[0018] The present invention also provides a system, comprising an arrayed solid surface, the solid surface com—
`prising an array of transcription factor binding targets; and a biological sample containing at least one transcription
`factor, the biological sample in communication with the array solid surface, and wherein the biological sample has been
`treated with a small molecule.
`
`[0019] The present invention further provides a system comprising a composition comprising an arrayed solid sur-
`face, the solid surface comprising an array of transcription factor polypeptides; at least one transcription factor binding
`target; and a cell lysate comprising at least one test compound, and the cell lysate in contact with the array solid surface.
`[0020]
`In some embodiments, the present invention provides a method of detecting biomolecular interactions, com-
`prising providing an arrayed solid surface, the solid surface comprising an array of transcription factor binding targets;
`at least one transcription factor polypeptide; and a detection apparatus in communication with the arrayed solid surface;
`and contacting the at least one transcription factor polypeptide with the array of transcription factor targets under
`conditions such that the apparatus detects interactions between the array of transcription factor targets and the at least
`one transcription factor polypeptide. In some embodiments, the array of transcription factor binding targets comprises
`at least 20, preferably at least 50, even more preferably at least 100, and still more preferably, at least 1000 distinct
`target n ucleic acid sequences. In other embodiments, the array comprises one of the transcription factor binding targets.
`In some embodiments, the at lest one transcription factor polypeptide comprises at least 20, preferably at least 50,
`even more preferably at least 100, and still more preferably at least 1000 distinct transcription factors.
`In some em-
`bodiments, the method further comprises the step of contacting the at least one transcription factor polypeptide and
`the array of transcription factor targets with competitor DNA, wherein the competitor DNA has an identical nucleic acid
`sequence as the transcription factor binding targets. In some embodiments, the method further comprises the step of
`providing an antibodythat specifically recognizes the at least on transcription factor polypeptide. In some embodiments,
`the antibody is conjugated to a gold particle. In some embodiments, the method furthercomprises the step of detecting
`the interaction between the transcription factor polypeptide and the transcription factor binding target by detecting the
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`EP 1 363125 A2
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`In some
`binding of the antibody to transcription factor polypeptide bound to the transcription factor binding target.
`embodiments, the method furthercomprises the step of providing a second transcription factortarget sequence, where-
`in a portion of the second transcription factor target sequence is complementary to the arrayed transcription factor
`target sequences. In other embodiments, the method further comprises the step of, prior to the contacting, the step of
`contacting the second transcription factor target with the transcription factor polypeptide under conditions such that
`the second transcription factor target and the transcription factor polypeptide interact. In some preferred embodiments,
`the solid surface is an SPR surface (9.9., an SPR prism).
`In some embodiments, the solid surface further comprises
`a plurality of microfluidics channels (9.9., one dimensional ortwo dimensional arrays). In other embodiments, the solid
`surface further comprises a plurality of etched microchannels. In some embodiments, the detection apparatus is con-
`figured for label-free detection. In other embodiments, the detection apparatus is configured for detection of a label.
`[0021] The present invention further provides a method of detecting biomolecular interactions, comprising providing
`an arrayed solid surface, the solid surface comprising an array of transcription factors; at least one transcription factor
`binding target; and a detection apparatus in communication with the arrayed solid surface; and contacting the at least
`one transcription factor binding target with the array of transcription factors under conditions such that the apparatus
`detects interactions between the array of transcription factors and the at least one transcription factor binding target.
`[0022] The present invention also provides a method for measuring biomolecular interactions, comprising providing
`an arrayed solid surface, the solid surface comprising an array of transcription factorbinding targets; a biological sample
`containing at least one transcription factor; and a detection apparatus; and contacting the biological sample with the
`arrayed solid surface targets under conditions such that the apparatus detects interactions between the array of tran-
`scription factor targets and the at least one transcription factor contained in the biological sample.
`[0023] The present invention additionally provides a method for measuring the effect of small molecules on biomo-
`lecular interactions, the method comprising providing an arrayed surface, the surface comprising an array oftranscrip-
`tion factor binding targets; a test compound; a biological sample comprising at least one transcription factor; a detection
`apparatus; and treating the biological sample with the small molecule; and contacting the treated biological sample
`with the array of transcription factortargets under conditions such that the apparatus detects interactions between the
`array of transcription factortargets and the at least one transcription factor contained in the biological sample. In some
`embodiments, priorto the step ofcontacting treated biological sample with the array of transcription factor, the biological
`sample is treated with a test compound. In some embodiments, the test compound is a drug. In some embodiments,
`the method further comprises the step of comparing the interactions between the array of transcription factor targets
`and the at least one transcription factor polypeptide in the presence of the test compound to the interactions in the
`absence of the test compound. In some embodiments, the test compound inhibits the interaction of the array of tran-
`scription factor targets and the at least one transcription factor polypeptide. In other embodiments, the test compound
`enhances the interaction of the array of transcription factor targets and the at least one transcription factor polypeptide.
