EP2411530A2 - Solid phase based nucleic acid assays combining high affinity capturing and detection by specific hybridization - Google Patents

Solid phase based nucleic acid assays combining high affinity capturing and detection by specific hybridization

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Publication number
EP2411530A2
EP2411530A2 EP07759429A EP07759429A EP2411530A2 EP 2411530 A2 EP2411530 A2 EP 2411530A2 EP 07759429 A EP07759429 A EP 07759429A EP 07759429 A EP07759429 A EP 07759429A EP 2411530 A2 EP2411530 A2 EP 2411530A2
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EP
European Patent Office
Prior art keywords
hybridization
target
probes
specific
assay
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EP07759429A
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German (de)
French (fr)
Inventor
Jens Burmeister
Ingmar Dorn
Brian Warner
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Bayer Intellectual Property GmbH
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Bayer Technology Services GmbH
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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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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

  • This invention relates to methods for detection of nucleic acids on a solid phase with high affinity and high specificity. More particularly, the invention relates to methods combining high-affinity hybridization-based capturing with highly specific hybridization-based discrimination in solid phase based nucleic acid assays. This invention further relates to kits containing the reagents necessary for carrying out the disclosed assays.
  • the detection of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is of importance in human or veterinary diagnostics, food control, environmental analysis, crop protection, biochemical/pharmacological research, or forensic medicine.
  • capture oligonucleotides are immobilized on a solid support.
  • the labeled or unlabeled nucleic acid target is specifically hybridized to the capture probes.
  • the hybridization event can be detected using e.g. optical, electrical, mechanical, magnetic etc. readout technology.
  • optical, electrical, mechanical, magnetic etc. readout technology e.g. optical, electrical, mechanical, magnetic etc. readout technology.
  • the high specificity of base pairing interactions between stands of nucleic acids is used in these methods to differentiate between different targets.
  • Using a solid phase enables facile multiplexing of nucleic acid hybridization assays by spatially separating different capture oligonucleotides having different sequences. A huge number of different supports e.g.
  • Chips planar surfaces
  • beads or gel matrices can be used as solid phases.
  • Methods for preparation of DNA oligonucleotide arrays are summarized e.g. in S.L. Beaucage, Curr. Med. Chem. 2001, 8, 1213-1244 or M. C. Pirrung, Angew. Chem. 2002, 114, 1326-1341).
  • Solid phase based nucleic acid hybridization assays are widely used e.g. for analysis of single nucleotide polymorphisms (SNPs), expression profiling or viral detection (for a summary see e.g. J. Wang, Nucl. Acids Res. 2000, 28, 3011-3016.
  • the present invention is directed to a method for combining high specificity with high sensitivity in order to enable nucleic acid analysis on a solid surface from biological sources without prior amplification.
  • WO95/16055 An example of a nucleic acid assay which employs multiple hybridization reactions for combination of affinity and specificity is given in WO95/16055.
  • capture probes are bound to a surface.
  • One or more capture extender molecules are employed, each containing a target specific binding sequence and a support binding sequence able to hybridize to the surface bound capture probes.
  • the capture extender sequences are used to bind the target to the support with high affinity.
  • For detection e.g. amplification multimers are hybridized to the target in order to amplifiy signals.
  • Different sequences can be discriminated by specific hybridization of capture extenders containing sequences specific to different target regions. In case of targets with closely related sequences (e.g. targets containing mutations like single nucleotide polymorphisms) this approach does not work for more than one capture extender, because the differences in thermodynamic stabilities and thus melting temperatures are too small for effective discrimination.
  • US 2004/0009506 Al describes a nucleic acid hybridization assay that enables highly sensitive target capturing by using an immobilized capture polymer.
  • the capture polymer consists of target-specific capture sequences that are either directly or indirectly bound to a solid support.
  • the captured nucleic acid sequences are detected by hybridization of label probes containing two or more units of label bound to the label probe.
  • the disclosed assay type does not provide methods for high sensitivity detection of closely related sequences, e.g. sequences differing by a single base.
  • a similar assay type, combining high sensitivity capturing with highly specific enzymatic discrimination is disclosed in WO 2004/020654 A2.
  • target-specific capture sequences that are bound to a solid support either directly or indirectly capture a target from solution with high affinity.
  • Closely related sequences e.g. sequences differing by a single base
  • enzymes that incorporate the label during e.g. ligation or extension reactions.
  • This highly sensitive assay type enables qualitative and quantitative detection of closely related sequences from genomic samples without target amplification, it relies on the discriminative power of enzymes.
  • the incorporation of enzymes into the assay workflow adds a process step, increases assay cost and makes integration into fully- automated devices complicated.
  • nucleic acid assay formats that make use of a hybridization reaction of a target probe to a capture probe immobilized on a solid phase suffer from either sensitivity or selectivity or require enzymatic steps. Therefore, problems occur if e.g. single nucleotide polymorphisms must be detected in samples without prior target amplification in a fast, simple and inexpensive way.
  • Methods and kits are provided for detecting nucleic acids with high sensitivity and high specificity on a solid support.
  • the methods combine high affinity capture using one or more target specific oligonucleotides with highly specific hybridization-based discrimination methods.
  • Preferred methods include the use of one or more capture extender molecules for capturing the target with high affinity, in combination with a set of two or more hybridization probes that are specific for a particular variant of the target (e.g. wild type or single base mutated target).
  • the different hybridization probes are encoded by a particular type (e.g. a specific color) of label.
  • Figure 1 Summary of the assay in a preferred embodiment.
  • a target (5) containing a single base mutation (8) is hybridized to mixtures of target-specific capture probes (1) that are immobilized on a solid support (6) with or without a spacer (7) and are complementary to sequences of the target (2).
