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EP1090138A1 - Procede de detection de nucleotide - Google Patents

Procede de detection de nucleotide

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Publication number
EP1090138A1
EP1090138A1 EP99930469A EP99930469A EP1090138A1 EP 1090138 A1 EP1090138 A1 EP 1090138A1 EP 99930469 A EP99930469 A EP 99930469A EP 99930469 A EP99930469 A EP 99930469A EP 1090138 A1 EP1090138 A1 EP 1090138A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
nucleotide
product
oligonucleotide
hybridization
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99930469A
Other languages
German (de)
English (en)
Other versions
EP1090138A4 (fr
Inventor
Michael Phillip Glaxo Wellcome Inc. WEINER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glaxo Group Ltd
Original Assignee
Glaxo Group Ltd
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Filing date
Publication date
Application filed by Glaxo Group Ltd filed Critical Glaxo Group Ltd
Publication of EP1090138A1 publication Critical patent/EP1090138A1/fr
Publication of EP1090138A4 publication Critical patent/EP1090138A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention provides methods for rapid detection of single nucleotide polymorphisms (SNPs) in a nucleic acid sample.
  • the present invention further provides a novel read-out method for improving the use of mobile solid support-based read-out technologies for detection of nucleic acid polymorphisms in a target nucleic acid
  • the methods can be utilized to detect SNPs in genomic DNA as well as amplified DNA, RNA, etc., thus making them useful for a variety of purposes, including genotyping (such as for disease mutation detection and for parentage determinations in humans and other animals), pathogen detection and identification, and differential gene expression.
  • the present invention further provides a method for identifying a nucleic acid utilizing a run-off sequencing reaction of a relatively short portion of the nucleic acid.
  • the method can be utilized, for example, to identify an EST from only a small portion of the EST and in an analysis of nucleotide polymorphisms.
  • the reactions can be multiplexed to increase data readout capacity.
  • the FlowMetrix system currently consists of uniformly-sized 5 micron polystyrene-divinylbenzene beads stained in eight concentrations of two dyes (orange and red). The matrix of the two dyes in eight concentrations allows for 64 differently-colored beads (8 2 ) that can each be differentiated by a FACScalibur suitably modified with the Luminex PC computer board.
  • covalently-linked to a bead is a short (approximately 18-20 bases) "target" oligodeoxynucleotide (oligo).
  • target oligo oligo
  • the nucleotide positioned at the center of the target oligo encodes the polymorphic base.
  • a pair of beads are synthesized; each bead of the pair has attached to it one of the polymorphic oligonucleotides.
  • a PCR of the region of DNA surrounding the to-be analyzed SNP is performed to generate a PCR product. Conditions are established that allow hybridization of the PCR product preferentially to the bead on which is encoded the precise complement.
  • the PCR product In one format (“without competitor”), the PCR product itself incorporates a flourescein dye and it is the gain of the flourescein stain on the bead, as measured during the FACScalibur run, that indicates hybridization.
  • a second format (“with competitor,") the beads are hybridized with a competitor to the PCR product. The competitor itself in this case is labeled with flourescein.
  • GPA Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms. Nikiforov T T; Rendle R B; Goelet P; Rogers Y H; Kotewicz M L; Anderson S; Trainor G L; Knapp M R. NUCLEIC ACIDS RESEARCH, (1994) 22 (20) 4167-75].
  • specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate- modified primer.
  • the double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer.
  • This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E.
  • the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates.
  • Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper also describes biochemical features of this method in detail.
  • GSA Genetic Bit Analysis
  • the enzyme activity was, however, completely inhibited by the presence of four phosphorothioates.
  • a method for the conversion of double-stranded PCR products into full-length, single-stranded DNA fragments was developed.
  • one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified.
  • the double-stranded product is treated with T7 gene 6 exonuclease.
  • the phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed.
  • the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate.
  • a simple and efficient method for the immobilization of relatively short ohgonucleotides to microtiter plates with a hydrophilic surface in the presence of salt was also described.
  • DNA analysis based on template hybridization (or hybridization plus enzymic processing) to an array of surface-bound ohgonucleotides is well suited for high density, parallel, low cost and automatable processing [Fluorescence detection applied to non-electrophoretic DNA diagnostics on oligonucleotide arrays. Ives, Jeffrey T.; Rogers, Yu Hui; Bogdanov, Valery L.; Huang, Eric Z.; Boyce-Jacino, Michael; Goelet, Philip L.L.C., Proc. SPIE-Int. Soc. Opt. Eng., 2680 (Ultrasensitive Biochemical Diagnostics), 258-269 (1996)].
  • Direct fluorescence detection of labeled DNA provides the benefits of linearity, large dynamic range, multianalyte detection, processing simplicity and safe handling at reasonable cost.
  • the Molecular Tool Corporation has applied a proprietary enzymatic method of solid phase genotyping to DNA processing in 96-well plates and glass microscope slides. Detecting the fluor- labeled GBA dideoxynucleotides requires a detection limit of approx. 100 mols./ ⁇ m 2 . Commercially available plate readers detect about 1000 mols./ ⁇ m 2 , and an experimental setup with an argon laser and thermoelectrically-cooled CCD can detect approximately 1 order of magnitude less signal. The current limit is due to glass fluorescence.
  • SNP analysis by hybridization is a powerful method, it has several disadvantages. These include; i) a need to synthesize two targets, and possibly two competitor ohgonucleotides for each allelic pair, ii) the establishment of the hybridization parameters (buffer content, temperature, time) that will efficiently discriminate between alleles, and iii) an avoidance of regions containing secondary structure that may effect the hybridization parameters.
  • the present invention provides a novel system for using a GBA single base chain extension (SBCE) which takes advantage of the powerful matrixing capabilities of a mobile solid support system having multiple dye color/concentration capabilities (e.g., the FlowMetrix system) to overcome the described disadvantages.
  • the present invention further provides a method to improve the detection of reaction products from such polymorphism identification methods.
