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  • B01J2219/00614—Delimitation of the attachment areas
  • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
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  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
  • B01J2219/00644—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being present in discrete locations, e.g. gel pads
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00718—Type of compounds synthesised
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  • B01J2219/00722—Nucleotides
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  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
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  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

• This invention relates to the detection of nucleic acid mutations and genetic polymorphisms by single base extension analysis.
• the invention relates to an apparatus and methods for the detection of single base extension of a particular oligonucleotide in an oligonucleotide microarray following hybridization between oUgonucleotides bound to defined regions of a polymeric hydrogel-based microarray and nucleic acids in a biological sample.
• nucleic acid-based assays also play an important role in identifying infectious microorganisms such as bacteria and viruses, in assessing levels of both normal and defective gene expression, and in detecting and identifying mutant genes associated with disease such as oncogenes. Improvements in the speed, efficiency, economy, and specificity of such assays are thus significant needs in the medical arts.
• nucleic acid assays should be sensitive, specific, and easily amenable to automation.
• Efforts to improve sensitivity in nucleic acid assays are known in the prior art.
• the polymerase chain reaction (Mullis, U.S. Patent No. 4,683,195, issued July 28, 1987) provides the capacity to produce useful amounts (about 1 ⁇ g) of a specific nucleic acid in a sample in which the original amount of the specific nucleic acid is substantially smaller (about 1 pg).
• the prior art has been much less successful in improving specificity of nucleic acid hybridization assays.
• nucleic acid assays The specificity of nucleic acid assays is determined by the extent of molecular complementarity of hybridization between probe and target sequences. Although it is theoretically possible to distinguish complementary targets from one or two mismatched targets under rigorously-defined conditions, the dependence of hybridization on target/probe concentration and hybridization conditions limits the extent to which hybridization mismatch can be used to reliably detect, ter alia, mutations and genetic polymorphisms.
• U.S. Patent No. 5,925,520 disclosed a method for detecting genetic polymorphisms using single base extension and capture groups on oligonucleotide probes using at least two types of dideoxy, chain-terminating nucleotide triphosphates, each labeled with a detectable and distinguishable fluorescent labeling group.
• U.S. Patent No. 5,710,028 disclosed a method of determining the identity of nucleotide bases at specific positions in nucleic acids of interest, using detectably- labeled chain-terminating nucleotides, each detectably and distinguishably labeled with a fluorescent labeling group.
• U.S. Patent No. 5,547,839 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using chain- terminating nucleotides comprising a photoremovable protecting group.
• U.S. Patent No. 5,534,424 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using each of four aliquots of a target nucleic acid annealed to an extension primer and extended with one of four chain-terminating species, and then further extended with all four chain- extending nucleotides, whereby the identity of the nucleotide at the position of interest is identified by failure of the primer to be extended more that a single base.
• U.S. Patent No. 4,988,617 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, by annealing two adjacent nucleotide primers to a target nucleic acid and providing a linking agent such as a ligase that covalently links the two oUgonucleotides to produce a third, combined oligonucleotide only under circumstances wherein the two oUgonucleotides are perfectly matched to the target nucleic acid at the 3' extent of the first oligonucleotide and at the 5' extent of the second oligonucleotide.
• a linking agent such as a ligase that covalently links the two oUgonucleotides to produce a third, combined oligonucleotide only under circumstances wherein the two oUgonucleotides are perfectly matched to the target nucleic acid at the 3' extent of the first oligonucle
• U.S. Patent No. 4,656,127 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using primer extension with a chain-terminating or other nucleotide comprising an exonuclease-resistant linkage, followed by exonuclease treatment of the plurality of extension products to detect the resistant species therein.
• Biochip microarrays are generally comprised of a solid supporting substrate, an attachment matrix, and a plurality of biomolecular probes immobilized to the attachment matrix.