  • Allelic discrimination is achieved by hybridization of probes that contain an allele-specific site (4) and are labeled with different labeling entities (3).
  • FIG. 2 Summary of the assay in a preferred embodiment.
  • a target (5) containing a single base mutation (8) is hybridized to a mixture of immobilized capture probes (1) and an allele-specific discrimination probe containing an allele- specific site (4) on a solid support (6) with or without a spacer (7).
  • the capture probes are hybridized to sequences on the target (2) outside the single base mutation.
  • the target (6) is labeled with labeling entities (3) that are directly or indirectly attached to the target.
  • the invention combines high-affinity oligonucleotide capture with highly specific hybridization-based discrimination on a solid support, preferably for the detection of single nucleotide polymorphisms in multiplex assays without prior amplification of genomic DNA.
  • the invention makes use of two or more target- specific capture probes that are directly or indirectly attached to a solid support.
  • the capture probes can be spotted as mixtures of different oligonucleotides and covalently or noncovalently attached to a support. Alternatively, they can be hybridized to universal capture sequences immobilized as arrays on planar substrates.
  • each target can be separately captured in defined spots on the surface. This enables a multiplex detection of e.g. different gene fragments of a gene of interest in different spots on the surface. If more than one capture sequence hybridizes to the same target, cooperative binding leads to a significant increase in sensitivity thereby enabling detection of targets directly from genomic DNA without prior amplification.
  • the capture probes are immobilized on a solid support.
  • a spacer can be introduced between the surface and the hybridizing sequence.
  • This spacer can be composed of units that are capable of hybridization such as nucleotides and nucleotide analogs.
  • the spacer can be composed of units that are not capable of hybridization such as a hydrocarbon chain.
  • the hydrocarbon chain can also contain atoms taken from the 5 th or 6 th group of the periodic system, preferably nitrogen, phosphorous, oxygen or sulfur.
  • the spacer can also be composed of both nucleotide and non nucleotide units.
  • the capture probes are covalently immobilized on the planar surface via their 3'- or 5'-termini without a spacer on a planar substrate that is coated with a polymeric layer.
  • the polymeric layer ensures effective hybridization of the capture probes to the target.
  • the capture probes are covalently immobilized on a planar surface via their 3'- or 5 '-termini with a short spacer containing 2-20 units capable of hybridization, particularly preferred 2-10 units capable of hybridization on a planar substrate that is coated with a polymeric layer.
  • a variant in which the capture probes are covalently immobilized on a planar surface via their 3'- or 5'-termini with a spacer containing 3-30 ethylene glycol units is particularly preferred.
  • spacer molecules that can be attached to the 5'-terminus of capture probes include the commercially available Spacer Phosphoramidite 9 (3 ethylene glycol units and one phosphate unit), 18 (6 ethylene glycol units and one phosphate unit) and C3 (propanol unit and phosphate unit) as well as the Spacer Cl 2 CE Phosphoramidite (dodecanol unit and phosphate unit) from Glen Research, Sterling, Virginia, USA.
  • the capture probes are covalently immobilized on a solid support via a 5'-terminal spacer that contains 3-10 nucleotide units and 3-30 ethylene glycol units).
  • capture extenders are provided for each target and are covalently or non-covalently immobilized on a planar substrate as mixtures resulting in an array of mixtures of immobilized capture probes wherein a particular target can be captured within a particular spot.
  • the capture probes comprise a sequence of 5-100 nucleotides, preferably 10-50 nucleotides that is complementary to a particular target.
  • the targets captured by mixtures of capture probes that are arrayed on a solid substrate, are labeled during hybridization of label probes.
  • These label probes can be chosen from the group of molecules capable of hybridization that are known to persons skilled in the art e.g. DNA, DNA analogs, LNA and PNA.
  • the length and sequence of the label probes depend on the target to be detected.
  • mutations e.g. single nucleotide polymorphisms and deletions
  • targets are detected by differential hybridization of allele-specific hybridization probes.
  • targets are captured on an array of immobilized mixtures of capture probes, whereupon both alleles of a gene fragment containing a single nucleotide polymorphism are captured within the same spot.
  • allele-specific label probes are hybridized to the targets.
  • the hybridization probes are encoded with different labels such that each allele corresponds to a particular type of label.
  • two allele-specific label probes per target each encoded by a specific label compete during hybridization to a particular sequence containing a mutation.
  • the disclosed invention combines two levels of multiplexing: spatially resolved immobilization of mixtures of capture probes that are specific for particular gene fragments and specific hybridization of label-encoded probes that can distinguish between different variants of a target.
  • different hybridization probes are encoded by different labels. Therefore, two or more types of labels are needed for one preferred embodiment. Labels or groups enabling labeling reactions can be e.g. fluorophors, nanoparticles, redox active moieties, antibodies, antibody fragments, biotin, aptamers, peptides, proteins, mono- or polysaccharides, nucleic acids, nucleic acid analogs, complexing agents, cyclodextrins, crown ethers, anticalins, receptors etc. In one particularly preferred embodiment two or more hybridization probes are encoded by two or more different fluorescent dyes that emit fluorescence of different wavelengths.
  • two or more hybridization probes are encoded by two or more different fluorescent nanoparticles that emit fluorescence of different wavelengths. If the invention is carried out with one type of label, the sample to be analyzed must be split into two separate hybridization reactions, carried out on two different arrays. In a particular embodiment of this invention, two different alleles of a set of targets are detected on two different arrays, whereupon the labeled probe specific for one allele competes with the unlabeled probe of the other allele for hybridization to the target.
  • An alternative embodiment of the invention that also uses one type of label makes use of immobilized mixtures of probes, each of the hybridizing to a specific target.