  • Various detection methods as described herein and as known in the art, can be enhanced by utilizing the present detection method.
  • Such methods can be combined with the present invention to provide a read out format that is time- and cost-efficient as it provides a means of using any given bead for use, individually, with many primers.
  • This read-out method can be utilized also with many polymorphism detection methods, such as SBCE, OLA and cleavase reaction/ signal release (Invader methods, Third Wave Technologies).
  • the present invention provides a method whereby a mobile solid support, such as a bead, which is detectably tagged, such as with a dye, a radiolabel, a magnetic tag, or a Quantum Dot® (Quantum Dot Corp.), is utilized in a nucleic acid read out procedure, either a direct readout onto a mobile solid support-linked nucleic acid such as SBCE, OLA or cleavase reaction/signal release (Invader methods, Third Wave Technologies, Madison, Wisconsin) or an indirect readout (in solution) which is then captured by an intermediate nucleic acid such as by a zipcode attached to a mobile solid support, and the readout product is then analyzed on a selected platform, such as by passing the mobile solid support over a detector (such as a laser detection device) or by passing a detector over the mobile solid support.
  • a mobile solid support such as a bead
  • a detector such as a laser detection device
  • the present invention provides a novel system for SNP readout using an encoded mobile solid support which takes advantage of the powerful matrixing capabilities of a mobile solid support system.
  • the system uses a GBA single base chain extension (SBCE).
  • SBCE GBA single base chain extension
  • the system utilizes an oligonucleotide ligation assay.
  • the system uses an enzymatic or chemical read-out method whereby an enzyme or chemical is used to modify or endonucleolytically cleave a mismatched base at the polymorphic site, resulting in the loss of an attached reporter or said modification resulting in a labeling means for the identification of the modification.
  • the system utilizes an endonuclease cleavase/signal release method (Invader methods, Third Wave Technologies) (see, e.g., Marshall et al. J. Clin. Microbiol. 35(12):3156- 3162 (1997); Brow, et al. J. Clin. Microbiol. 34(12):3129-3137 (1997)).
  • FET fluorescence energy transfer
  • target nucleic acid e.g., PCR product or genomic DNA
  • a cleavase enzyme recognizes the specific structure formed between the target nucleic acid, Invader probe, and Signal probe, and cleaves the Signal probe at the branch site and thereby releases the signal for detection.
  • Another Signal probe can then bind to the nucleic acid and the cleavase reaction begins anew. This process is repeated many times and thereby increases the signal amplification.
  • the essence for cleavase to work is the presence of an overlapping base of the Invader probe with the signal base.
  • Invader Squared two rounds of Invader are performed simultaneously.
  • the primary invader reaction involves using SNP-specific target DNA, the resulting cleavase-product becomes functional in a secondary Invader reaction with a universal signal probe and universal complementary target DNA. After the second round invader assay, a linear signal amplification of greater than 10 6 signal/target/hr is obtained.
  • the present invention further provides a novel read-out method for improving the use of mobile solid support-based read-out technologies for detection of nucleic acid polymorphisms in a target nucleic acid utilizing a target oligonucleotide having a first complementarity region complementary to the target nucleic acid and a second complementarity region, 5' to the first complementarity region, complementary to a capture oligonucleotide, which capture oligonucleotide is linked to a mobile solid support.
  • the improved method can be applied to any of several methods of identifying a nucleic acid polymorphism, such as oligonucleotide ligation assay (OLA) or single base chain extension (SBCE), as described herein.
  • the methods can be utilized to detect SNPs in genomic DNA as well as amplified DNA, RNA, etc., thus making it useful for a variety of purposes, including geno typing (such as for disease mutation detection and for parentage determinations in humans and other animals), pathogen detection and identification, and differential gene expression.
  • the present invention further provides the development of a simple method for multiplexing short sequencing reads (about 16 bases) in the same lane.
  • One application to which this method can be applied is high-throughput yeast two-hybrid analysis. In this analysis, it is desired to sequence short regions of the interacting proteins, and then use a large database to determine the hit identification. Because each bait analyzed generates approximately 100 hits, the present method to increase the efficiency of analysis was needed and therefore developed.
  • the invention can be utilized to analyze a nucleic acid sample that comprises genomic DNA, amplified DNA, such as a PCR product, cDNA, cRNA, a restriction fragment or any other desired nucleic acid sample.
  • genomic DNA typically the genomic DNA will be treated in a manner to reduce viscosity of the DNA and allow better contact of a primer or probe with the target region of the genomic DNA.
  • reduction in viscosity can be achieved by any desired method, which are known to the skilled artisan, such as DNase treatment or shearing of the genomic DNA, preferably lightly.
  • Amplified DNA can be obtained by any of several known methods.
  • Sources of genomic DNA are numerous and depend upon the purpose of performing the methods, but include any tissue, organ or cell of choice.
  • Ohgonucleotides can be generated by amplification or by de novo synthesis, for example.
  • Complementary nucleic acids i.e., cRNA (obtained from a process wherein DNA is primed with a T7- RNA polymerase/specific sequence primer fusion, then T7 RNA polymerase is added to amplify the first strand to create cRNA) and cDNA, can be obtained by standard methods known in the art.
  • nucleic acid includes any of, for example, an oligonucleotide, a 16s ribosomal RNA, a PCR product, a DNA fragment, an RNA molecule, a cDNA molecule or a cRNA molecule
  • the nucleic acid primer is an oligonucleotide, a PCR product, a DNA fragment, an RNA molecule, a cDNA molecule or a cRNA molecule.
  • a primer or a probe in an example is an oligonucleotide, but the source of the primers or probes is not so limited herein.
  • "a” and “an” can mean one or more, depending upon the context in which it is used.
  • a single oligonucleotide is attached to a detectably tagged, mobile solid support, such as a bead or a rod, preferably that can be processed for detection of the tag quickly once the desired reaction has taken place, such as by a FACS-type system.
