WO2000026412A1 - Analyse d'acides nucleiques par hybridation en tandem ciblee sur des sequences - Google Patents

Analyse d'acides nucleiques par hybridation en tandem ciblee sur des sequences Download PDF

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WO2000026412A1
WO2000026412A1 PCT/US1999/025693 US9925693W WO0026412A1 WO 2000026412 A1 WO2000026412 A1 WO 2000026412A1 US 9925693 W US9925693 W US 9925693W WO 0026412 A1 WO0026412 A1 WO 0026412A1
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probes
probe
labeled
hybridization
nucleic acid
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PCT/US1999/025693
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Kenneth Loren Beattie
Rogelio Maldonado Rodriguez
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Kenneth Loren Beattie
Rogelio Maldonado Rodriguez
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Publication of WO2000026412A1 publication Critical patent/WO2000026412A1/fr

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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • 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

Definitions

  • the present invention relates generally to the fields of molecular biology and nucleic acid analysis. More specifically, the present invention relates to a novel method of nucleic acid analysis using tandem hybridization approaches.
  • oligonucleotide arrays to analyze a nucleic acid sample is to immobilize numerous nucleic acid samples in a two-dimensional array, then to analyze the binding of a DNA probe to each of the arrayed analytes. Included in the latter approach are membrane hybridizations using size-separated nucleic acid fragments (as in Southern blots and Northern blots) and slot blots and dot blots in which each analyte is placed onto the membrane at a specific location.
  • Another approach to multiplex DNA hybridization is to immobilize each DNA probe to microbeads color-coded with a specific "signature" of fluorophores, then to hybridize the analyte nucleic acid labeled with a molecular tag with the bead mixture and analyze the mixture by flow cytometry, using the fluorescent signature to resolve each probe and the molecular tag to quantitate the binding of analyte to each probe (FlowMetrix method of Luminex, Inc.).
  • This problem may be especially difficult when short oligonucleotide probes are used, wherein the hybridization temperature is too low to disrupt some regions of intrastrand secondary structure.
  • One strategy for minimizing the secondary or higher order structure in the DNA target is to fragment the target sequence to very small size. However, such cleavage is difficult to control and does not solve the problem in the case of strong hairpin loops occurring within a short target sequence.
  • RNA targets can be cleaved to short pieces using 25 mM MgCl 2 at 95°C.
  • PNA peptide nucleic acid
  • PNA PNA is expensive, however, and the stabilizing effect is not uniform over all sequences, and furthermore, the discrimination against mismatches appears to be sacrificed in some sequences using PNA probes (Weiler et al, Nucl. Acids Res.. 25:2792- 2799, 1997).
  • a further inconvenience in hybridization-based nucleic acid analysis is the need to prepare isolated single-stranded target DNA prior to hybridization to surface-immobilized probes, in order to achieve optimal hybridization signals.
  • Various procedures for isolation of single-stranded targets are available, including the use of affinity columns and strand-specific nuclease digestion, but these added steps are costly, time consuming and inconvenient.
  • An additional inconvenience in array hybridization analysis is the need to label each nucleic acid analyte prior to hybridization to the DNA probe array.
  • a number of techniques are available for introduction of labels or tags into nucleic acid strands, including (i) the enzymatic incorporation of label from ⁇ -labeled ATP into the 5 '- terminus of DNA fragments, using polynucleotide kinase; (ii) incorporation of labeled nucleotides into the target nucleic acid by a polymerase in a "nick translation” or “random primer” labeling reaction, in an in vitro transcription reaction, or in a "reverse transcriptase” reaction; and (iii) direct chemical labeling of DNA or RNA, involving covalent reactions which incorporate fluorescent tags, ligands or haptens into the nucleobases.
  • sequence-targeted nucleic acid analysis by oligonucleotide array hybridization is that more than one complementary sequence may exist within the nucleic acid analyte for any given oligonucleotide probe, making it difficult to target the analysis to unique sites.
  • oligonucleotide hybridization base mismatches at the terminal positions of the probe are difficult to discriminate against, while multiple mismatches are readily discriminated against and single internal mismatches are discriminated against to an intermediate extent and sometimes poorly.
  • short oligonucleotide hybridization is known to be highly influenced by base composition, nearest neighbor and probe length, so that a large amount of experimentation is required in order to identify oligonucleotide probes that yield reliable and interpretable hybridization results, and furthermore, if an extensive oligonucleotide array is used, the numerous probes must be designed to form duplex structures (hybrids) of very similar thermal stability.
  • tandem hybridization approaches of the prior art are inoperable in the analysis of extensive nucleic acid targets, such as complex mixtures of PCR fragments, expressed sequences or total genomes.
  • nucleic acid targets such as complex mixtures of PCR fragments, expressed sequences or total genomes.
  • Due to deficiencies of the prior art there is a need for improved methods for analysis of numerous known mutations or DNA sequence polymorphisms , using short oligonucleotide probes immobilized on a solid surface.
  • technologies that minimize the influence of probe length and sequence in short oligonucleotide hybridization analysis There is furthermore a need for improved techniques for analysis of nucleic acid samples of high genetic complexity, using sequence-targeted oligonucleotide array hybridization.
  • Figure 1 depicts schematically the concept of the disclosed invention in a preferred embodiment of nucleic acid sequence analysis by tandem hybridization on sequence-targeted genosensor arrays.
  • Figure 2 displays the electrophoretic analysis of the ⁇ F508 mutation, a 3-base deletion in the human CFTR gene.
  • the results show the distinct electrophoretic banding patterns formed by PCR fragments bearing wild-type, homozygous mutant, and heterozygous (wild- type/mutant) allelic status at the ⁇ F508 mutational site.
  • Figure 3 characterizes the annealing of auxiliary oligonucleotide CF164 with single-stranded target DNA, showing the quantity of auxiliary oligonucleotide required to form a duplex structure with a given quantity of target strand prior to hybridization to the oligonucleotide array.