  • capture probes are immobilized within the spots, that are specific to a particular variant of a target, e.g. a particular allele.
  • each allele of a target binds to a specific spot only if both the target- specific capture probes and the allele-specific capture probes become hybridized to the target.
  • the target can be labeled prior, during or after hybridization to the array using a method known to a person skilled in the art. Examples for labeling reactions include, but are not limited to, incorporation of dyes during primer extension, hybridization of labeling probes or chemical attachment of dyes.
  • fragmentation can be performed using different methods known to a person skilled in the art. Examples for fragmentation methods include, but are not limited to, sonication, enzymatic digestion, chemical fragmentation e.g. using radicals (as described e.g. in Y. Zhang et al., Nucleic Acids Res. 2001, 29, 13, e66). Fragmentation allows for the detection of a small (i.e. 50-1000 bp) sequence stretch within a target gene on one spot of the array. Thus, close-by mutations can be detected on different gene fragments in different spots of an array.
  • readout methods can be used to assess the result of the assay.
  • readout methods include optical, electrical, mechanical or magnetic detection. More specifically, fluorophores can be detected using e.g. planar optical waveguides as disclosed in US 5959292, total reflection on interfaces as disclosed in DE 196 28 002 or using optical fibers as disclosed in US 4815843. Nanoparticle labels can be detected e.g. via optical methods or e.g. by direct electrical detection after autometallographic enhancement as disclosed in US patents US 4794089, US 5137827 and US 5284748.
  • the disclosed invention requires a solid phase which is used to immobilize the capture probes. Examples for solid phases include, but are not limited to, beads, planar surfaces, metallic particles and gel matrices.
  • HET samples were obtained from the Coriell Cell Repository (Coriell Institute, Camden, NJ, USA): NAl 1497 (containing the SNP G542X, heterozygote, sample 2) and NA 12585 (containing the SNP R1162X, heterozygote, sample 3).
  • VAR sample was also obtained from the Coriell Institute: NAl 1496 (G542X, variant, sample 4). 60 ⁇ g genomic DNA from each sample was dissolved in Tris-
  • the samples were cooled on ice and sonified using a Branson Sonifier B 12 (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a microtip (3mm diameter) under the conditions of power 3 and 50% duty cycle for a total of 10 min.
  • the sonicated samples were precipitated by addition of 20 ⁇ l aqueous 5M NaCl and 2 ⁇ l glycogen solution (20mg/ml in water) and ImI of ice cold ethanol. Samples were incubated for 30 min. at -2O 0 C. Subsequently, the solutions were centrifuged 15 min at 13000 g.
  • the supernatant was removed and 400 ⁇ l of 70% ethanol were added to the precipitate. Subsequently the samples were centrifuged for 5 min. at 13000 g. The supernatant was removed, the pellets were dried under vacuum and redissolved in 40 ⁇ l of distilled water.
  • biochips (Unaxis AG, Balzers, Liechtenstein) with outer dimensions of 2x1 cm were obtained.
  • the chips consisted of glass and an optical grating with 18 nm deepness etched into the glass. They were coated with a 155nm thick layer of Ta2O5.
  • the chips were cleaned by the following protocol: 10 min sonication in 65% HNO 3 , rinsing by pure distilled water, 10 min sonication in 30% hydrogen peroxide, rinsing by pure distilled water, 10 min sonication in pure distilled water.
  • the PWG- chips were coated with aminopropyltriethoxysilane (APTES, Sigma, Deisenhofen, Germany) using the following protocol: drying of the cleaned chips @80°C for 5min, preparation of 100 ml silanization solution (1% APTES in acetone/water (95:5 v/v)), submerging the warm chips in the silanization solution, incubation for 15 min. at room temperature, rinsing of the chips 5 times by acetone, drying of the chips at 110 0 C for 45 min.
  • APTES aminopropyltriethoxysilane
  • the silanized chips were incubated in a 100ml solution of 1OmM suberic acid bis(N-hydroxysuccinimideester) in dimethylsulfoxide (DMSO) and 50 ⁇ L triethylamine for one hour. Thereafter, the chips were rinsed two times by DMSO and once by distilled water and blown dry in a stream of nitrogen. Two different mixtures of capture probes were prepared: mix 1 consisted of the following capture probe sequences:
  • CE002 CTAATTTTCTATTTTTGGTAATAGGAC ATTTTT-NH2
  • CE004 CAAGAATTTCTTTAGCAAGGTGAATAATTTTT-NH2
  • CE02 AAATAACAACATTTTTGTTTITAAGAATTTTTT-NH2
  • CE05 TCTCAATAATCATAACTTTCGAGAGTTTTTTT-NH2
  • AIl ten sequences were obtained from Thermo Hybaid Corporation, UIm, Germany and were synthesized with a 3 '-terminal amine (3 '-amino modifier by Glen Research, Sterlington, VA, USA).
  • the oligonucleotides were mixed in equal amounts to give solutions of mix 1 and mix 2 with a final DNA concentration of 5xlO-5M in potassion phosphate buffer, pH 7,2. These solutions were placed in small droplets (0.4 nl) onto the chip surface using a Biochip Arrayer (Perkin Elmer, Germany), creating two arrays of 4x4 spots on each chip and incubated overnight in a humid chamber.
  • Chips were rinsed once by 1 M NaOH solution and six times by distilled water. Capping of unreacted amino groups on the surface was performed by reaction with a solution of 0,4 mg/ml bis-sulfo-succinimidyl-suberate (BS3, Pierce, Rockford, IL, USA) in 0,1 M potassium phosphate buffer, pH 7,2 overnight. Chips were washed three times with distilled water, dried in a stream of nitrogen and used thereafter.