  • a detectably tagged, mobile solid support such as a bead or a rod
  • a detectably tagged, mobile solid support such as a bead or a rod
  • Detection systems can be used, preferably, however, wherein the mobile solid support is passed over a detection device, such as a laser detection device, capable of detecting and discerning the selected tags and labels (see, e.g., PCT publication WO 9714028). Detection systems can also be utilized wherein the mobile solid support, after performing any reactions, is fixed onto a two-dimensional surface and a detection device, such as a laser detection device, is passed over the fixed mobile solid support.
  • the mobile solid support can comprise any useful material, such as polystyrene-divinylbenzene. Detection of the mobile solid support and any nucleic acid or nucleotide associated with it, can be performed by FACS-based method, such as the Luminex FlowMetrixTM system.
  • the oligonucleotide is designed such that the 5' end is coupled to the bead.
  • the 3' base ends at a nucleotide chosen relative to the polymorphic base, depending upon the assay being performed.
  • the 3' base of this primer or probe can end at the nucleotide 5' to the polymorphic base, it can end with a base corresponding to the polymorphic base.
  • the length of the oligo in the SBCE method is not critical, but it does need to be long enough to support hybridization by a nucleic acid sample, such as a PCR product generated from a region surrounding the SNP.
  • the primer or probe can be designed wherein an exact match is required or it may be designed to allow some mismatch upon initial hybridization to the sample nucleic acid.
  • nucleotide capable of chain termination is utilized.
  • chain termination is a termination event that occurs before the same labeled base occurs again in the target sequence.
  • nucleotides are known in the art and include, for example, a dideoxynucleotide (when polymerase is used in the extension reaction), a thiol derivative (when polymerase is used in the extension reaction), a 3' deoxynucleotide (using reverse transcriptase in the extension reaction), or a 3' deoxyribonucleotide (using reverse transcriptase in the extension reaction).
  • nucleotides can be, for example, a dinucleotide, a trinucleotide, or a longer nucleic acid.
  • a bank of dinucleotides or longer nucleic acids such that within the bank one has optional nucleotides at more than one location.
  • the labeling step is typically performed in solution (thus providing efficient hybridization), and the analysis step can be performed either in solution or on a solid, non-mobile support.
  • the present invention therefore provides a method of identifying a selected nucleotide in a first nucleic acid comprising
  • nucleic acid primer comprises a region complementary to a section of the first nucleic acid that is directly 3' of and adjacent to the selected nucleotide, under hybridization conditions that allow the first nucleic acid and the nucleic acid primer to form a hybridization product;
  • a primer is designed such that its 3' base ends at the nucleotide immediately 5' of the polymorphic base. A set of 4 dideoxynucleotide triphosphate mixtures are generated.
  • Each mixture contains one of four labeled dideoxynucleotide molecules that have been chemically-coupled to a flourescein molecule (i.e., ddATP-F, ddCTP-F, ddGTP-F or ddTTP-F), and three non-labeled dideoxynucleotide triphosphates.
  • a flourescein molecule i.e., ddATP-F, ddCTP-F, ddGTP-F or ddTTP-F
  • the PCR product is added to the bead and the bead aliquoted into 2 or more tubes.
  • the chain-terminating mixtures are dispensed to the tubes and a polymerase is added to generate the SBCE reaction tubes.
  • the polymerase will extend a base onto the 3' end of the bead-attached oligo, this base being the complement of the base at the polymorphic site.
  • the reaction tubes are analyzed by FlowMetrix and the appearance of a label in a particular reaction tube on a particular bead will indicate the polymorphic base at the site.
  • a comparison of the present method with a hybridization method is illustrative of the utility of the present invention.
  • SNP analysis by hybridization 2 oligos on 2 beads in the same tube are used to generate the material to be read for analysis.
  • SCBE method the same oligo on the same bead is analyzed in 2 tubes with 2 different labeled dideoxynucleo tides.
  • the present methods can be performed wherein the chain terminating nucleotide is a dideoxynucleotide and the primer extension is performed in the presence of one labeled, identified dideoxynucleotide and three different, non-labeled dideoxynucleotides.
  • the chain terminating nucleotide is a dideoxynucleotide
  • the primer extension is performed in the presence of a first identified dideoxynucleotide labeled with a first detectable label, a second identified dideoxynucleotide labeled with a second detectable label, a third identified dideoxynucleotide labeled with a third detectable label and a fourth identified dideoxynucleotide labeled with a fourth detectable label
  • detection of the presence of the first, the second, the third or the fourth detectable label in the hybridization product indicates the identity of the nucleotide complementary to the selected nucleotide as the first, the second, the third or the fourth dideoxynucleotide, respectively.
  • the present invention provides a method of determining a selected nucleotide polymorphism in genomic DNA treated to reduce viscosity comprising (a) performing an amplification of the genomic DNA using a first nucleic acid primer comprising a region complementary to a section of one strand of the nucleic acid that is 5' of the selected nucleotide, and a second nucleic acid primer complimentary to a section of the opposite strand of the nucleic acid downstream of the selected nucleotide, under conditions for specific amplification of the region of the selected nucleotide between the two primers, to form a PCR product; (b) contacting the PCR product with a first nucleic acid linked at its 5 ' end to a detectably tagged mobile solid support, wherein the first nucleic acid comprises a region complementary to a section of one strand of the PCR product that is directly 5' of and adjacent to the selected nucleotide, under hybridization conditions to form a hybridization product;
  • the PCR product can be in single-stranded form.
  • the present invention further provides a method of determining a selected nucleotide polymorphism in genomic DNA treated to reduce viscosity comprising (a) performing an amplification of the genomic DNA using as a primer an oligonucleotide comprising a first region having a T7 RNA polymerase promoter and a second region complementary to a section of one strand of the nucleic acid that is directly 5' of the selected nucleotide, and using T7 RNA polymerase to amplify one strand into cRNA and using reverse transcriptase to amplify the second strand complementary to the cRNA strand, under conditions for specific amplification of the region of the nucleotide between the two primers, to form an amplification product;
  • the labeled chain -terminating nucleotide can be, for example, a 3 'deoxynucleotide, a 3 'deoxyribonucleotide, a thiol nucleotide derivative or a dideoxynucleotide.