  • Figure 4 shows a hybridization pattern which indicates the feasibility of using the strategy of preannealing an unlabeled target strand with labeled auxiliary oligonucleotides, in mutation analysis by oligonucleotide array hybridization.
  • Figure 5 displays the hybridization of wild-type target DNA, preannealed with different auxiliary oligonucleotides, to an array of glass-tethered 9mer probes. The results show that contiguous base stacking between a 9mer capture probe and a longer labeled auxiliary oligonucleotide yields a strong hybridization signal and enables efficient discrimination between wild-type and mutant target sequences .
  • Figure 6 displays the hybridization of partially duplex wild- type, homozygous ⁇ F508 and heterozygous wild-type/ ⁇ F508 target DNA, preannealed with auxiliary oligonucleotides, to an array of 9rner probes tethered to glass. The results show that normal (wild-type), homozygous mutant, and heterozygous allelic states can be distinguished using the disclosed invention of tandem hybridization on genosensor arrays.
  • Figure 7 depicts the effect of hybridization temperature on mutation detection by hybridization of target DNA (preannealed with labeled auxiliary oligonucleotide) to an array of glass-tethered 9mer probes. The results further illustrate the utility of introducing a label into the target strand by preannealing with labeled oligonucleotide and indicate that superior mismatch discrimination using 9mer probes is achieved at 25°C hybridization temperature compared with 15°C.
  • Figure 8 illustrates efficient mismatch discrimination at the end of a glass-tethered oligonucleotide probe using the tandem hybridization approach (contiguous stacking with labeled auxiliary oligonucleotide) of the disclosed invention.
  • Figure 8A lists the sequences of the probes and labeled auxiliary oligonucleotide targets.
  • Figure 8B shows the results of the tandem hybridization approach to achieve efficient mismatch discrimination.
  • Figure 9 shows the effect of hybridization temperature and washing duration on mismatch discrimination by stacking hybridization using an array of glass-tethered 9mer probes. The results further illustrate that improved mismatch discrimination can be obtained at higher hybridization temperature and reveal that in the tandem hybridization method of the disclosed invention mismatch discrimination occurs primarily at the hybridization step rather than the washing step.
  • Figure 9A shows the hybridization dots after 3 hr hybridization at each temperature, followed by 5 min washing at the same temperature.
  • Figure 9B shows the results after continued washing for successive 1-hr periods at 30, 35, 40 and ending with 45°C.
  • Figure 10 illustrates the influence of the length of glass- tethered capture probe (7mer versus 8mer here, in comparison with 9mer data of Figure 8) on mismatch discrimination by tandem hybridization performed at 25°C. The results indicate that improved mismatch discrimination at the end of the capture probe can be achieved using shorter capture probes in the disclosed invention.
  • Figure 1 1 shows the influence of capture probe length
  • Figure 12 shows the influence of mismatch position within the capture probe on mutation detection by tandem hybridization.
  • Figure 12A is a listing the sequences, the mismatch type and the mismatch position of the capture probes.
  • Figure 12B indicates that with 7mer glass-tethered capture probes, the best mismatch discrimination is achieved when the capture probes are designed to place the mismatched site at positions 2, 3 and 4 from the free end of the capture probe.
  • Figure 13 illustrates in schematic form the utility of annealing auxiliary oligonucleotides on both sides of the site within the target strand hybridizing to the capture probe, and optionally to the opposite strand, to enable analysis of denatured double-stranded DNA by the disclosed tandem hybridization invention, without prior isolation of single-stranded target DNA.
  • Figure 14 illustrates schematically the use of tandem hybridization to analyze short tandem repeat polymorphisms (STRPs) using allele-specific stacking probes ( Figure 14A) and allele-specific capture probes ( Figure 14B).
  • each STRP marker is represented by a capture probe tethered to a specific location on the oligonucleotide array.
  • a different label for each STRP allele analyzed in a given hybridization reaction is needed. For markers containing numerous alleles, several hybridization reactions may be performed. In 1 the target sequence contains four repeat units; the stacking probe hybridizes in tandem with the capture probe yielding a positive hybridization signal with the label (* ). In 2 the target sequence contains five repeat units; no stabilizing stacking interaction occurs yielding little or no hybridization signal with the label (*). In Figure 14B the capture probe shown will give contiguous stacking with the labeled stacking probe only if the target contains eight repeat units; thus, little or no label binds at this site in the array. The target shown will give a hybridization signal at an array location containing a six- repeat capture probe.
  • Figure 15 illustrates in schematic form various embodiments of the disclosed invention using bead technology.
  • tandem hybridization is performed using capture probes tethered to color-coded polystyrene beads, which may be individually recognized and quantitated using flow cytometry and spectroscopic techniques.
  • the short capture probe on the color-coded bead is specific for a known sequence at the 3 '-end of mRNA.
  • the bead-immobilized capture probe is allele-specific, designed to hybridize in tandem with the longer labeled stacking probe designed for the sequence polymorphism or mutation.
  • the longer labeled stacking probe may be gene-specific; ie, one labeled stacking probe for each sequence region analyzed, or it may be a universal stacking probe; e.g., oligo(dT)-NNN for analysis of 3 ' -end of eukaryotic mRNAs.
  • the relative level of binding of label to each color-coded bead quantitated by flow cytometric with spectral analysis indicates the relative abundance of mRNA species (transcriptional profiling) or the allelic status at each DNA marker or mutational site.
  • a sequence-specific capture probe of a length sufficient to hybridize to the nucleic acid analyte at a single unique position hybridizes with the target nucleic acid in conjunction with a short labeled probe which itself is designed to hybridize in tandem with the given bead-tethered capture probe.