• Polymeric hydrogel pads especially polyacrylamide gel pads, have been used as an attachment matrix in a variety of biochip microarrays (see U.S. Patent Nos. 5,552,270; 5,616,478; 5,736,257; and 5,741,700).
• Microarrays fabricated from polymeric hydrogel pads offer distinct advantages over other types of microarrays in that microarrays fabricated from polymeric hydrogel pads are more economical and permit a wider diversity of functional groups to be used for biomolecular immobilization.
• the polymeric hydrogel provides a three-dimensional structure thereby permitting for the attachment of a larger number of biomolecular probes, which in turn increases the likelihood of interactions between the immobilized probes and target molecules in a biological sample.
• the present invention provides for the detection of nucleic acid mutations and genetic polymorphisms by single base extension analysis. Specifically, the invention provides an apparatus and methods for the detection of single base extension of a particular oligonucleotide in an oligonucleotide microarray following hybridization between oUgonucleotides bound to defined regions of a polymeric hydrogel-based microarray and nucleic acids in a biological sample.
• This invention provides methods and apparatus for detecting mutations and genetic polymorphisms in a biological sample containing a nucleic acid of interest. Detection of single base extension using the methods and apparatus of the invention is achieved by sequence-specific incorporation of labeled chain-terminating nucleotide species. In preferred embodiments, single base extension is performed using hybridization to an oligonucleotide array, most preferably an addressable array wherein the sequence of each oligonucleotide in the array is known and associated with a particular address in the array.
• the invention provides an array of oligonucleotide probes immobilized via linker moieties to porous, polymeric pads.
• the sequence of each oligonucleotide at each particular identified test site (or "address") in the array is known and at least one of said oUgonucleotides is complementary to a sequence in a nucleic acid contained in the biological sample to be assayed (termed the "target” or “target nucleic acid”).
• the sequence of at least one oligonucleotide is selected to hybridize to a position immediately adjacent to the nucleotide position in the sample nucleic acid that is to be interrogated, i.e., for the purpose of determining whether there is a mutation or genetic polymorphism at that nucleotide position.
• adjacent in this context is intended to encompass positions that are one nucleotide base upstream of base to be interrogated, i.e. in the 3' direction with respect to the template strand of the target DNA.
• Hybridization of oligonucleotide probes in the array to nucleic acid in the sample is performed at test sites comprising said oligonucleotide probes, and in a hybridization buffer, for a time and at a temperature that permits hybridization to occur between nucleic acid in the sample and oligonucleotide probes in tUe array that are complementary to said nucleic acid.
• Single base extension is performed using a polymerase, most preferably a thermally stable polymerase, in the presence of labeled chain-terminating primer extension units.
• each chain-terminating nucleotide species (for example, dideoxy(dd)ATP, ddGTP, ddCTP and ddTTP) is conjugated to a different label, most preferably a radioactive label, an electrochemical label or a fluorescent dye.
• a different label most preferably a radioactive label, an electrochemical label or a fluorescent dye.
• Single base extension is detected by determining the identity of the label incorporated into the oligonucleotide using, e.g. conventional optical detection methods.
• the sequence of at least one oligonucleotide probe is selected to hybridize to a target nucleic acid so that the 3 ' residue of the oligonucleotide corresponds to the nucleotide position in the target nucleic acid that is to be interrogated for mutation or genetic polymorphism.
• oligonucleotide probes having sequence identify to target nucleic acids hybridize at the 3' residue and are capable of being extended in the polymerase-mediated single base extension step as described below.
• Hybridization of the oUgonucleotides to target nucleic acids polymo ⁇ hic at the position corresponding to the 3' residue of the oligonucleotide also hybridize (because hybridization conditions are chosen to permit such hybridization), but there will be a "mismatch" at the 3' residue of the oligonucleotide that does not hybridize at the mismatch.
• only oligonucleotide hybridized to target nucleic acid species that are properly-hybridized at the oUgonucleotides' 3' residue will be extended, using a polymerase that will not recognize the mismatch.