  • the amplification product can be in single-stranded form.
  • primers can be the primers used to i) make the first strand cDNA, and, ii) with a set that has attached to it the T7 RNA polymerase, can be used to make cRNA.
  • the primers can be made FITC-labeled for the cDNA.
  • the present method further provides a method of determining a selected nucleotide polymorphism in genomic DNA treated to reduce viscosity comprising
  • the DNA can be in single-stranded form.
  • the labeled chain -terminating nucleotide can be, for example, a 3 'deoxynucleotide, a 3 'deoxyribonucleotide, a thiol nucleotide derivative or a dideoxynucleotide.
  • the hybridization time should be of a length sufficient to allow hybridization of the first primer to the genomic DNA since the genomic DNA has not been amplified in this specific embodiment.
  • relatively long hybridization times may be utilized, such as, for example, 12 hours, 24 hours, 48 hours, as is known in the art for hybridization to genomic DNA (see, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1989).
  • alternative polymerases can be employed, such as a polymerase with a temperature condition for function, or a polymerase with a particular specificity for nucleotides, such as a polymerase that preferentially incorporates dideoxynucleotides (see, e.g., Sambrook, et al).
  • a polymerase with a temperature condition for function or a polymerase with a particular specificity for nucleotides, such as a polymerase that preferentially incorporates dideoxynucleotides (see, e.g., Sambrook, et al).
  • the skilled artisan is familiar with such polymerases, and new polymerases, as they are discovered, can be incorporated into the methods, given the teachings herein..
  • the present invention additionally provides the use of the beads in an oligonucleotide-ligation assay (OLA) format, i.e., in which one can hybridize genomic DNA, cRNA or PCR product to a first nucleic acid attached to the bead, then come in with a second nucleic acid with a fluorescent label, then add ligase, and wherein the second nucleic acid has at it's 3' end the polymorphic bases.
  • OLA oligonucleotide-ligation assay
  • the present invention provides a method of identifying a selected nucleotide in a first nucleic acid comprising (a) contacting the first nucleic acid with (i) a second nucleic acid linked at its 5' end to a detectably tagged mobile solid support wherein the second nucleic acid comprises a region complementary to a section of the first nucleic acid that is directly 3' of the selected nucleotide and wherein the second nucleic acid terminates at its 3 ' end in a test nucleotide positioned to base-pair with the selected nucleotide, and (ii) a third, fluorescently labeled nucleic acid, wherein the third nucleic acid comprises a region complementary to a section of the second nucleic acid that is directly 5' of and adjacent to the selected nucleotide, under hybridization conditions that allow the first nucleic acid and the second nucleic acid to form a hybridization product and the first nucleic acid and the third nucleic acid to form a hybridization product;
  • This oligonucleotide ligation assay can be performed both (a) wherein the polymorphic base is located at the 5' side of either the reporter or acceptor oligonucleotide, or (b) wherein the polymorphic base is located at the 3' side of either the reporter or acceptor oligonucleotide.
  • the first nucleic acid can be genomic DNA (treated to reduce viscosity, e.g., by DNase treatment or by shearing), amplified nucleic acid such as a PCR product, an oligonucleotide, a 16s ribosomal RNA, a DNA fragment, an RNA molecule, a cDNA molecule, a cRNA molecule, restriction enzyme-generated DNA fragment, size- selected DNA, Bridge-amplified DNA, 16S RNA, 16S DNA or any other desired nucleic acid.
  • Any selected ligase can be used, such as T4 DNA ligase.
  • a thermostable ligase would be particularly useful. See, generally Wu and Wallace, Genomics 4: 560-569 (1989).
  • the present invention additionally encompasses the use in the OLA readout of degenerate reporter ohgonucleotides, preferably the use of 8-mer ohgonucleotides wherein 6 of the bases are chosen to be specific to the target nucleic acid and 2 of the bases are variable, or wobble or degenerate, positions.
  • the degeneracy can be placed in any position in the reporter oligonucleotide; however, preferable positions can be positions 3, 4, 5, and 6.
  • Preferable variable position combinations in a selected oligonucleotide can be positions 3 and 6, positions 4 and 5, and positions 3 and 4.
  • the present invention includes an OLA readout wherein the reporter oligonucleotide is an 8-base complementary 8-mer conjugated to a reporter molecule or hapten to which a reporter molecule can be conjugated by means of a hapten- recognizing intermediary (e.g., antibody, avidin, strep tavidin).
  • a hapten- recognizing intermediary e.g., antibody, avidin, strep tavidin
  • the present invention further includes an OLA readout wherein the reporter oligonucleotide is an 6-base complementary 8-mer ("6+2 -mer") conjugated to a reporter molecule or hapten to which a reporter molecule can be conjugated by means of a hapten-recognizing intermediary.
  • the two non-complementary bases can be any of the four natural bases or can be a non-natural derivative capable of forming a non-helix disturbing duplex structure.
  • the non-complementary bases can preferably be located at positions 3 and 6 or positions 4 and 5.
  • Non-natural base derivatives and/or 6+2-mers can be components of a kit for use in performing the detection methods described herein. Further, one can employ a 'Taqman' approach wherein one incorporates Dye quenchers and Dye acceptors into the attached oligos and asks for the polymerase to remove the dye quencher in a repair reaction.
  • the invention further employs hybridization methods wherein two nucleic acids are hybridized to the sample nucleic acid but the step of ligation can be omitted and a match instead detected by fluorescence energy transfer between the two nucleic acids hybridized to the sample nucleic acid.
  • the two hybridizing nucleic acids are designed such that the 3' end of the nucleic acid linked to the bead is a test base, and when it is complementary to the polymorphic base, and a single wavelength of light is directed onto the sample, one can detect a transfer of energy, read as a second wavelength of light. A second reader can be employed for this detection of this second wavelength.