  • the relative level of label associated with each color-coded bead quantitated by flow cytometry with spectral analysis is indicative of the relative abundance of mRNA/cDNA species, or relative abundance of different sequence variants.
  • Unlabeled nucleic acid analyte (the "target sequence") is denatured and annealed or hybridized with a molar excess of two or more oligonucleotide probes, at least one of which is labeled, and which bind to target sequences in one or more regions of known sequence, to form a partially duplex structure in which at least two oligonucleotide probes bind to the target sequence in tandem, forming a duplex region in which binding of at least one probe is stabilized by uninterrupted contiguous base stacking with the tandemly hybridizing probe.
  • the target sequence Unlabeled nucleic acid analyte
  • At least one of the oligonucleotides can be designed to disrupt interfering secondary or higher order structures, or to cover up alternative hybridization sites that any of the sequence-detecting probes may have within the nucleic acid analyte.
  • This multiple probe strategy is designed to improve the reliability of hybridization analyses, and avoids the inconvenient and costly labeling of numerous nucleic acid samples.
  • the label is introduced only into specific target molecules that are targeted by the surface-tethered capture probes, the problem of nonspecific binding or imperfect hybridization is minimized, particularly with nucleic acid analytes of high genetic complexity.
  • auxiliary oligonucleotides preannealed to different nucleic acid samples
  • a multiplicity of nucleic acid samples can be simultaneously analyzed in a single hybridization assay.
  • the nucleic acid target is a heat-denatured double-stranded DNA
  • the competing reassociation reaction of target strands can be minimized by preannealing the denatured target with a molar excess of oligonucleotides selected to bind to the target on one or both sides of the capture probe.
  • each labeled probe is designed to anneal to a unique site on the target strand, in tandem with a shorter surface- tethered "capture probe.”
  • Hybridization is carried out at elevated temperature or other increased stringency conditions, such that the short capture probe will not by itself form a stable duplex structure with the target sequence. Only if uninterrupted, contiguous base stacking occurs between labeled probe and surface-tethered capture probe, providing sufficient stability to the short duplex (capture probe paired with target strand), will a strong hybridization signal be seen.
  • the labeled longer oligonucleotide is preferably preannealed to the target nucleic acid, however it can alternatively be added to the analyte nucleic acid at the time of hybridization to the array of surface-tethered capture probes.
  • Increased site specificity is achieved because the analysis is targeted to a unique region on the nucleic acid analyte, complementary to the combined sequence of capture probe plus tandemly hybridizing labeled probe. Mismatches in the middle of the short capture probe or at any position extending to its junction with the tandemly hybridizing labeled probe will decrease the stability of the short duplex formed between target and capture probe, and will therefore reduce or eliminate the hybridization signal.
  • a collection of labeled "auxiliary" oligonucleotides, annealing on the target strand in tandem with a set of surface-tethered capture probes, arrayed at separate regions of a hybridization substrate provides a robust means for simultaneous analysis of a multiplicity of nucleic acid sequences, and simultaneously, provides a convenient means for labeling of the analyte nucleic acid.
  • An important feature of the disclosed invention is that since the longer labeled probes serve to position the hybridization of shorter capture probes to unique sites along the target, nucleic acids of high genetic complexity can be analyzed.
  • auxiliary oligonucleotides preannealed to the single-stranded target nucleic acid to form a partially duplex target molecule, offers several advantages in the analysis of nucleic acid sequences by hybridization to genosensor arrays or "DNA chips".
  • These advantages include (i) a convenient means for introducing one or more labels into the target; (ii) prevention of short-range secondary structure that can interfere with hybridization to the surface-tethered oligonucleotide probes; (iii) masking of redundant sequences in the target strand to insure that a given capture probe interrogates a single site within the target; (iv) contiguous base stacking with the capture probe through tandem hybridization on the target strand, which enhances the hybridization signal and gives improved mismatch discrimination near the end of the capture probe; (v) ability to target the hybridization analysis to unique sites in nucleic acids of high genetic complexity; and (vi) improved ability to analyze double- stranded DNA targets by preannealing with a molar excess of oligonucleotides binding to the target adjacent to the capture probe.
  • the hybridization was carried out in two steps: The target DNA was first hybridized to decanucleotide probes (l Omers) covalently attached within a thin polyacrylamide gel matrix, to place the mutant site adjacent to the l Omer duplex, then fluorescently labeled shorter contiguous stacking pentanucleotide probes (5mers) were applied to the gel matrix to detect point mutations within the target sequence immediately adjacent to the 10-base duplex.
  • decanucleotide probes l Omers
  • 5mers fluorescently labeled shorter contiguous stacking pentanucleotide probes
  • oligonucleotide probes are covalently attached in different positions on a solid surface, then the nucleic acid target, preannealed with one or more longer labeled oligonucleotides, each of which binds to the target in a unique position and may act as contiguous stacking oligonucleotides, is hybridized to the array.
  • the Mirzabekov group proposed that the "stabilizing" effect of contiguous stacking can be used to improve the efficiency of de novo DNA sequencing by hybridization (Parinov et al, Nucl. Acids Res. 24:2998-3004, 1996) and to detect point mutations (Yershov et al, Proc. Natl. Acad. Sci., U.S.A. 93:4913-4918, 1996).
  • CSH contiguous stacking hybridization
  • the tandem hybridization strategy disclosed herein is designed to detect known sequence variations, in a single hybridization reaction on the oligonucleotide array.
  • the tandem hybridization approach disclosed herein has a more limited range of applications than Mirzabekov' s CSH, it is much simpler, yet applicable to many important DNA diagnostic tests, where the relevant alleles are known from previous research.
  • the auxiliary oligonucleotides annealed to the target nucleic acid in the hybridization strategy intoduced here may serve to remove secondary structure from the target strand, such as interfering hairpin structures, and may also facilitate analysis of duplex target DNA.