• single base extension is directed at a site adjacent to the polymo ⁇ hic site, and most preferably comprises a non- polymo ⁇ hic site that can be addressed using only a single, labeled chain-terminating species.
• the present invention provides methods for the detection of nucleic acid mutations and genetic polymo ⁇ hisms, most preferably single nucleotide polymo ⁇ hism (SNP), by single base extension analysis.
• the invention provides an apparatus and methods for the detection of single base extension of a particular oligonucleotide in an oligonucleotide microarray following hybridization between oUgonucleotides bound to defined regions of a polymeric hydrogel-based microarray and nucleic acids in a biological sample.
• array refers to an ordered spatial arrangement, particularly an arrangement of immobilized biomolecules at a plurality of test sites.
• the term "addressable array” refers to an array wherein the individual test sites have precisely defined x- and y-coordinates, so that a given test site at a particular position in the array can be identified.
• probe and “biomolecular probe” refer to a biomolecule, specifically an oligonucleotide, used to detect a complementary biomolecule (referred to herein as a target molecule), preferably a nucleic acid.
• microarray refers to an ordered spatial arrangement of immobilized biomolecular probes arrayed at test sites on a solid supporting substrate.
• Biochips encompass substrates containing arrays or microarrays, preferably ordered arrays and most preferably ordered, addressable arrays, of biological molecules that comprise one member of a biological binding pair.
• the microarrays of the present invention are preferably oligonucleotide arrays comprising a nucleotide sequence that is complementary to at least one sequence that may be or is expected to be present in a biological sample.
• test site refers to a predefined region on a substrate to which a plurality probe molecules are immobilized.
• the test site may have any convenient shape, e.g. , circular, rectangular, elliptical, or wedge-shaped.
• the test sites have an area of about 1 cm .
• the test sites have an area of less than 1 mm 2 , less than 0.5 mm 2 , less than about 10,000 ⁇ m 2 , or less than 100 ⁇ m 2 .
• the devices of the invention are particularly useful for analyzing target nucleic acids for the diagnosis of infectious and genetic disease.
• the target nucleic acid is generally a portion of a gene having a known nucleotide sequence that is associated with an infectious agent or genetic disease; more specifically, the disease is caused by a single nucleotide (or point) mutation.
• the device inco ⁇ orates a nucleic acid oligonucleotide array specific for the target gene, and means for detecting and determining the identity of a specific single base in the target sequence adjacent to the hybridization site of at least one probe in the oligonucleotide array (termed the "3 ' offset method") or encompassing the 3' residue of at least one oligonucleotide probe in the array (termed the "3' inclusive method”).
• the present invention provides an array of oligonucleotide probes immobilized to a polymeric hydrogel-based attachment matrix.
• the sequence of each oligonucleotide at each address in the array is known and at least one oligonucleotide in said oligonucleotide array is complementary to part of a sequence in a nucleic acid in the sample to be assayed.
• the sequence of at least one oligonucleotide is most preferably selected to extend to a position immediately adjacent to the nucleotide position in the sample nucleic acid that is to be interrogated, i.e., for mutation or genetic polymo ⁇ hism.
• the oligonucleotide is selected to encompass the site of mutation or genetic polymo ⁇ hism; in these latter embodiments, it is generally preferred to provide a single oligonucleotide, most preferably having the sequence of the wildtype allele or species, or alternatively a multiplicity of oUgonucleotides having one of each possible nucleotide at the polymo ⁇ hic position to ensure hybridization of at least one of the oUgonucleotides in the array to nucleic acid in the sample.
• Hybridization and extension reactions are performed in a reaction chamber and in a hybridization buffer for a time and at temperature that permits hybridization and single base extension to occur between nucleic acid in the sample and the oUgonucleotides in the array complementary thereto.