  • the present invention provides a method of identifying a selected nucleotide in a first nucleic acid comprising (a) contacting the first nucleic acid with (i) a second nucleic acid linked at its 5' end to a detectably tagged mobile solid support and linked at its 3 ' end to a fluorescent label, wherein the second nucleic acid comprises a region complementary to a section of the first nucleic acid that is directly 3' of the selected nucleotide and wherein the second nucleic acid terminates at its 3' end in a test nucleotide positioned to base-pair with the selected nucleotide, and
  • a third nucleic acid fluorescently labeled at its 5 ' end wherein the third nucleic acid comprises a region complementary to a section of the second nucleic acid that is directly 5' of and adjacent to the selected nucleotide, under hybridization conditions that allow the first nucleic acid and the second nucleic acid to form a hybridization product and the first nucleic acid and the third nucleic acid to form a hybridization product;
  • the present invention also provides a method for determining the sequence of a polymorphic base comprising: a first nucleic acid attached at a 5' end to a mobile solid support and having a 3' end adjacent to a polymorphic base on a second nucleic acid; a third nucleic acid with an attached reporter moiety that is complementary to a region adjacent to the polymorphic base of the second nucleic acid; the first nucleic acid and the third nucleic acid together defining a gap opposite the polymorphic base; a nucleotide that is complementary to one of a set of two possible polymorphic bases, a polymerase, and a ligase; wherein the polymerase is able to polymerize the nucleotide across the gap if the nucleotide is complementary to the polymorphic base; the ligase is able to ligate the newly polymerized nucleotide to the reporter-attached third nucleic acid; and a means for detecting the reporter covalently linked to
  • a second nucleic acid linked at its 5' end to a detectably tagged mobile solid support, wherein the second nucleic acid comprises a region complementary to a section of the first nucleic acid that is directly 3' of and immediately adjacent to the selected nucleotide, and
  • a third nucleic acid fluorescently labeled wherein the third nucleic acid comprises a region complementary to a section of the second nucleic acid that is directly 5' of and adjacent to the selected nucleotide, under hybridization conditions that allow the first nucleic acid and the second nucleic acid to form a hybridization product and the first nucleic acid and the third nucleic acid to form a hybridization product, wherein the first, second and third nucleic acids form a hybridization product that defines a gap opposite the selected nucleotide;
  • the polymerase can preferably be a non-strand displacing polymerase. Further, it can be a thermostable polymerase.
  • the ligase can be a DNA ligase. Further, it can be a thermostable ligase.
  • the present invention further provides a method of detecting a single base polymo ⁇ hism comprising using an enzyme or chemical to modify or endonucleolytically cleave a mismatched base at the polymo ⁇ hic site in a nucleic acid, resulting in the loss of an attached reporter or in a modification, and detecting a loss of the reporter or detecting the modification, thus resulting in a labeling means for the identification of the modification.
  • an end-labeled (such as with FITC) genomic fragment or a labeled (such as with FITC) PCR fragment is hybridized to an oligonucleotide and attached to a bead, then the construct is treated with an enzyme that recognizes and/or restricts mispaired DNA (such as FITC-labeled recA, mutS or T7 enzyme) and analyzed for the addition or loss of the label.
  • an enzyme that recognizes and/or restricts mispaired DNA such as FITC-labeled recA, mutS or T7 enzyme
  • a chemical recognizing single stranded regions of DNA and capable of modifying the region is utilized, and the modification is detected.
  • any of the herein described methods can be utilized in a method for quantitating expression of a selected nucleic acid in a sample.
  • it can be used, for example, for differential gene expression wherein the expression of a selected gene is quantitated and compared to a standard or some other reference.
  • a gene fragment from a region of interest or a region that distinguishes the gene (or allele or haplotype or polymo ⁇ hism) of interest is linked at its 5' end to a detectably labeled mobile solid support; message (e.g., RNA, cDNA, cRNA) is hybridized to the fragment, and fluorescence is quantitated by performing a primer extension reaction, a ligase reaction or a hybridization/fluorescence energy transfer reaction, such as that described herein.
  • the nucleic acid probe can comprise a region complementary to a section of the selected nucleic acid unique to the nucleic acid.
  • a standard such as that from a normal subject, or a diseased/ afflicted subject, or a particular tissue or organ, or a particular species, can be used as a comparison reference to draw conclusion regarding the quantity detected in the sample.
  • the present invention provides a method of detecting a result from an identification reaction to identify a selected nucleotide in a target nucleic acid comprising: a) contacting a target oligonucleotide comprising a first complementarity region and a second complementarity region, wherein the second complementarity region is 5' of the first complementarity region and wherein the first complementarity region comprises a region complementary to a section of the target nucleic acid that is directly 3' of and adjacent to the selected nucleotide, with a sample comprising the target nucleic acid, under hybridization conditions that allow the formation of a hybridization product between the first complementarity region of the target oligonucleotide and a region of the target nucleic acid complementary to the first complementarity region of the target oligonucleotide, to form a first hybridization product; b) performing a selected identification reaction with the first hybridization product to determine the identity of the selected nucleotide wherein a selectively labele
  • the basic method thus involves the use of a capture oligonucleotide, linked to a mobile solid support (such as a bead), to isolate a reaction product from a reaction.
  • a "target oligonucleotide” is designed which comprises, in addition to a first complementarity region, which is a region complementary to a region of the target nucleic acid, a second complementarity region, which is located 5' of the first complementarity region, and which is complementary to the nucleotide sequence of the capture oligonucleotide.
  • the capture oligonucleotide can be utilized in a hybridization reaction to isolate the target oligonucleotide in its reacted form (e.g., as a ligation product or as a primer extension product).
  • a reaction such as SBCE or OLA
  • the capture oligonucleotide can be utilized in a hybridization reaction to isolate the target oligonucleotide in its reacted form (e.g., as a ligation product or as a primer extension product).