  • a duplex DNA sample prior to hybridization to an array of capture probes, can simply be heat-denatured, then preannealed with a molar excess of auxiliary oligonucleotides, at least one serving as a contiguously stacking probe, the other annealing nearby to the DNA target on the other side of the capture probe, and one or both introducing the label, enabling detection of hybridization signal across the array of capture probes. Formation of these additional duplex regions flanking the test site should dominate over the competing reannealing reaction of complementary strands.
  • auxiliary oligonucleotide probes can eliminate two costly and time consuming steps that are normally conducted with each sample prior to hybridization, isolation of single-stranded target DNA and labeling of the target.
  • the CSH strategy used by the Mirzabekov laboratory to identify mutations involved a first round of hybridization of a target DNA to an array of l Omer probes immobilized within thin sections of polyacrylamide gel, followed by subsequent rounds of hybridization with fluorescent-labeled shorter (5mer) probes, which hybridized to the target strand in tandem with the longer (lOmer) "capture probe.”
  • a different approach is used, in which unlabeled target DNA is preannealed with a longer "stacking oligonucleotide,” which also functions to introduce the label into the target molecule, then the partially duplex DNA target is hybridized with an array of shorter oligonucleotide probes tethered to a solid surface.
  • mismatch discrimination occurs within the short stacking probe added in a subsequent round of hybridi zation oj] Ul ⁇ oli gonucleotide array. whereas in the method disclosed herein, the mismatch discrimination occurs within the short glass-tethered probe, in a single hybridization reaction on the oligonucleotide array.
  • sequence variations at each position of interest are represented within the short "capture probes” that are immobilized at separate locations on the surface, whereas in Mirzabekov' s CSH approach, the sequence differences are represented in the short "stacking probes" added in subsequent rounds of hybridization.
  • the immobilized capture probes serve to position the target strand adjacent to the diagnostic fluorescent-labeled stacking probes added in one or more subsequent rounds of hybridization.
  • tandem hybridization method In the tandem hybridization method disclosed herein it is the longer stacking oligonucleotide (annealed to the target) which serves to place the target strand in register with the shorter sequence-interrogating capture probe, and therefore the support-bound oligonucleotide and stacking oligonucleotides serve opposite roles in the two approaches.
  • the stacking hybridization method disclosed herein has several advantages over the more common oligonucleotide array approaches of the prior art, in which point mutations or single base polymorphisms are identified using immobilized allele-specific oligonucleotides (ASOs) without the stacking oligomer (Conner et al, Proc. Natl. Acad. Sci., USA. 80:278-282, 1983; Pease et al, Proc. Natl. Acad. Sci., U.S.A. 91 :5022-5026, 1994; Hacia et al, Nature Genetics 14:441 -447, 1996).
  • ASOs immobilized allele-specific oligonucleotides
  • a given nucleotide residue in the target is interrogated using probes differing at a single position in the middle of the oligomer sequence, and it is well known that in this system mismatch discrimination is efficient only in a central position of the probe.
  • mismatch discrimination is efficient at the terminal position of the immobilized oligonucleotide, adjacent to the stacking oligomer, as well as at internal positions within the surface-tethered capture probe.
  • sequence changes can be detected over a greater stretch of sequence targeted by a given oligonucleotide probe using the stacking hybridization technique disclosed herein.
  • n L/4 P
  • a labeled stacking oligonucleotide of greater length eg. 15mer
  • a labeled stacking oligonucleotide of greater length eg. 15mer
  • total bacterial DNA is preannealed (at high stringency) with total bacterial DNA, such that upon hybridization with an array of short "capture probes,” the labeled DNA binds specifically to an array element containing a short capture probe which hybridizes to the target strand in tandem with the labeled stacking oligonucleotide.
  • hybridization of analyte nucleic acid to the oligonucleotide array is carried out under conditions (eg., elevated temperature) in which the short surface-tethered capture probes do not form stable duplex structures with complementary sequences that may be present at distal locations within the target nucleic acid, not subject to stabilization by contiguous base stacking.
  • the stacking hybridization approach enables direct analysis of target sequences of high genetic complexity.
  • the disclosed tandem hybridization strategy enables analysis of numerous sequence variations within a target sequence of high genetic complexity. Similarly, the disclosed strategy may be used to quantitatively detect any expressed sequence in a bulk mRNA preparation.
  • tandem hybridization invention is the use of short capture probes with long stacking probes
  • the invention also encompasses the use of short capture probes with short stacking probes, and the use of long capture probes with short or long stacking probes.
  • an array of long surface- tethered oligonucleotides (each of length sufficient to hybridize with a single, unique sequence within the nucleic acid) may be used to first capture a unique sequence at each array element (such as a specific mRNA species, a DNA fragment derived from a specific gene transcript by reverse transcription, or a specific genomic region), then one or more labeled stacking probes may be hybridized to the array to reveal the relative abundance of target sequences bound to each array element (such as a transcriptional profile) or to reveal the allele status ⁇ of a collection of polymorphic markers.
  • the labeled stacking probes and arrayed capture probes may alternatively be hybridized to the nucleic acid sample in a single step.
  • the disclosed invention additionally includes the use of DNA ligase to covalently join the surface-tethered capture probe to the labeled stacking probe following hybridization to the oligonucleotide array.
  • This ligation step preferentially stabilizes the hybridization signal that arises from contiguous stacking between capture and stacking probes, while having no effect on label bound to the array through any other means, including nonspecific hybridization and isolated hybridization of capture and labeled probes to different (noncontiguous) sites on the target strand.
  • the ligation approach is particularly advantageous when nucleic acids of high genetic complexity are analyzed using short capture probes.