  • BS3 bis-sulfo-succinimidyl-suberate
  • Hybridization of genomic DNA to arrays of mixed capture probes Hybridization chambers were mounted onto the four biochips, thereby creating eight discrete chambers with a volume of approximately 100 ⁇ l each.
  • Eight different Allele Specific Hybridization probes (ASH-probes) were obtained from Thermo Hybaid Corporation, UIm, Germany.
  • the ASH-probes comprised the following oligonucleotides:
  • each of the sonicated DNA samples was mixed with an equal volume of denaturing diluent (taken from the Versant 3.0 bDNA assay kit, Bayer HealthCare LLC, Tarrytown, NY, USA) and incubated for Ih at 65°C.
  • ASH probes Two different mixtures of ASH probes were prepared: A wild type probe mix containing fA, B, fC and D at 5xl0-5M concentration each in a 1 :1 solution of neutralization solution (taken from the Versant 3.0 bDNA assay kit, Bayer HealthCare LLC, Tarrytown, NY, USA) and HIV 3.0 lysis solution (taken from the Versant 3.0 bDNA assay kit, Bayer HealthCare LLC, Tarrytown, NY, USA) supplemented with 5x Denhardt's solution (Sigma, Deisenhofen, Germany).
  • the variant probe mix consisted of the ASH probes fB, A, fD and C with the same buffers and additives as above.
  • the DNA samples (40 ⁇ l each) were mixed with 40 ⁇ l of the ASH probe mixes to give a total volume of 80 ⁇ l.
  • Each DNA sample was prepared in two different reaction mixtures containing the two different sets of ASH probes.
  • the resulting solutions were pipetted into the eight different hybridization chambers, sealed tightly and were incubated overnight at 54°C.
  • the chips were read out on a fluorescence reader, type Minifluo IV (Bayer Technology Services, Leverkusen Germany). The reader enabled to couple laser light into the optical grating and image the fluorescence with a CCD camera. Fluorescent intensity of each spot on the array was quantified and the mean of fluorescence for all mix 1 and mix 2 spots was calculated. The results are summarized in the table below:
  • the ratio of mean fluorescence intensities for each sample was calculated by dividing the signal for the wild type probe mix by the signal for the variant probe mix for the respective type of spots (spots mix 1 and spots mix 2). The results are given in the table below, expressed as log (wild type RFU / variant RFU):

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Abstract

This invention relates to methods for detection of nucleic acids on a solid phase with high affinity and high specificity. More particularly, the invention relates to methods combining high-affinity hybridization-based capturing with highly specific hybridization-based discrimination in solid phase based nucleic acid assays. This invention further relates to kits containing the reagents necessary for carrying out the disclosed assays. The detection of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is of importance in human or veterinary diagnostics, food control, environmental analysis, crop protection, biochemical/pharmacological research, or forensic medicine.

Description

SOLID PHASE BASED NUCLEIC ACID ASSAYS
COMBINING HIGH AFFINITY CAPTURING AND DETECTION
BY SPECIFIC HYBRIDIZATION
Field of the Invention
This invention relates to methods for detection of nucleic acids on a solid phase with high affinity and high specificity. More particularly, the invention relates to methods combining high-affinity hybridization-based capturing with highly specific hybridization-based discrimination in solid phase based nucleic acid assays. This invention further relates to kits containing the reagents necessary for carrying out the disclosed assays. The detection of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is of importance in human or veterinary diagnostics, food control, environmental analysis, crop protection, biochemical/pharmacological research, or forensic medicine.
Background of the Invention
In a typical solid phase based nucleic acid assay, capture oligonucleotides are immobilized on a solid support. The labeled or unlabeled nucleic acid target is specifically hybridized to the capture probes. After hybridization and, if necessary, labeling, the hybridization event can be detected using e.g. optical, electrical, mechanical, magnetic etc. readout technology. Generally, the high specificity of base pairing interactions between stands of nucleic acids is used in these methods to differentiate between different targets. Using a solid phase enables facile multiplexing of nucleic acid hybridization assays by spatially separating different capture oligonucleotides having different sequences. A huge number of different supports e.g. planar surfaces ("chips"), beads or gel matrices can be used as solid phases. Methods for preparation of DNA oligonucleotide arrays are summarized e.g. in S.L. Beaucage, Curr. Med. Chem. 2001, 8, 1213-1244 or M. C. Pirrung, Angew. Chem. 2002, 114, 1326-1341). Solid phase based nucleic acid hybridization assays are widely used e.g. for analysis of single nucleotide polymorphisms (SNPs), expression profiling or viral detection (for a summary see e.g. J. Wang, Nucl. Acids Res. 2000, 28, 3011-3016.
Methods for genotyping single nucleotide polymorphisms are described e.g. in P.-Y. Kwok, Annu. Rev. Genomics Hum. Gen. 2001, 2, 235-258. One current general method for detection of SNPs relies on a three step procedure: purification of genomic DNA from biological material, amplification of the desired gene fragment e.g. by PCR and subsequent detection by allele specific hybridization, enzymatic reactions etc. Due to the current lack of highly sensitive nucleic acid detection methods, the amplification step is unavoidable. However, this step is very laborious, time consuming, expensive, difficult to multiplex and difficult to integrate into folly automated devices as they are needed for nucleic acid analysis at the point of care.
In summary, there is a need for fast, simple and inexpensive assays that allow for highly sensitive, highly selective detection of nucleic acids, e.g. comprising SNPs, directly from genomic DNA, without prior amplification. These assays are especially needed for genotyping in a point-of-care environment.
The present invention is directed to a method for combining high specificity with high sensitivity in order to enable nucleic acid analysis on a solid surface from biological sources without prior amplification.