• the apparatus comprises a supporting substrate comprising an array of test sites; a plurality of porous, polymeric pads in contact with the supporting substrate at the test sites; a plurality of linker moieties in contact with the porous, polymeric pads at the test sites; a plurality of oligonucleotide probes immobilized to the linker moieties, wherein said oligonucleotide probes specifically bind to or interact with nucleic acids in a biological sample; a reaction solution in contact with the porous, polymeric pads; linker moieties; and oligonucleotide probes, wherein the reaction solution comprises a hybridization buffer, a polymerase, a plurality of primer extension units further comprising chain-terminating nucleotide species, wherein each different chain-terminating nucleotide species is conjugated to a distinguishable label; and a means for detecting the labeled chain-terminating nucleotide species.
• the supporting substrate of the apparatus of the invention is advantageously made from any solid material, including but not limited to glass, silicon, silicon nitride, plastic, rubber, fabric, ceramics, printed circuit board, compound semiconductors (e.g., GaAs), or combinations thereof.
• the supporting substrate of the apparatus of the present invention is composed of silicon or glass.
• the supporting substrate has a surface area between about 0.01 ⁇ m and about 5 cm containing from 1 to about 10 test sites.
• the supporting substrate has a surface area of about 10,000 ⁇ m 2 and contains about 10 4 test sites.
• the test sites are arranged on the supporting substrate so that they are separated by a distance of from about 0.05 ⁇ m to 0.5 mm.
• test sites are regularly spaced on the solid supporting substrate with a uniform spacing there between.
• the oligonucleotide probes at any particular test site are identical to each other, while each test site comprises oligonucleotide probes that are unique to that test site.
• the porous, polymeric pads of the apparatus of the invention are composed of materials including, but not limited to, polyacrylamide gel, agarose gel, polyethylene glycol, cellulose gel, sol gel, polypyrrole, carbon, carbides, oxides, nitrides, or other porous, polymeric materials known to those with skill in the art.
• the porous, polymeric pads comprise polyacrylamide gel.
• the porous, polymeric hydrogel matrix of the apparatus can be fabricated, and biomolecular probes immobilized thereto, using methods as described in co-owned and co-pending U.S. Patent Application Serial Nos. 09/344,217; 09/344,620; and 09/438,209, each of which is inco ⁇ orated herein by reference.
• the probe used in the present invention is preferably an oligonucleotide, having upper and lower limits of length that are empirically determined.
• the lower limit of oligonucleotide length is the length required for stable hybridization, since it is known in the art that probes that are too short do not form thermodynamically-stable duplexes sufficient for single base extension under the hybridization conditions of the assay.
• the upper limit of oligonucleotide length is the length required to produce a duplex in a region other than that of the predetermined interrogation target, leading to artifactual inco ⁇ oration of labeled primer extension unit(s).
• Preferred oligonucleotide probes used in the present invention have a length of from about 8 to about 50, more preferably from about 10 to about 40, even more preferably from about 12 to about 30, and most preferably from about 15-25 nucleotides. However, longer probes, i.e. longer than 40 nucleotides, may also be used.
• the oUgonucleotides are immobilized to the porous, polymeric pads at their 5' ends, leaving the 3' end of the oligonucleotide available for single base extension using a polymerase as described herein.
• the linker moieties comprise a conjugated polymer or copolymer film.
• Such conjugated polymer or copolymer film is composed of materials including, but not limited to, polypyrrole, polythiophene, polyaniline, polyfuran, polypyridine, polycarbazole, polyphenylene, poly(phenylenvinylene), polyfluorene, or polyindole, or their derivatives, copolymers, or combinations thereof.
• the linker moieties comprise a neutral pyrrole matrix.
• the linker moieties comprise thiol linkers.
• the linker moieties further comprise streptavidin (and the probe molecules are biotinylated).