  • a washing step can be carried out at elevated temperature, to remove absolutely all label except that that which has bound to the array via contiguous stacking hybridization. Furthermore, since the ligation reaction is well known to be inhibited by base mismatches at the ligation junction, the strategy of ligation followed by washing at elevated temperature can improve the discrimination against mismatched bases at or adjacent to the termini of capture and stacking probe in the disclosed tandem hybridization approach. Consequently, the combined tandem hybridization/ligation strategy can improve the identification of mutations and DNA sequence polymorphisms.
  • the ligation approach can be applied in a variety of embodiments using different numbers and lengths of capture and stacking probes, including the use of multiple stacking probes flanking (on one or both sides) the capture probe; the use o short capture probes with either short or long stacking probes; and the use of long capture probes with either short or long stacking probes.
  • capture and stacking probes including the use of multiple stacking probes flanking (on one or both sides) the capture probe; the use o short capture probes with either short or long stacking probes; and the use of long capture probes with either short or long stacking probes.
  • the 5'-phosphate group can be incorporated in a variety of ways well known to the practioner, including chemical phosphorylation during the chemical synthesis of oligonucleotides using the standard phosphoramidite procedure, and phosphorylation of oligonucleotides using polynucleotide kinase.
  • the kinase reaction can be applied to free oligonucleotides in solution, or can be carried out "in situ" with oligonucleotides immobilized at their 3'-ends on a solid surface.
  • each hybridization site serves to purify a specific sequence among a complex mixture of sequences.
  • the purified sequences are available for further analytical steps, which can include one or more additional hybridization steps on the array substrate, or elution of the bound (purified) sequences from one or more array elements, for further analysis or manipulation (including sequencing, cloning, and additional hybridization reactions).
  • the quantity of sequence captured at each array element and the elution/recovery of bound material may be optimized through the use of a flowthrough hybridization substrate comprised of any high surface area support material, including but not limited to glass fiber filters, micromachined or etched silicon structures, microchannel glass, porous plastics, arrays of encapsulated microbeads, etc.
  • RNA/DNA hybrid a term originally used to denote the formation of a duplex structure between complementary strands of RNA and DNA
  • RNA/DNA hybrid a term originally used to denote the formation of a duplex structure between complementary strands of RNA and DNA
  • RNA/DNA hybrid a term originally used to denote the formation of a duplex structure between complementary strands of RNA and DNA
  • RNA/DNA hybrid a term originally used to denote the formation of a duplex structure between complementary strands of RNA and DNA
  • RNA/RNA hybrid any duplex formation between complementary strands, whether DNA/DNA, DNA/RNA, or RNA/RNA, carried out either in solution or in the solid phase, wherein one of the two strands is immobilized onto a solid surface or matrix.
  • Annealing - in the field of nucleic acid analysis a term originally used to describe the process of duplex formation in which two nucleic acids are mixed together, heated to denature the duplex structure, then incubated at slowly decreasing temperature to allow complementary sequences to find themselves and form new duplex structures, accommodating a range of base compositions and strand lengths.
  • the term is used more recently (including herein) to mean incubation of a single-stranded or heat-denatured duplex nucleic acid analyte with an oligonucleotide probe or primer, under hybridization conditions enabling the probe or primer to bind to its complementary ⁇ sequence within the analyte nucleic acid, either at slowly decreasing temperature or at a single temperature.
  • Analyte or analyte nucleic acid - the class of compound in a sample which is the object of analysis, for example a nucleic acid extracted from a biological sample.
  • Label also known as "tag" - a substituent that can be attached to a nucleic acid analyte which enables its detection/and or quantitation.
  • radiolabels such as 32 P, 33 P and 35 S ; fluorescent tags, chemiluminescent tags, enzymes that catalyze formation of a fluorescent, chemiluminescent or colored compound, ligands such as biotin, and chemical groups that are distinguishable by mass or other spectroscopic properties.
  • the label may be introduced into the analyte nucleic acid by a variety of means, including chemical reaction, incorporation of labeled nucleotide by enzymatic reaction (including polymerase, kinase or ligase), or by hybridization or annealing of a labeled probe with the analyte nucleic acid.
  • Probe - a nucleic acid sequence used as a reagent to bind its complementary sequence within the analyte nucleic acid, via a hybridization reaction.
  • Sequence a string of bases within a nucleic acid, comprising A, G, C, T residues in DNA or A, G, C, U residues in RNA, linked together in a specific order and chain length.
  • a sequence can contain any or all of the four bases.
  • Compl ementary or compl em entary sequence - two sequences are said to be complementary if they are capable of forming a two-stranded (duplex) structure in which all of the bases in one strand form specific Watson-Crick base pairs (A»T or G»C in DNA; A «U and G»C in RNA) with the opposing bases in the opposite strand.
  • the term can also be used at the single base pair level: A is complementary to T or U; T or U are complementary to A; G is complementary to C; C is complementary to G.
  • Noncomplementary Q ⁇ noncomplementary sequence - two sequences are said to be noncomplementary if they do not form a perfectly Watson-Crick base-paired duplex structure.
  • Imperfectly base paired duplex structures sometimes form (though usually less stable than a perfectly paired duplex) in which a small fraction of the opposing bases are noncomplementary (A opposite G, C; G opposite A, T or G; C opposite T, A or C; or T opposite G, C or T).
  • Labeled probe - as used herein, an oligonucleotide bearing one or more detectable labels or tags, which is capable of binding to its complementary sequence within a nucleic acid analyte, enabling the detection and/or quantitation of said analyte nucleic acid.
  • Capture probe - as used herein, an oligonucleotide of specific sequence bound at one end (tethered) to a solid surface, enabling the capture of a nucleic acid analyte containing a complementary sequence onto said solid surface, in a hybridization reaction.
  • Stacking probe - as used herein an oligonucleotide designed to bind to its complementary sequence within a nucleic acid analyte, immediately adjacent to (in tandem with) the complementary sequence within the nucleic acid analyte which hybridizes with a capture probe.