An example of a nucleic acid assay which employs multiple hybridization reactions for combination of affinity and specificity is given in WO95/16055. In this approach, capture probes are bound to a surface. One or more capture extender molecules are employed, each containing a target specific binding sequence and a support binding sequence able to hybridize to the surface bound capture probes. The capture extender sequences are used to bind the target to the support with high affinity. For detection, e.g. amplification multimers are hybridized to the target in order to amplifiy signals. Different sequences can be discriminated by specific hybridization of capture extenders containing sequences specific to different target regions. In case of targets with closely related sequences (e.g. targets containing mutations like single nucleotide polymorphisms) this approach does not work for more than one capture extender, because the differences in thermodynamic stabilities and thus melting temperatures are too small for effective discrimination.
A nucleic acid hybridization assay combining affinity and specificity has been described by Wanda L.B. White (Poster-title "SNP determination by dual hybridization with DNA and PNA probes" at the Cambridge Healthtech Institute
Conference on Nucleic Acid Based Technologies, Washington D.C., 2002). An immobilized 40mer DNA is used to capture the target with high affinity, while a short PNA probe is used for allele-specific hybridization. Drawbacks of this assay principle are that long capture sequences are expensive in synthesis, not easily accessible in sufficient purity and often not specific enough for multiplexed assays.
US 2004/0009506 Al describes a nucleic acid hybridization assay that enables highly sensitive target capturing by using an immobilized capture polymer. The capture polymer consists of target-specific capture sequences that are either directly or indirectly bound to a solid support. The captured nucleic acid sequences are detected by hybridization of label probes containing two or more units of label bound to the label probe. The disclosed assay type does not provide methods for high sensitivity detection of closely related sequences, e.g. sequences differing by a single base. A similar assay type, combining high sensitivity capturing with highly specific enzymatic discrimination is disclosed in WO 2004/020654 A2. Here, target-specific capture sequences that are bound to a solid support either directly or indirectly capture a target from solution with high affinity. Closely related sequences, e.g. sequences differing by a single base, are discriminated by using enzymes that incorporate the label during e.g. ligation or extension reactions. Although this highly sensitive assay type enables qualitative and quantitative detection of closely related sequences from genomic samples without target amplification, it relies on the discriminative power of enzymes. The incorporation of enzymes into the assay workflow adds a process step, increases assay cost and makes integration into fully- automated devices complicated.
In summary, many nucleic acid assay formats that make use of a hybridization reaction of a target probe to a capture probe immobilized on a solid phase suffer from either sensitivity or selectivity or require enzymatic steps. Therefore, problems occur if e.g. single nucleotide polymorphisms must be detected in samples without prior target amplification in a fast, simple and inexpensive way.
Summary of the Invention
Methods and kits are provided for detecting nucleic acids with high sensitivity and high specificity on a solid support. In general, the methods combine high affinity capture using one or more target specific oligonucleotides with highly specific hybridization-based discrimination methods. Preferred methods include the use of one or more capture extender molecules for capturing the target with high affinity, in combination with a set of two or more hybridization probes that are specific for a particular variant of the target (e.g. wild type or single base mutated target). The different hybridization probes are encoded by a particular type (e.g. a specific color) of label.
Brief Description of the Drawings
Figure 1: Summary of the assay in a preferred embodiment. A target (5) containing a single base mutation (8) is hybridized to mixtures of target-specific capture probes (1) that are immobilized on a solid support (6) with or without a spacer (7) and are complementary to sequences of the target (2). Allelic discrimination is achieved by hybridization of probes that contain an allele-specific site (4) and are labeled with different labeling entities (3).
Figure 2: Summary of the assay in a preferred embodiment. A target (5) containing a single base mutation (8) is hybridized to a mixture of immobilized capture probes (1) and an allele-specific discrimination probe containing an allele- specific site (4) on a solid support (6) with or without a spacer (7). The capture probes are hybridized to sequences on the target (2) outside the single base mutation. The target (6) is labeled with labeling entities (3) that are directly or indirectly attached to the target. Detailed Description of the Invention
The invention combines high-affinity oligonucleotide capture with highly specific hybridization-based discrimination on a solid support, preferably for the detection of single nucleotide polymorphisms in multiplex assays without prior amplification of genomic DNA. The invention makes use of two or more target- specific capture probes that are directly or indirectly attached to a solid support. The capture probes can be spotted as mixtures of different oligonucleotides and covalently or noncovalently attached to a support. Alternatively, they can be hybridized to universal capture sequences immobilized as arrays on planar substrates. By spotting or hybridizing mixtures of capture sequences each target can be separately captured in defined spots on the surface. This enables a multiplex detection of e.g. different gene fragments of a gene of interest in different spots on the surface. If more than one capture sequence hybridizes to the same target, cooperative binding leads to a significant increase in sensitivity thereby enabling detection of targets directly from genomic DNA without prior amplification.