  • a bead specifically for each oligonucleotide e.g., the "first complementarity region of the target oligonucleotide in the present invention
  • the present invention additionally encompasses the use in the OLA readout of degenerate reporter ohgonucleotides, preferably the use of 6+2-mers as described herein.
  • reporter ohgonucleotides can be a component of a useful kit for performing the detection methods herein.
  • the capture oligonucleotide can be designed such that it does not specifically hybridize, i.e., is not sufficiently complementary for specific hybridization to occur, to the target nucleic acid. For example, it can include nucleotide usage not typically found in the target species (such as human). If the target sequence is fully known, the capture sequence can be selected as a sequence which does not occur in the target sequence.
  • a capture oligonucleotide can be of any desired length so long as it is sufficiently long so as to selectively hybridize to a first complementarity region of a target oligonucleotide (under selective hybridization conditions, e.g., stringent hybridization conditions, as known to one skilled in the art), and not so long as to interfere with either the identification reaction being performed with the target oligonucleotide or the hybridization reaction between the capture oligonucleotide and the target oligonucleotide.
  • the capture oligonucleotide length selected can also be a function of how many different capture ohgonucleotides one desires to use in any selected use.
  • the capture oligonucleotide can be 8, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 or more nucleotides.
  • a preferred length is around 25 nucleotides, such as 23, 24, 25, 26, 27 or 28 nucleotides.
  • other oligonucleotide lengths can be utilized.
  • Optimal length for any specific use can be determined according to the specific nucleic acid composition, as will be known to those skilled in the art.
  • a bank of complementary regions can be maintained for use in generating target ohgonucleotides for any specific target nucleic acid.
  • the present invention provides a method of detecting a reaction product to identify a selected nucleotide in a target nucleic acid comprising: a) contacting a target oligonucleotide comprising a first complementarity region and a second complementarity region, wherein the first complementarity region comprises the oligonucleotide primer and the second complementarity region comprises a nucleic acid sequence complementary to a capture oligonucleotide, and wherein the oligonucleotide primer comprises a region complementary to a section of the target nucleic acid that is directly 3' of and adjacent to the selected nucleotide, with a sample comprising the target nucleic acid, under hybridization conditions that allow the formation of a hybridization product between the first complementarity region of the target oligonucleotide and a region of the target nucleic acid complementary to the first complementarity region of the target oligonucleotide, to form a first hybridization product; b) performing a
  • the target oligonucleotide is designed such that the 5' end comprises second complementarity region and later allows for hybridization to a complementary capture oligonucleotide linked to a mobile solid support, and the 3' end comprises a first complementarity region complementary a region of the target nucleic acid just 3' of the polymo ⁇ hic base.
  • the 3' base ends at a nucleotide chosen relative to the polymo ⁇ hic base, depending upon the assay being performed.
  • the 3' base of this target oligonucleotide can end at the nucleotide 5' to the polymo ⁇ hic base, or it can end with a base corresponding to the polymo ⁇ hic base.
  • the present invention additionally provides the use of the beads in an oligonucleotide- ligation assay (OLA) format, i.e., in which one can hybridize genomic DNA, cRNA or PCR product to a target oligonucleotide having a first complementarity region that is complementary to a section of the target nucleic acid that is directly 3' of the selected nucleotide, then come in with a reporter oligonucleotide having a fluorescent label, then add ligase, and wherein the target oligonucleotide has at its 3' end the polymo ⁇ hic bases.
  • OLA oligonucleotide- ligation assay
  • the reagents can comprise: a target oligonucleotide containing two regions of complementarity; a first complementarity region of the target oligo is complementary to a region immediately adjacent to a single nucleotide polymo ⁇ hism to be analyzed, a second complementarity region of the target oligonucleotide which is complementary to a capture oligonucleotide; a capture oligonucleotide that is covalently coupled to a mobile solid support; a reporter oligonucleotide complementary to the the region overlapping the SNP and containing a means for readout, and a 3' base on the strand opposite the SNP position; a ligase capable of ligating the reporter and the target if the base on the reporter that is opposite the SNP is complementary.
  • the ligation reaction is then added to the capture-oligonucleotide- coupled mobile solid support and hybridization of the second complementarity region to the bead is allowed to occur under standard hybridization conditions.
  • Readout of the reporter could be performed using a Luminex LXlOO-type machine.
  • the advantages to this system include the reduced number of bead sets needed to analyze many different SNPs, i.e., if given 100 bead colors, then one could synthesize only 100 capture ohgonucleotides and use them over and over again in the different wells.
  • the present invention provides a method of detecting a result from an identification reaction (OLA) to identify a selected nucleotide in a target nucleic acid comprising: a) hybridizing (i) a target oligonucleotide comprising a first complementarity region and a second complementarity region, wherein the first complementarity region comprises a region complementary to a section of the target nucleic acid that is directly 3' of and adjacent to the selected nucleotide and the second complementarity region comprises a nucleic acid sequence complementary to a capture oligonucleotide, and (ii) a fluorescently labeled reporter oligonucleotide comprising a region complementary to a section of the target nucleic acid that is directly 5' of and adjacent to the selected nucleotide, to a sample comprising the target nucleic acid, under hybridization conditions that allow specific hybridization between the first complementarity region of the target oligonucleotide and a region of the target nucleic acid
  • the target nucleic acid can be genomic DNA treated to reduce viscosity, an oligonucleotide, a 16s ribosomal RNA, a 16S DNA, a PCR product, a DNA fragment, a restriction enzyme-generated DNA fragment, size-selected DNA, Bridge-amplified DNA, an RNA molecule, a cDNA molecule or a cRNA molecule.