  • the stacking probe and capture probe hybridize in tandem with the target strand to form a duplex region of length equal to the sum of the lengths of stacking and capture probes, in which all of the bases in one strand are Watson-Crick base- paired with opposing bases on the opposite strand.
  • At the junction between stacking ancT capture probes there is uninterrupted base stacking interaction between the terminal residues of stacking and capture probes.
  • the stacking probe is normally labeled and is normally of length sufficient to have a single, unique binding site (complementary sequence) within the nucleic acid analyte.
  • the base stacking interactions propagating from the stacking probe into the capture probe results in stabilization of the binding of target strand to the surface-tethered capture probe, yielding an effective duplex stability similar to that which would be obtained using a (longer) capture probe of length equal to the combined lengths of capture and stacking probes.
  • the stacking probe is normally designed to be of length sufficient to possess a single, unique complementary sequence within the analyte nucleic acid.
  • Partially duplex structure a nucleic acid molecule that is partially single-stranded and partially duplex, such as the structure formed upon annealing of an oligonucleotide with a longer single- stranded nucleic acid strand.
  • Target, target sequence, target strand or target nucleic acid - a nucleic acid sequence whose presence in the analyte is the object of detection, for example (as used herein), through hybridization with a specific DNA probe.
  • target nucleic acid refers to a specific molecule, strand or fragment (such as a single mRNA species or a specific PCR fragment) that is the object of detection, for example via hybridization to a labeled DNA probe.
  • target sequence is sometimes used in a broad sense to mean the nucleic acid molecule or fragment bound by a DNA probe, or can be used in a restricted sense to mean the specific nucleotide sequence within the target nucleic acid which binds to the DNA probe via complementary base pairing.
  • an oligonucleotide probe (typically, a labeled stacking probe) is said to bind (or hybridize) to a unique position within the target nucleic acid when the oligonucleotide is of length sufficient to have only a single complementary sequence within the analyte nucleic acid, thus is capable of binding at a single, unique position within the analyte nucleic acid.
  • a DNA probe (typically an oligonucleotide) is said to be surface-tethered or tethered to a surface if it is bound at one end with the surface, through a covalent bond or otherwise strong bond formed between a functional group on the surface and a functional group at one end of the DNA probe.
  • Base stacking the major force accounting for the stability of duplex nucleic acid structures, comprising electronic interactions between adjacent planar bases within each strand. Base stacking is normally an intrastrand force which propagates along a strand and is much stronger when two strands are Watson-Crick base paired in a duplex structure. The longer the duplex region, the stronger the stabilizing stacking interactions within each strand, which largely accounts for the well known length dependence of duplex stability.
  • Tandem hybridization a hybridization reaction carried out using a surface-tethered oligonucleotide (the ⁇ "capture probe") and an auxiliary oligonucleotide (the “stacking probe,” which is typically labeled and preannealed with the target), wherein the capture probe and stacking probe bind at adjacent (non- overlapping but contiguous) sites on the target strand, forming a contiguous duplex structure in which stacking and capture probes interact with one another through base stacking interactions.
  • the capture probe and stacking probe bind at adjacent (non- overlapping but contiguous) sites on the target strand, forming a contiguous duplex structure in which stacking and capture probes interact with one another through base stacking interactions.
  • Solid phase hybridi ation - a hybridization reaction conducted in which one of the two "reactant strands" participating in formation of a duplex structure is immobilized on a solid support.
  • auxiliary functions include labeling of the analyte nucleic acid (performed by the labeled probe); stabilization of the duplex formed between target strand and capture probe (performed by the stacking probe); and enabling the binding of the capture probe to a single, unique position within the target nucleic acid (also performed by the stacking probe).
  • DNA sequence polymporphi sm naturally occurring variation in the DNA sequence within a population of a given species, generally considered useful as DNA markers if the frequency of a minor allele is greater than about 10% in the population.
  • Oligonucleotide - short nucleic acid (DNA or RNA) strand which can be chemically synthesized, typically of length up to abouf 100 nucleotides.
  • Gene - a unit of genetic function, including sequences encoding a protein or functional RNA (eg., rRNA or tRNA), intronic (noncoding) sequences interpersed within a gene, and additional sequences functioning in the regulation of the gene.
  • RNA eg., rRNA or tRNA
  • intronic (noncoding) sequences interpersed within a gene
  • additional sequences functioning in the regulation of the gene.
  • Genome the entire complement of genes, intergenic sequences and other genetic elements that comprise an organism or autonomously replicating entity.
  • Amplicon - a fragment of DNA amplified using the polymerase chain reaction.
  • Genotype A collection of detectable DNA sequence variations (polymorphisms or markers) that may distinguish one individual from another; also a verb meaning the act of determining a genotype .
  • RNA species individual mRNA, rRNA and tRNA "transcripts" from individual genes, typically catalyzed by RNA polymerase. In vivo transcription occurs to yield a wide range of abundancies of individual gene transcripts, from none to many millions of copies per cell, depending on cell type and physiological state.
  • Gene expression the process of biosynthesis of gene products from genes of an organism, including the processes of transcription, intron splicing (in eukaryotes), translation of mRNA into protein, and posttranslational modification of proteins to give altered activity or function.
  • Multiplex - in the field of nucleic acid analysis refers to the ability to detect a mixture of simultaneously occurring reactions or entities, such as formation and detection of multiple PCR products in a ⁇ single reaction, and detection of multiple nucleic acids in a mixture, through use of distinguishable tags or through spatial separation into distinct sites.
  • DNA chip or genosensor chip - a two-dimensional array of surface-tethered DNA probes formed on a surface, enabling simultaneous analysis of a multiplicity of hybridization reactions.
  • the "chip” also termed “microarray”
  • microarray is typically in a miniaturized format, with individual DNA probes arrayed at center-to-center spacing of one millimeter or less.