In a preferred embodiment of the disclosed invention the capture probes are immobilized on a solid support. In order to ensure effective hybridization of the capture probes to the target, a spacer can be introduced between the surface and the hybridizing sequence. This spacer can be composed of units that are capable of hybridization such as nucleotides and nucleotide analogs. Alternatively, the spacer can be composed of units that are not capable of hybridization such as a hydrocarbon chain. The hydrocarbon chain can also contain atoms taken from the 5th or 6th group of the periodic system, preferably nitrogen, phosphorous, oxygen or sulfur. The spacer can also be composed of both nucleotide and non nucleotide units. In one particularly preferred embodiment the capture probes are covalently immobilized on the planar surface via their 3'- or 5'-termini without a spacer on a planar substrate that is coated with a polymeric layer. In this embodiment, the polymeric layer ensures effective hybridization of the capture probes to the target. In another particularly preferred embodiment, the capture probes are covalently immobilized on a planar surface via their 3'- or 5 '-termini with a short spacer containing 2-20 units capable of hybridization, particularly preferred 2-10 units capable of hybridization on a planar substrate that is coated with a polymeric layer. A variant in which the capture probes are covalently immobilized on a planar surface via their 3'- or 5'-termini with a spacer containing 3-30 ethylene glycol units is particularly preferred. Examples for spacer molecules that can be attached to the 5'-terminus of capture probes include the commercially available Spacer Phosphoramidite 9 (3 ethylene glycol units and one phosphate unit), 18 (6 ethylene glycol units and one phosphate unit) and C3 (propanol unit and phosphate unit) as well as the Spacer Cl 2 CE Phosphoramidite (dodecanol unit and phosphate unit) from Glen Research, Sterling, Virginia, USA. One example for a spacer molecule that can be attached to the 3'-terminus of the capture probes is the 3' Spacer 9 CPG (3 ethylene glycol units) which is also commercially available from Glen Research, Sterling, Virginia, USA. In a further preferred embodiment of the disclosed invention the capture probes are covalently immobilized on a solid support via a 5'-terminal spacer that contains 3-10 nucleotide units and 3-30 ethylene glycol units).
In order to capture the targets with high sensitivity and high specificity, it is necessary to provide two or more capture probes that can hybridize to one target. In a preferred embodiment of the disclosed invention 2-10 capture extenders are provided for each target and are covalently or non-covalently immobilized on a planar substrate as mixtures resulting in an array of mixtures of immobilized capture probes wherein a particular target can be captured within a particular spot. The capture probes comprise a sequence of 5-100 nucleotides, preferably 10-50 nucleotides that is complementary to a particular target.
The targets, captured by mixtures of capture probes that are arrayed on a solid substrate, are labeled during hybridization of label probes. These label probes can be chosen from the group of molecules capable of hybridization that are known to persons skilled in the art e.g. DNA, DNA analogs, LNA and PNA. The length and sequence of the label probes depend on the target to be detected. In a preferred embodiment of the disclosed invention mutations (e.g. single nucleotide polymorphisms and deletions) within targets are detected by differential hybridization of allele-specific hybridization probes. In a preferred embodiment of the disclosed invention targets are captured on an array of immobilized mixtures of capture probes, whereupon both alleles of a gene fragment containing a single nucleotide polymorphism are captured within the same spot. Before, during or after hybridization of the targets to the array, allele-specific label probes are hybridized to the targets. To discriminate between alleles, the hybridization probes are encoded with different labels such that each allele corresponds to a particular type of label. In a particularly preferred embodiment of the present invention two allele-specific label probes per target each encoded by a specific label compete during hybridization to a particular sequence containing a mutation. This competing hybridization (also termed differential hybridization) increases the specificity and is especially useful if single nucleotide polymorphisms are going to be detected. Thus in one preferred embodiment the disclosed invention combines two levels of multiplexing: spatially resolved immobilization of mixtures of capture probes that are specific for particular gene fragments and specific hybridization of label-encoded probes that can distinguish between different variants of a target.
In one preferred embodiment of the invention, different hybridization probes are encoded by different labels. Therefore, two or more types of labels are needed for one preferred embodiment. Labels or groups enabling labeling reactions can be e.g. fluorophors, nanoparticles, redox active moieties, antibodies, antibody fragments, biotin, aptamers, peptides, proteins, mono- or polysaccharides, nucleic acids, nucleic acid analogs, complexing agents, cyclodextrins, crown ethers, anticalins, receptors etc. In one particularly preferred embodiment two or more hybridization probes are encoded by two or more different fluorescent dyes that emit fluorescence of different wavelengths. In a second particularly preferred embodiment two or more hybridization probes are encoded by two or more different fluorescent nanoparticles that emit fluorescence of different wavelengths. If the invention is carried out with one type of label, the sample to be analyzed must be split into two separate hybridization reactions, carried out on two different arrays. In a particular embodiment of this invention, two different alleles of a set of targets are detected on two different arrays, whereupon the labeled probe specific for one allele competes with the unlabeled probe of the other allele for hybridization to the target. An alternative embodiment of the invention that also uses one type of label makes use of immobilized mixtures of probes, each of the hybridizing to a specific target. In addition, capture probes are immobilized within the spots, that are specific to a particular variant of a target, e.g. a particular allele. In this particularly preferred embodiment each allele of a target binds to a specific spot only if both the target- specific capture probes and the allele-specific capture probes become hybridized to the target. In this assay configuration the target can be labeled prior, during or after hybridization to the array using a method known to a person skilled in the art. Examples for labeling reactions include, but are not limited to, incorporation of dyes during primer extension, hybridization of labeling probes or chemical attachment of dyes.
If the disclosed assays are used to detect DNA- or RNA-Sequences without prior amplification of the respective target it may be beneficial to fragment the nucleic acids prior to hybridization to the planar surface. Fragmentation can be performed using different methods known to a person skilled in the art. Examples for fragmentation methods include, but are not limited to, sonication, enzymatic digestion, chemical fragmentation e.g. using radicals (as described e.g. in Y. Zhang et al., Nucleic Acids Res. 2001, 29, 13, e66). Fragmentation allows for the detection of a small (i.e. 50-1000 bp) sequence stretch within a target gene on one spot of the array. Thus, close-by mutations can be detected on different gene fragments in different spots of an array.