`[0024] The present invention further provides a method for measuring biomolecular interactions, comprising provid-
`ing an arrayed solid surface, the solid surface comprising an array of transcription factors; a biological sample com—
`prising at least one test compound; at least one transcription factor binding target; and a detection apparatus; and
`contacting the biological sample and the at least one binding target with the arrayed solid surface under conditions
`such that the detection apparatus detects interactions between the array of transcription factors and the at least one
`transcription factor binding target.
`[0025] The present invention also provides a method for measuring the effect of small molecules on biomolecular
`interactions, comprising providing an arrayed solid surface, the surface comprising an array of transcription factor
`binding targets; at least one small molecule test compound; a biological sample containing at least one transcription
`factor; a detection apparatus; and treating the biological sample with the at least one small molecule test compound
`to generate a treated biological sample; contacting the treated biological sample with the array of transcription factor
`targets under conditions such that the apparatus detects interactions between the array of transcription factor targets
`and the at least one transcription factor contained in the biological sample.
`
`DESCRIPTION OF THE FIGURES
`
`[0026]
`
`Figure 1 shows an overview of the creation of a photopatterned MUAM surface on an SPR-capable gold coated
`glass slide.
`Figure 2 shows SPR data for the binding of AP2 DNA to AP2 protein. The top image shows real-time change in
`SPR signal measured on AP2 DNA target spots after addition of AP2 protein. The middle image shows the final
`difference image after AP2 binding to the arrayed AP2 oligonucleotides. The bottom image is a schematic repre-
`sentation of the array surface used in these experiments.
`Figure 3 shows SPR data for AP2 protein binding in high salt concentration conditions.
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`DEFINITIONS
`
`EP 1 363125 A2
`
`[0027] As used herein, the term "solid surface" refers to any solid surface suitable for the attachment of biological
`molecules and the performance of molecular interaction assays. Surfaces may be made of any suitable material (9.9.,
`including, but not limited to, metal, glass, and plastic) and may be modified with coatings (9.9., metals or polymers).
`[0028] As used herein, the term "substrate" refers to any material with a surface that may be coated with a film.
`[0029] As used herein, the phrase "coated with a film" in regard to a substrate refers to a situation where at least a
`portion of a substrate surface has a film attached to it (9.9. through covalent or non-covalent attachment).
`[0030] As used herein, the term "microarray" refers to a solid surface comprising a plurality of addressed biological
`macromolecules (9.9., nucleic acids or antibodies). The location of each of the macromolecules in the microarray is
`known, so as to allow for identification of the samples following analysis.
`[0031] As used herein, the term "array of transcription factor binding targets" refers to an microarray of nucleic acid
`sequences that are known to, or are suspected of, binding to a transcription factor, arrayed on a solid support.
`[0032] As used herein, the term "array of transcription factors" refers to an microarray of transcription factor polypep-
`tides on a solid support.
`[0033] As used herein, the term "SPR surface" refers to a solid surface that is suitable for use in SPR detection. In
`some embodiments, "SPR surfaces" are "SPR prisms."
`[0034] As used herein, the term "disposable arrayed SPR prism" refers to a prism that is suitable for use in SPR
`detection, comprises an arrayed surface (9.9., a microarray), and is not intended to be reused for multiple detection
`assays. In some embodiments, the disposable arrayed prisms are those disclosed herein.
`[0035] As used herein, the term "coated on one face" when used in reference to an SPR prism, refers to a prism
`with a coating on one of the main faces of the prism. For example, in some embodiments, triangular prisms are coated
`on the upward facing surface. The term "face" is not intended to encompass the small facets on each face of a prism
`that reflect light.
`[0036] As used herein, the term "SPR capable metal film" refers to any metallic film that is suitable for use in SPR
`detection. Examples include, but are not limited to, gold, silver, chrome, and aluminum.
`[0037] As used herein, theterm "microfluidics channels" or "etched microchannels" refers to three-dimensional chan-
`nels created in material deposited on a solid surface. In some embodiments, microchannels are composed of a polymer
`(9.9., polydimethylsiloxane). Exemplary methods for constructing microchannels include, but are not limited to, those
`disclosed herein.
`
`[0038] As used herein, the term "one-dimensional line array" refers to parallel microfluidic channels on top of a
`surface that are oriented in only one dimension.
`[0039] As used herein, the term "two dimensional arrays" refers to microfluidics channels on top of a surface that
`are oriented in two dimensions. In some embodiments, channels are oriented in two dimensions that are perpendicular
`to each other.
`
`[0040] As used herein, the term "microchannels" refers to channels etched into a surface. Microchannels may be
`one—dimensional or two—dimensional.
`
`[0041] As used herein, the term "biological macromolecule" refers to large molecules (9.9., polymers) typically found
`in living organisms. Examples include, but are not limited to, proteins, nucleic acids, lipids, and carbohydrates.