  • Secondary structure any double-stranded structure formed between two complementary or largely complementary sequences.
  • the term includes interstrand duplex formation, such as in annealing and hybridization reactions, as well as intrastrand duplex formation, such as hairpin loops.
  • Hairpin loop or stem-loop a type of intrastrand secondary structure formed when two inverted repeat sequences occur near each other (eg., AGCCTGtatCAGGCT) - the inverted repeat sequences (denoted here by capital letters) fold back on each other to form a duplex region (hairpin stem) and the short sequence between the inverted repeats (denoted here by lower case letters) forms the single- stranded loop at the top of the stem.
  • Intrastrand formation of stable hairpin loops within a target nucleic acid can interfere with the availability of the target sequence to hybridize with a surface-tethered oligonucleotide that is complementary to a sequence within the hairpin-loop .
  • Mismatch the existence of one or more base mispairings (or “noncomplementary base oppositions") within a stretch of otherwise complementary duplex-forming (or potentially duplex- forming) sequences.
  • the existence of a single mismatched base pair within a short oligonucleotide duplex normally destabilizes the duplex.
  • Mismatch discrimination as used herein, the ability of a surface-tethered oligonucleotide probe to hybridize specifically to a fully complementary sequence, and not to a mismatch-containing (nearly complementary) sequence.
  • Reverse transcriptase a DNA polymerizing enzyme which polymerizes deoxynucleoside triphosphates to form a complementary DNA (“cDNA”) strand using an RNA template strand hybridized with an appropriate primer strand (which is elongated during the "reverse transcription”) .
  • Primer - a nucleic acid sequence (such as an oligonucleotide) possessing a free 3'-OH terminus, which is base paired with a "template strand" and thus can be elongated by a polymerase enzyme.
  • an oligonucleotide primer annealed with a DNA template can serve as a substrate (along with deoxynucleoside 5' - triphosphates) for a DNA polymerase, resulting in "primer extension,” as in the PCR reaction.
  • Genetic complexity the total length of nonrepetitive, unique sequence within a nucleic acid analyte.
  • An example of high genetic complexity is an entire genome and an example of low genetic complexity is a single PCR fragment.
  • Thermal stability The stability of a duplex nucleic acid structure as a function of temperature in a given salt/buffer solution. It is well known that thermal stability increases with increased length of a nucleic acid duplex. It is also well known that thermal stability of s short duplex region (formed by hybridization of an oligonucleotide probe with a single-stranded target) can be substantially increased by contiguous base stacking with a tandemly hybridized oligonucleotide. For example, the thermal stability of two tandemly hybridized 7mer oligonucleotides (contiguously stacked on a complementary strand) is similar to that of a single 14mer hybridized to its complement.
  • PCR fragment - a fragment of DNA of defined length (defined by the spacing between priming sites on the template) formed by the polymerase chain reaction
  • Expressed sequence an RNA molecule formed by transcription of a coding sequence(s) within a gene (typically mRNA but also including rRNA, tRNA), or alternatively, a complementary DNA (cDNA) copy of the RNA, formed by an in vitro reverse transcription reaction.
  • a coding sequence(s) within a gene typically mRNA but also including rRNA, tRNA
  • cDNA complementary DNA
  • preanneal - as used herein, the annealing (or hybridization) of a heat-denatured analyte nucleic acid with one or more auxiliary oligonucleotides prior to hybridization of the analyte to surface- tethered capture probes.
  • Allele - a specific member of a collection of naturally occurring sequence variants (detectable within a population of individuals) at a specific genomic locus or marker.
  • Homozygous - in a diploid genome the occurrence of an identical allele on both copies of the relevant chromosome.
  • Heterozygous - in a diploid genome the occurrence of different alleles on the two copies of the relevant chromosome.
  • DNA marker - a defined genomic site containing naturally occurring DNA sequence variation (detectable within a population of individuals) which can be analyzed using biochemical techniques to determine the allele status, including homozygous/heterozygous state and any other properties related to the sequence variation (such as length of restriction fragment or PCR fragment, or sequence identity as revealed by hybridization analysis or DNA sequencing). Redundant residue - a position within a sequence (for example, an oligonucleotide) which is occupied by a mixture of two or more (and typically all four) bases.
  • Repetitive sequence a sequence that exists in numerous copies within a genome, for example SINE (including Alu) sequences in the human genome and short tandem repeat sequences (including
  • ACACACACAC scattered throughout the genomes of higher eukaryotes .
  • nucleic acid chain containing a nucleotide with a non-esterified carbon-5 on its deoxyribose (or ribose).
  • the most extensively studied mammalian SINE is the Alu sequences, comprising a 282 consensus sequence, typically followed by an A-rich region and flanked by direct repeat sequence representing the duplicated insertion site. Alus are repeated on average, every 3,000 base pairs in the human genome.
  • the auxili ary oligonucleotide annealing/tandem hybridization strategy of the disclosed invention offers improved performance in many important oligonucleotide hybridization applications, including: (i) repetitive analysis of known mutations or sequence polymorphisms in numerous genomic samples; (ii) simultaneous analysis of numerous known mutations or sequence polymorphisms in single genes, a multiplicity of genes, or on a genome-wide scale; (iii) identification of species, strains or individuals through the use of oligonucleotide probes and auxiliary oligonucleotides targeted to nucleotide sequences known to be unique for said species, strains or individuals; (iv) analysis of gene expression
  • a Microlab 2200 robotic fluid-delivery system (Hamilton, Reno, NV), supplied with a four-needle delivery head, was used to place submicroliter droplets onto glass slides.
  • the Microlab 2200 system was programmed (using resident software) to deliver droplets of 200 nL onto each slide as previously described (Beattie et al, Molec. Biotechnol. 4:213-225, 1995; Beattie et al, Clin. Chem. 41 :700-706, 1995 ) .