Depending on the type of label that has been introduced during the hybridization-based detection step, different readout methods can be used to assess the result of the assay. Examples for readout methods include optical, electrical, mechanical or magnetic detection. More specifically, fluorophores can be detected using e.g. planar optical waveguides as disclosed in US 5959292, total reflection on interfaces as disclosed in DE 196 28 002 or using optical fibers as disclosed in US 4815843. Nanoparticle labels can be detected e.g. via optical methods or e.g. by direct electrical detection after autometallographic enhancement as disclosed in US patents US 4794089, US 5137827 and US 5284748. The disclosed invention requires a solid phase which is used to immobilize the capture probes. Examples for solid phases include, but are not limited to, beads, planar surfaces, metallic particles and gel matrices.
Examples
Example 1: Shearing of human genomic DNA samples
Four different samples of genomic DNA, representing wild type (WT), variant (VAR) and heterozygous (HET) samples for two different SNP sites were acquired. As WT sample human genomic DNA (Roche, Switzerland) was used
(sample 1). HET samples were obtained from the Coriell Cell Repository (Coriell Institute, Camden, NJ, USA): NAl 1497 (containing the SNP G542X, heterozygote, sample 2) and NA 12585 (containing the SNP R1162X, heterozygote, sample 3). VAR sample was also obtained from the Coriell Institute: NAl 1496 (G542X, variant, sample 4). 60 μg genomic DNA from each sample was dissolved in Tris-
EDTA buffer (10 mM Tris base, ImM EDTA, pH=8.0) to give a volume of 400μl. The samples were cooled on ice and sonified using a Branson Sonifier B 12 (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a microtip (3mm diameter) under the conditions of power 3 and 50% duty cycle for a total of 10 min. The sonicated samples were precipitated by addition of 20 μl aqueous 5M NaCl and 2 μl glycogen solution (20mg/ml in water) and ImI of ice cold ethanol. Samples were incubated for 30 min. at -2O0C. Subsequently, the solutions were centrifuged 15 min at 13000 g. The supernatant was removed and 400 μl of 70% ethanol were added to the precipitate. Subsequently the samples were centrifuged for 5 min. at 13000 g. The supernatant was removed, the pellets were dried under vacuum and redissolved in 40 μl of distilled water.
Example 2: Preparation of DNA arrays with mixed capture probes
Four biochips (Unaxis AG, Balzers, Liechtenstein) with outer dimensions of 2x1 cm were obtained. The chips consisted of glass and an optical grating with 18 nm deepness etched into the glass. They were coated with a 155nm thick layer of Ta2O5.
The chips were cleaned by the following protocol: 10 min sonication in 65% HNO3, rinsing by pure distilled water, 10 min sonication in 30% hydrogen peroxide, rinsing by pure distilled water, 10 min sonication in pure distilled water. Then, the PWG- chips were coated with aminopropyltriethoxysilane (APTES, Sigma, Deisenhofen, Germany) using the following protocol: drying of the cleaned chips @80°C for 5min, preparation of 100 ml silanization solution (1% APTES in acetone/water (95:5 v/v)), submerging the warm chips in the silanization solution, incubation for 15 min. at room temperature, rinsing of the chips 5 times by acetone, drying of the chips at 1100C for 45 min.
For immobilization of capture probes, the silanized chips were incubated in a 100ml solution of 1OmM suberic acid bis(N-hydroxysuccinimideester) in dimethylsulfoxide (DMSO) and 50 μL triethylamine for one hour. Thereafter, the chips were rinsed two times by DMSO and once by distilled water and blown dry in a stream of nitrogen. Two different mixtures of capture probes were prepared: mix 1 consisted of the following capture probe sequences:
CEOOl : TCAGATTGAGCATACTAAAAGTGACTCTTTTTT-NH2
CE002 : CTAATTTTCTATTTTTGGTAATAGGAC ATTTTT-NH2
CE003: TCTCCAAGTTTGCAGAGAAAGACATTTTT-NH2
CE004: CAAGAATTTCTTTAGCAAGGTGAATAATTTTT-NH2
CE005: CTAATTATTGGTCTAGCAAGCATTTTT-NH2
Mix 2 consisted of the following five sequences:
CEOl : GGeAGAeAATTTCAeA7VTTAGTTTGTTTTT^NH2
CE02: AAATAACAACATTTTTGTTTITAAGAATTTTTT-NH2
CE03: GACTTGGTAGGTTTACCTTCTGTTGTTTTT-NH2
CE04: GGCCATTCTTGTATGGTTTGGTTTTTTT-NH2
CE05: TCTCAATAATCATAACTTTCGAGAGTTTTTTT-NH2 AIl ten sequences were obtained from Thermo Hybaid Corporation, UIm, Germany and were synthesized with a 3 '-terminal amine (3 '-amino modifier by Glen Research, Sterlington, VA, USA). The oligonucleotides were mixed in equal amounts to give solutions of mix 1 and mix 2 with a final DNA concentration of 5xlO-5M in potassion phosphate buffer, pH 7,2. These solutions were placed in small droplets (0.4 nl) onto the chip surface using a Biochip Arrayer (Perkin Elmer, Germany), creating two arrays of 4x4 spots on each chip and incubated overnight in a humid chamber. Thereafter, the chips were rinsed once by 1 M NaOH solution and six times by distilled water. Capping of unreacted amino groups on the surface was performed by reaction with a solution of 0,4 mg/ml bis-sulfo-succinimidyl-suberate (BS3, Pierce, Rockford, IL, USA) in 0,1 M potassium phosphate buffer, pH 7,2 overnight. Chips were washed three times with distilled water, dried in a stream of nitrogen and used thereafter.