  • the present invention further provides a method of determining a selected nucleotide polymo ⁇ hism in genomic DNA treated to reduce viscosity comprising a) performing an amplification of the genomic DNA using a first nucleic acid primer comprising a region complementary to a section of one strand of the nucleic acid that is 5' of the selected nucleotide, and a second nucleic acid primer complimentary to a section of the opposite strand of the nucleic acid downstream of the selected nucleotide, under conditions for specific amplification of the region of the selected nucleotide between the two primers, to form a PCR product; b) contacting the PCR product with a target oligonucleotide comprising a first complementarity region and a second complementarity region, wherein the first complementarity region is complementary to a section of one strand of the PCR product that is directly 5' of and adjacent to the selected nucleotide, under hybridization conditions to form a first hybridization product; c) performing
  • the present invention further provides a method of determining a selected nucleotide polymo ⁇ hism in genomic DNA treated to reduce viscosity comprising a) performing an amplification of the genomic DNA using as a primer an oligonucleotide comprising a first region having a T7 RNA polymerase promoter and a second region complementary to a section of one strand of the nucleic acid that is directly 5' of the selected nucleotide, and using T7 RNA polymerase to amplify one strand into cRNA and using reverse transcriptase to amplify the second strand complementary to the cRNA strand, under conditions for specific amplification of the region of the nucleotide between the two primers, to form an amplification product; b) contacting the amplification product with a target oligonucleotide comprising a first complementarity region and a second complementarity region, wherein the first complementarity region is complementary to a section of one strand of the PCR
  • the labeled chain -terminating nucleotide can be, for example, a 3 'deoxynucleotide, a 3 'deoxyribonucleotide, a thiol nucleotide derivative or a dideoxynucleotide.
  • the amplification product can be in single-stranded form. Furthermore, one can design and synthesize some primers to sit just downstream of the target ohgonucleotides.
  • the present method further provides a method of determining a selected nucleotide polymo ⁇ hism in genomic DNA treated to reduce viscosity comprising a) contacting the genomic DNA with a target oligonucleotide comprising a first complementarity region and a second complementarity region, wherein the first complementarity region is complementary to a section of one strand of the PCR product that is directly 5' of and adjacent to the selected nucleotide and wherein the second complementarity region is 5' to the first complementarity region, under hybridization conditions for forming a specific first hybridization product; b) performing a primer extension reaction with the specific first hybridization product and a detectably labeled, identified chain-terminating nucleotide under conditions for primer extension; c) isolating the primer extension product by contacting the primer extension product with a capture oligonucleotide that is covalently coupled to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary
  • the DNA can be in single-stranded form.
  • the labeled chain -terminating nucleotide can be, for example, a 3 'deoxynucleotide, a 3 'deoxyribonucleotide, a thiol nucleotide derivative or a dideoxynucleotide.
  • the hybridization time should be of a length sufficient to allow hybridization of the first primer to the genomic DNA since the genomic DNA has not been amplified in this specific embodiment.
  • relatively long hybridization times may be utilized, such as, for example, 12 hours, 24 hours, 48 hours, as is known in the art for hybridization to genomic DNA (see, e.g., Sambrook, et al).
  • any selected ligase can be used, such as T4 DNA ligase.
  • a thermostable ligase would be particularly useful. See, generally Wu and Wallace, Genomics 4: 560-569 (1989).
  • the invention further employs hybridization methods wherein two nucleic acids are hybridized to the sample nucleic acid but the step of ligation can be omitted and a match instead detected by fluorescence energy transfer between the two nucleic acids hybridized to the sample nucleic acid.
  • the two hybridizing nucleic acids are designed such that the 3' end of the target oligonucleotide is a test base, and when it is complementary to the polymo ⁇ hic base, and a single wavelength of light is directed onto the sample, one can detect a transfer of energy, read as a second wavelength of light. A second reader can be employed for this detection of this second wavelength.
  • the present invention provides a method of identifying a selected nucleotide in a target nucleic acid comprising a) contacting the target nucleic acid with i. a target oligonucleotide comprising a first complementarity region and a second complementarity region, wherein the first complementarity region is complementary to a section of the first nucleic acid that is directly 5' of the selected nucleotide, wherein the target oligonucleotide terminates at its 3' end in an identified test nucleotide positioned to base-pair with the selected nucleotide, and wherein the second complementarity region is 5' to the first complementarity region, and ii.
  • a fluorescently labeled reporter oligonucleotide wherein the reporter oligonucleotide comprises a region complementary to a section of the target nucleic acid that is directly 3' of and adjacent to the selected nucleotide, under hybridization conditions that allow the target nucleic acid and the target oligonucleotide to hybridize and the target nucleic acid and the reporter oligonucleotide to hybridize, thus forming a first hybridization product; b) adding to the first hybridization product a ligase under ligation conditions; c) isolating the first hybridization product by contacting the first hybridization product with a capture oligonucleotide that is covalently coupled to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementarity region of the target oligonucleotide, under hybridization conditions to form an isolated second hybridization product; and detecting the presence or absence of the fluorescent label, after dissoci
  • the present invention additionally provides a method of identifying a selected nucleotide in a target nucleic acid comprising a) contacting the target nucleic acid with i. a target oligonucleotide linked at its 3' end to a fluorescent label, wherein the target oligonucleotide comprises a first complementarity region that is complementary to a section of the target nucleic acid that is directly 3' of the selected nucleotide, wherein the target oligonucleotide terminates at its
  • a reporter oligonucleotide fluorescently labeled at its 5' end
  • the reporter oligonucleotide comprises a region complementary to a section of the target nucleic acid that is directly 5' of and adjacent to the selected nucleotide, under hybridization conditions that allow the target nucleic acid and the target oligonucleotide to hybridize and the target nucleic acid and the reporter oligonucleotide to hybridize, to form a first hybridization product
  • the present invention also provides a method for determining the sequence of a polymo ⁇ hic base in a target nucleic acid which can utilize a kit comprising one or more of the following: a target oligonucleotide, wherein the target oligonucleotide comprises a first complementarity region and a second complementarity region 5' of the first complementarity region, wherein the first complementarity region is complementary to a section of the target nucleic acid having a 3' end adjacent to and directly 5' of the polymo ⁇ hic base on the target nucleic acid; a reporter oligonucleotide with an attached reporter moiety that is complementary to a region immediately adjacent to and 3' of the polymo ⁇ hic base of the target nucleic acid; the target oligonucleotide and the reporter oligonucleotide together defining a gap opposite the polymo ⁇ hic base; a capture oligonucleotide that is covalently linked to a mobile solid support (
  • the present invention provides a method of identifying a selected nucleotide in a target nucleic acid comprising a) contacting the target nucleic acid with i. a target oligonucleotide, wherein the target oligonucleotide comprises a first complementarity region and a second complementarity region 5' of the first complementarity region, wherein the first complementarity region is complementary to a section of the target nucleic acid that is directly 3' of and immediately adjacent to the selected nucleotide, and ii.