• Preferred polymerases for performing single base extensions using the methods and apparatus of the invention are polymerases having little or no exonuclease activity. More preferred are polymerases that tolerate and are biosynthetically-active at temperatures greater than physiological temperatures, for example, at 50°C or 60°C or 70°C or are tolerant of temperatures of at least 90°C to about 95°C.
• Preferred polymerases include Taq polymerase from T. aquaticus (commercially available from Perkin-Elmer Cetus, Foster City, CA), Sequenase ® and ThermoSequenase ® (commercially available from U.S. Biochemical, Cleveland, OH), and Exo(-)Pfu polymerase (commercially available from New England Biolabs, Beverley, MA).
• the apparatus further comprises a plurality of wells wherein each well encompasses a porous, polymeric pad, wherein a plurality of oligonucleotide probes is immobilized to linker moieties that are in contact with the porous, polymeric pad.
• wells is used herein in its conventional sense, to describe a portion of the supporting substrate in which the porous, polymeric pad is contained in a defined volume; said wells can protrude from the surface of the supporting substrate, or be embedded therein.
• the oligonucleotide probes in any particular well are identical to each other, while each well comprises oligonucleotide probes unique to that well.
• the inventive methods for SNP detection generally comprise: (1) preparing a sample containing the target nucleic acid(s) of interest to obtain single-stranded nucleic acid that spans the specific position (typically by denaturing the sample); (2) contacting the single- stranded target nucleic acid with an oligonucleotide probe of known sequence that hybridizes with a portion of the nucleotide sequence in the target nucleic acid immediately adjacent to the nucleotide base to be interrogated (thereby forming a duplex between the primer and the target such that the nucleotide base to be interrogated is the first unpaired base in the target immediately 5 ' of the nucleotide base annealed with the 3 '-end of the primer in the duplex; this oligonucleotide is preferably a specific oligonucleotide occupying a particular address in an addressable array); (3) contacting the duplex with a reagent which includes a buffer solution, a polymerase
• the primer extension reaction catalyzed by the polymerase results in the inco ⁇ oration of the extension moiety of the primer extension unit at the 3 '-end of the primer, and the extension of the primer by a single base; (4) removing the uninco ⁇ orated primer extension unit(s); and (5) determining the identity of the inco ⁇ orated primer extension unit in the extended duplex by its unique label or tag.
• Preferred labels or tags include, for example, radioactive labels, electrochemical labels and fluorescent dyes.
• the oligonucleotide is constructed so that the 3' residue is complementary only to one of a plurality of polymo ⁇ hic species of target nucleic acids.
• target nucleic acids that are complementary to the full extent of the oligonucleotide, including the 3 ' residue support single base extension of the oligonucleotide and inco ⁇ oration of a labeled primer extension unit therein, whereas target nucleic acids that are not complementary to the 3 ' residue of the oligonucleotide do not support single base extension of the oligonucleotide and labeled primer extension units are not inco ⁇ orated.
• the inco ⁇ orated primer extension units are preferably complementary to a site immediately adjacent to the polymo ⁇ hic site, which site is preferably non-polymo ⁇ hic.
• the extension moiety in the primer extension unit is preferably a chain- terminating moiety, most preferably dideoxynucleoside triphosphates (ddNTPs), such as ddATP, ddCTP, ddGTP, and ddTTP; however other terminators known to those skilled in the art, such as acyclonucleotide analogs or arabinoside triphosphates, are also within the scope of the present invention.
• ddNTPs differ from conventional deoxynucleoside triphosphates (dNTPs) in that they lack a hydroxyl group at the 3' position of the sugar component. This prevents chain extension of inco ⁇ orated ddNTPs, and thus terminates the chain.
• the present invention provides primer extension units labeled with, for example, electrochemical labels, fluorescent dyes or radioactive labels, which can be detected using electrochemical, optical or radioactivity detectors.
• electrochemical labels for example, electrochemical labels, fluorescent dyes or radioactive labels
• any label that does not interfere with the inco ⁇ oration of the ddNTP into the nucleotide chain may be used.