Example 3: Hybridization of genomic DNA to arrays of mixed capture probes Hybridization chambers were mounted onto the four biochips, thereby creating eight discrete chambers with a volume of approximately 100 μl each. Eight different Allele Specific Hybridization probes (ASH-probes) were obtained from Thermo Hybaid Corporation, UIm, Germany. The ASH-probes comprised the following oligonucleotides:
fA: CY5-ttatatcaagaacctcttccacctt
A: ttatatcaagaacctcttccacctt
ffi: CY5-ttatatcaagaaactcttccacctt
B: ttatatcaagaaactcttccacctt
fC: CY5- gatctgtgagccgagtctttaag
C: gatctgtgagccgagtctttaag
fD: CY5- gatctgtgagctgagtctttaag
D: gatctgtgagctgagtctttaag As indicated, four oligonucleotides were modified at their 5 'end using a CY5- modifier (Glen Research, Sterling, VA, USA).
20 μl each of the sonicated DNA samples (see example 1) was mixed with an equal volume of denaturing diluent (taken from the Versant 3.0 bDNA assay kit, Bayer HealthCare LLC, Tarrytown, NY, USA) and incubated for Ih at 65°C. Two different mixtures of ASH probes were prepared: A wild type probe mix containing fA, B, fC and D at 5xl0-5M concentration each in a 1 :1 solution of neutralization solution (taken from the Versant 3.0 bDNA assay kit, Bayer HealthCare LLC, Tarrytown, NY, USA) and HIV 3.0 lysis solution (taken from the Versant 3.0 bDNA assay kit, Bayer HealthCare LLC, Tarrytown, NY, USA) supplemented with 5x Denhardt's solution (Sigma, Deisenhofen, Germany). The variant probe mix consisted of the ASH probes fB, A, fD and C with the same buffers and additives as above. After incubation, the DNA samples (40μl each) were mixed with 40μl of the ASH probe mixes to give a total volume of 80 μl. Each DNA sample was prepared in two different reaction mixtures containing the two different sets of ASH probes. The resulting solutions were pipetted into the eight different hybridization chambers, sealed tightly and were incubated overnight at 54°C. Following incubation, the chips were read out on a fluorescence reader, type Minifluo IV (Bayer Technology Services, Leverkusen Germany). The reader enabled to couple laser light into the optical grating and image the fluorescence with a CCD camera. Fluorescent intensity of each spot on the array was quantified and the mean of fluorescence for all mix 1 and mix 2 spots was calculated. The results are summarized in the table below:
For genotype determination, the ratio of mean fluorescence intensities for each sample was calculated by dividing the signal for the wild type probe mix by the signal for the variant probe mix for the respective type of spots (spots mix 1 and spots mix 2). The results are given in the table below, expressed as log (wild type RFU / variant RFU):
The results are consistent with the assumed genotypes of WT/ WT (sample 1), WT/HET (sample 2), VAR/WT (sample 3) and HET/WT (sample 4). In this example the DNA samples analyzed were split into two separate mixtures in order to perform the experiment with only one type of label. In case of two types of fluorescent labels ("two colors"), the wild type and variant probes can be labeled differently and the analysis of one sample can be performed on one array.

Claims

ClaimsWe claim:
1. A solid phase based nucleic acid assay combining high sensitivity and high specificity, comprising (a) capturing one or more targets by high-affinity hybridization to mixtures of capture probes immobilized on a solid support, (b) discriminating different variants of the targets by hybridization of labeled probes and (c) detecting the result exploiting the physical properties of the labels.
2. A solid phase based nucleic acid assay combining high sensitivity and high specificity, comprising capturing the target by high-affinity hybridization to mixtures of target-specific capture probes and capture probes specific for a variant of the respective target immobilized on a solid support.
3. The assay of claim 2 wherein the targets to be detected are labeled directly or indirectly prior, during or after hybridization of the targets to the array.
4. The assay of claims 1 and 2 wherein mixtures of two or more target-specific capture probes per target are immobilized directly or indirectly on a solid support, forming an array.
5. The assay of claim 1 wherein the hybridization probes that are specific for certain variants of a target are encoded by a specific label so that each allele of a target can be identified by an allele-specific hybridization probe.
6. The assay of claims 1-5 wherein fluorescent molecules or particles are used for labeling.
7. The assay of claim 5 wherein differential hybridization of differently labeled hybridization probes that compete for hybridization is used to enhance discrimination between different variants of a target.
8. The assay of claim 5 wherein the sample to be analyzed is separated in two parts and one part is hybridized to labeled probes that are specific for a first variant of the target in the presence of non labeled probes that are specific for a second variant of the target and that compete with the labeled probes for hybridization and wherein the second part of the sample is hybridized to labeled probes that are specific for a second variant of the target in the presence of non labeled probes that are specific for a first variant of the target and that compete with the labeled probes for hybridization.
9. The assay of claim 4 wherein the mixtures of capture probes are immobilized on a solid support via a short spacer of 1-20 nucleotides between the attaching unit and the hybridizing sequence.
10. The assay of claim 4 wherein the mixtures of capture probes are immobilized on a solid support that is coated with a polymeric layer with a spacer of 0-20 atoms between the hybridizing sequence and the group that is used for attachment to the support.
11. The assays of claims 1-10 wherein planar optical waveguides are used for reading out the labels during or after hybridization to the array.
12. The assays of claims 1-11 wherein the target is fragmented prior to hybridization to the array.
13. The assays of claims 1-12 wherein the targets contain mutations selected from the group of single base substitutions, single nucleotide polymorphisms, insertions, deletions or duplications.
14. The assays of claims 1-13 wherein the solid support is selected from the group consisting of: beads, planar surfaces, metallic particles and gel matrices as supports for multiplexing.
15. The assays of claims 1-14 wherein optical, electrical, mechanical, magnetic methods are used for discriminating sequences.
16. Kits for performing the assays of claims 1-15.
17. An array according to claims 1-16.
EP07759429A 2006-04-26 2007-03-27 Solid phase based nucleic acid assays combining high affinity capturing and detection by specific hybridization Withdrawn EP2411530A2 (en)

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