`[0042] As used herein, the term "target molecule" refers to a molecule in a sampleto be detected. Examples of target
`molecules include, but are not limited to, oligonucleotides (9.9. containing a particular DNA binding domain recognition
`sequence), viruses, polypeptides, antibodies, naturally occurring drugs, synthetic drugs, pollutants, allergens, affector
`molecules, growth factors, chemokines, cytokines, and Iymphokines. As used herein, the term "target nucleic acid
`sequence" refers to a nucleic acid sequence known to be, or suspected of being, a transcription factor recognition
`target sequence.
`[0043] As used herein, the term "binding partners" refers to two molecules (9.9., proteins) that are capable of, or
`suspected of being capable of, physically interacting with each other. As used herein, the terms "first binding partner"
`and "second binding partner" refer to two binding partners that are capable of, or suspected of being capable of,
`physically interacting with each other.
`[0044] The term "sample" as used herein is used in its broadest sense and includes, but is not limited to, environ-
`mental, industrial, and biological samples. Environmental samples include material from the environment such as soil
`and water. Industrial samples include products or waste generated during a manufacturing process. Biological samples
`may be animal, including, human, fluid (9.9., blood, plasma and serum), solid (9.9., stool), tissue, liquid foods (9.9.,
`milk), and cell Iysates (9.9., cultured cell Iysates).
`[0045] The term "test compound" refers to any chemical entity, pharmaceutical, drug, and the like that is suspected
`of altering the affinity of a transcription factorfor its target sequence. Test compounds comprise both compounds known
`to alter such interactions, and those suspected to. Atest compound can be determined to be active in altering binding
`interactions by screening using the screening methods of the present invention.
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`EP 1 363125 A2
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`[0046] The term "signal" as used herein refers to any detectable effect, such as would be caused or provided by an
`assay reaction. For example, in some embodiments of the present invention, signals are SPR or fluorescent signals.
`[0047] As used herein, the term "label free detection" refers to the detection of a binding interaction between unla-
`beled transcription factors and binding targets. Methods of label free detection include, but are not limited to, those
`disclosed herein.
`
`[0048] As used herein, the term "detection apparatus" refers to an apparatus configured for the detection of an
`interaction between a transcription factor and a nucleic acid target. In some embodiments, detection apparatus are
`configured for "label free detection." In other embodiments, they are configured for detection of a label (6.9., on a
`transcription factor or a DNA binding target).
`[0049] DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make
`oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is
`attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligo—
`nucleotides or polynucleotide, referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononu—
`cleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide
`pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide,
`also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to
`as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription
`proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements that direct transcription of
`a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their
`effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenyla—
`tion signals are located 3' or downstream of the coding region.
`[0050] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.
`e., a sequence of nucleotides) related by the base—pairing rules. For example, the sequence "5'—A—G—T—3'," is comple—
`mentary to the sequence "3'—T—C—A—5'." Complementarity may be "partial," in which only some of the nucleic acids'
`bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between
`the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency
`and strength of hybridization between nucleic acid strands.
`[0051] The term "homology" refers to a degree of complementarity. There may be partial homology or complete
`homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits a completely com-
`plementary sequence from hybridizing to a target nucleic acid and is referred to using the functional term "substantially
`homologous." The term "inhibition of binding," when used in reference to nucleic acid binding, refers to inhibition of
`binding caused by competition of homologous sequences for binding to a target sequence. The inhibition of hybridi-
`zation of the completely complementary sequence to the target sequence may be examined using a hybridization
`assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. Asubstantially
`homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homol—
`ogous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that
`non—specific binding is permitted; low stringency conditions require that the binding of two sequences to one another
`be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second
`target that lacks even a partial degree of complementarity (9.9., less than about 30% identity); in the absence of non-
`specific binding the probe will not hybridize to the second non-complementary target.
`[0052] The art knows well that numerous equivalent conditions may be employed to comprise low stringency con-
`ditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target
`(DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other
`components (9.9., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and
`the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equiv-
`alent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions
`of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in
`the hybridization solution, etc.).
`[0053] When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the
`term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded
`nucleic acid sequence under conditions of low stringency as described above.
`[0054] When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous"
`refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under
`conditions of low stringency as described above.
`[0055] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids.
`Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is im-
`pacted by such factors as the degree of complementary between the nucleic acids, stringency ofthe conditions involved,
`the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
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`EP 1 363125 A2
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`[0056] As used herein, the term "Tm" is used in reference to the "melting temperature." The melting temperature is
`the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single
`strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references,
`a simple estimate of the Tm value may be calculated by the equation: Tm = 81.5 + 0.41 (% G + C), when a nucleic acid
`is in aqueous solution at 1 M NaC (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid
`Hybridization [1985]). Other references include more sophisticated computations that take structural as well as se-
`quence characteristics into account forthe calculation of Tm.
`[0057] As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and
`the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted.
`Those skilled in the art will recognize that "stringency" conditions may be altered by varying the parameters just de-
`scribed either individually or in concert. With "high stringency" conditions, nucleic acid base pairing will occur only
`between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization
`under "high stringency" conditions may occur between homologs with about 85—100% identity, preferably about
`70-100% identity). With medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with
`an intermediate frequency of complementary base sequences (e.g., hybridization under "medium stringency" cond