• the target DNA in the sample to be investigated can be amplified by means of in vitro amplification reactions, such as the polymerase chain reaction (PCR) technique well known to those skilled in the art.
• PCR polymerase chain reaction
• Enriching the target DNA in a biological sample to be used in the methods of the invention provides more rapid and more accurate template-directed synthesis by the polymerase.
• a general formula of a preferred embodiment of the primer extension unit is: ddNTP-L-* where ddNTP represents a dideoxyribonucleotide triphosphate including ddATP, ddGTP, ddCTP, ddTTP, L represents an optional linker moiety, and * represents an appropriate label or tag.
• each chain-terminating nucleotide species (for example, dideoxy(dd)ATP, ddGTP, ddCTP and ddTTP) is labeled with a different label.
• the label comprising the chain-terminating nucleotides of the invention is optionally linked to the extension nucleotide through a linker (L), preferably having a length of from about 10 to about 20 Angstroms.
• the linker can be an organic moiety such as a hydrocarbon chain (CH 2 ) n , or can comprise an ether, ester, carboxyamide, or thioether moiety, or a combination thereof.
• the linker can also be an inorganic moiety such as siloxane (O-Si-O).
• the length of the linker is selected so that the label does not interfere with either nucleic acid hybridization between the bound oligonucleotide probe and target nucleic acid, or with polymerase-mediated chain extension.
• a reaction mixture is prepared containing at least one labeled chain-terminating nucleotide, a hybridization buffer compatible with the polymerase and having a salt concentration sufficient to permit hybridization between the target nucleic acid and oligonucleotide probes under the conditions of the assay, and a DNA polymerase such as Taq DNA polymerase or Thermo Sequenase.
• a DNA polymerase such as Taq DNA polymerase or Thermo Sequenase.
• Single stranded target nucleic acid for example, having been denatured by incubation at a temperature >90°C, is diluted to a concentration appropriate for hybridization in deionized water and added to the reaction mixture.
• the resulting hybridization mixture is exposed to the polymeric hydrogel pads of the apparatus of the invention, the pads having a plurality of probes possessing known sequences attached thereto. At least one of the probes has a nucleotide sequence capable of hybridizing with a portion of the nucleotide sequence of the target comprising the nucleotide base to be interrogated under the hybridization conditions employed in the assay.
• a duplex between the probe and the target is formed wherein the nucleotide base to be interrogated is the first unpaired base in the target nucleic acid immediately 5' of the nucleotide base that is annealed with the 3 '-end of the primer in the duplex.
• Single base extension of the 3' end of the annealed probe is achieved by inco ⁇ oration of a labeled chain-terminating nucleotide into the probe.
• the probe sequence and labeled chain-terminating nucleotide are chosen so that inco ⁇ oration of the nucleotide is informative of the identity (/. e., mutant, wildtype or polymo ⁇ hism) of the interrogated nucleotide in the target.
• the probe comprises a 3' terminal residue that corresponds to and hybridizes with the interrogated base.
• oUgonucleotides having a "mismatch" at the 3' terminal residue will hybridize but will not be extended by the polymerase. Detection of inco ⁇ oration of the primer extension unit by interrogating the label is then informative of the identity of the interrogated nucleotide base, provided that the sequence of the oligonucleotide probe is known at each position in the array.
• test site is washed at high stringency (i.e., in a low-salt solution, such as 0.1 X SSC, 0.015 M NaCl, 15 mM sodium citrate, pH 7.0), optionally including a detergent such as sodium dodecyl sulfate at temperature of between aboutlO-65°C for a time and at a temperature wherein the target nucleic acid is removed. Wash conditions vary depending on factors such as probe length and probe complexity. Detection of single base extension is then carried out using conventional detection means for detecting electrochemical, fluorescent or radioactive labels. It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

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