  • a reporter oligonucleotide fluorescently labeled wherein the reporter oligonucleotide comprises a region complementary to a section of the second nucleic acid that is directly 5' of and adjacent to the selected nucleotide, under hybridization conditions that allow the target nucleic acid and the target oligonucleotide to form a hybridization product and the target nucleic acid and the reporter oligonucleotide to form a hybridization product, wherein the target nucleic acid, target oligonucleotide and reporter oligonucleotide form a hybridization product that defines a gap opposite the selected nucleotide; b) adding an identified test nucleotide, a polymerase and a ligase, under conditions for polymerization and ligation; c) isolating the first hybridization product by contacting the first hybridization product with a capture oligonucleotide that is covalently coupled to a mobile solid support, wherein the capture oligon
  • the polymerase can preferably be a non-strand displacing polymerase. Further, it can be a thermostable polymerase.
  • the ligase can be a DNA ligase. Further, it can be a thermostable ligase.
  • any of the herein described methods can be utilized in a method for quantitating expression of a selected nucleic acid in a sample.
  • it can be used, for example, for differential gene expression wherein the expression of a selected gene is quantitated and compared to a standard or some other reference.
  • a gene fragment from a region of interest or a region that distinguishes the gene (or allele or haplotype or polymo ⁇ hism) of interest is selected for use as the first complementarity region of a target oligonucleotide; message (e.g., RNA, cDNA, cRNA) is hybridized to the target oligonucleotide, and fluorescence is quantitated by performing a primer extension reaction, a ligase reaction or a hybridization/fluorescence energy transfer reaction, such as that described herein.
  • message e.g., RNA, cDNA, cRNA
  • a corresponding capture oligonucleotide (complementary to a second complementarity region utilized in the target oligonucleotide) linked to a mobile solid support is utilized to capture the reaction product.
  • the first complementarity region of a target oligonucleotide can comprise a region complementary to a section of the selected nucleic acid unique to the nucleic acid.
  • a standard such as that from a normal subject, or a diseased/ afflicted subject, or a particular tissue or organ, or a particular species, can be used as a comparison reference to draw conclusions regarding the quantity detected in the sample.
  • the present invention provides a method of quantitating expression of a selected nucleic acid in a sample comprising a) contacting (i) message nucleic acid isolated from a selected source with (ii) a target oligonucleotide, wherein the target oligonucleotide comprises a first complementarity region and a second complementarity region 5' of the first complementarity region, wherein the first complementarity region comprises a region complementary to a section of the selected nucleic acid; b) performing a selected identification reaction with the first hybridization product to determine the identity of the selected nucleotide wherein a selectively labeled detection product comprising the second complementarity region of the target oligonucleotide can be formed; c) isolating the detection product by contacting the detection product with a capture oligonucleotide that is covalently coupled to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementarity
  • a sample can be, for example, any body sample that contains message, such as organ tissue and/or cells, such as blood, red or white blood cells, bone marrow, liver, kidney, brain, skin, heart, lung, spleen, pancreas, gall bladder, muscle, neuronal cells, neurons, precursor cells, ovaries, testicles, uterus, glands.
  • organ tissue and/or cells such as blood, red or white blood cells, bone marrow, liver, kidney, brain, skin, heart, lung, spleen, pancreas, gall bladder, muscle, neuronal cells, neurons, precursor cells, ovaries, testicles, uterus, glands.
  • kits for detecting a single base polymo ⁇ hism comprising a detectably tagged mobile solid support, such as a polystyrene-divinylbenzene bead, and one to four modified (chain-terminating) nucleotide(s), such as a 3' deoxynucleotide,m, a 3' deoxyribonucleotide, a thiol derivative, or a dideoxynucleotide.
  • the kit can additionally comprise a polymerase, and in particular, a polymerase that preferentially inco ⁇ orates the modified nucleotide.
  • the kit can additionally comprise a ligase.
  • the kit can also comprise one or more fluorescent label for labeling the nucleic acid(s).
  • the kit can further comprises a DNase for reducing the viscosity of the DNA.
  • the kit can further contain an array of combinations of dinucleotides and/or a collection of combinations of trinucleotides.
  • EP 754240 (based on WO 9521271) (Molecular Tool Inc.)
  • EP 736107 (based on WO 9517524) (Molecular Tool
  • EP 576558 (based on WO 9215712) (Molecular Tool

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Abstract

L'invention concerne un procédé permettant d'identifier un nucléotide sélectionné dans un premier acide nucléique à l'aide d'un support solide mobile, ainsi qu'un nouveau procédé de lecture permettant d'améliorer l'utilisation de technologies de lecture à support solide mobile permettant de détecter des polymorphismes d'acide nucléique dans un acide nucléique cible.
EP99930469A 1998-06-24 1999-06-22 Procede de detection de nucleotide Withdrawn EP1090138A4 (fr)

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US9050398P 1998-06-24 1998-06-24
US90503P 1998-06-24
US9072098P 1998-06-26 1998-06-26
US90720P 1998-06-26
US10070398P 1998-09-17 1998-09-17
US100703P 1998-09-17
US10801898P 1998-11-12 1998-11-12
US108018P 1998-11-12
PCT/US1999/013928 WO1999067414A1 (fr) 1998-06-24 1999-06-22 Procede de detection de nucleotide

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