  • Oli gonucleotides were synthe sized at Geno sys Biotechnologies (The Woodlands, TX) by means of the standard phosphoramidite procedure (49) and an efficient multiple synthesis strategy (Beattie & Frost, U.S. Patent No. 5, 175,209, 1992; Beattie et al, Appl. Biochem. Biotechnol. 10:510-521 , 1988 ; Beattie & Hurst, In Innovation and Perspectives in Solid Phase Synthesis, Proc. 3rd International Symposium on Solid Phase Synthesis, Epton, Ed., Mayflower Worldwide Ltd., Birmingham, U.K. , pp.
  • Phosphoramidites for introduction of 5 ' -amino linker into oligonucleotides were obtained from Glen Research (Sterling, VA). Glass microscope slides were epoxysilanized for probe attachment as previously described (Beattie et al, Molec. Biotechnol. 4:213-225, 1995 ; Beattie et al, Clin. Chem. 41 :700-706, 1995). Oligonucleotide probes containing 5 '-terminal amino modification were dissolved in H2O to a final concentration of 20 ⁇ M, and 200 nL droplets of each probe were applied to the epoxysilanized glass slides using a Hamilton Microlab 2200 station equipped with a multiprobe head. Rows of three droplets of each probe were attached to observe the reproducibility of the results. Before hybridization the slides were soaked for 1 hr at room temperature with blocking agent (lOmM tripolyphosphate) and then rinsed with water and air dried.
  • blocking agent laOmM tripolyphosphate
  • Genomic DNA was isolated from peripheral blood leukocytes from normal individuals and from cystic fibrosis (CF) patients. Mutation ⁇ F508 was originally tested by Southern analysis. The haplotypes of allele ⁇ F508 in the DNA samples were confirmed by the mobility of the products formed by the polymerase chain reaction (PCR) in 10.5% polyacrilamide gel electrophoresis.
  • PCR polymerase chain reaction
  • CF163 5'-GCACAGTGGAAGAATTTCATTCTG (SEQ ID NO: 2)
  • CF164 5'-ACCTCTTCTAGTTGGCATGCTTTG (SEQ ID NO: 3 )
  • This fragment contains four of the most frequent sites of mutations causing cystic fibrosis, Q493X, ⁇ I507, ⁇ F508 and V520F (Tsui, Trends Genet. 8:392-398, 1992).
  • primer CF164 labeled with biotin at the 5' end was used.
  • the PCR product was processed with a Millipore (Bedford, MA) Ultrafree spin- ' filter (30,000 M r cutoff) to remove the excess of PCR primers, and the material retained was applied in two portions to AffiniTipTM Strep 20 columns (Genosys Biotechnol).
  • the single-stranded DNA fragment was eluted with 20 ⁇ L of 0.2 N NaOH per column and the solution was neutralized with 4 ⁇ l of IN HC1.
  • auxiliary oligonucleotides (CF164, CF168, CF169 and CF170) were preannealed to the single-stranded PCR fragment, as follows. Five pmol of oligonucleotide CF164 or CF170 was labeled by kinasing with an excess of ⁇ 2 P-ATP (23mM, specific activity 7000 Ci/mmol). Aliquots of single-stranded target DNA were annealed either with CF164, with all the auxiliary oligonucleotides, or with all the auxiliary oligonucleotides except CF164.
  • the annealing mixture contained 50 ⁇ l 20X SSC, 10 ⁇ l 1M Tris-HCl (pH 8.0), 3 ⁇ l 0.5M EDTA, one or more prelabeled auxiliary oligonucleotide (0.23 pmol each), 10 ⁇ l single-stranded target DNA, and HPLC-pure H 2 0 to 90 ⁇ l.
  • the mixture was incubated at 95°C for 5 min, 45°C for 5 min, then 6°C 5 min. Excess ⁇ 3 2 P-ATP was removed by microcentrifugation through an
  • oligonucleotides designed to produce partially duplex structure across the target sequence included CF164 (sequence listed above), CF168 (5'-ATGAAATTCTTCCACTGTGC (SEQ ID NO: 4)),
  • CF169 (5'-TTCTTTAATGGTGCCAGGCATAATCCAGGA (SEQ ID NO: 5)
  • CF170 (5'-GTATCTATATTCATCATAGGAA (SEQ ID NO: 6)). These oligonucleotides anneal to the single-stranded 138-base PCR product derived from exon 10 of the human CFTR gene.
  • Each of four possible CF mutations in the 138-b target was represented on the slide by a pair of probes, one complementary to the wild type allele and the other complementary to the mutant allele.
  • Each probe was derivatized to carry a primary amino group at the 5'- terminus, which covalently bound to the epoxysilanized glass.
  • the sequences of the eight 9-mer probes were as follows (the 5 '-amino group is denoted by the character, "@".
  • CF170 (CF13W)ttcgcagta@ (CF164) (CF13M) tcg a agta g@
  • the normal target sequence is shown in capital letters, with positions of mutations underlined.
  • the "forward primer” (CF163) is indicated ' at the 5 ' -end. Aligned below the target sequence are the auxiliary oligonucleotides and the glass-tethered probes with 5 '-amine denoted by " @ .”
  • the subscript letters in the probe sequences correspond to positions of mismatch with the normal target.
  • DNA samples were subjected to PCR amplification using primers CF163 and CF164 and the products were analyzed by electrophoresis in 10.5% polyacrylamide gels.
  • the DNA sample with homozygous ⁇ F508 mutation yielded a PCR product (lane 1) of greater mobility than the normal DNA (lane 2).
  • the heterozygous DNA sample yielded both bands plus a third band with lower mobility (lane 3).
  • the third band was positively amplified with nested primers, suggesting that the third band corresponds to a hybrid duplex DNA fragment formed by one strand of normal sequence paired with a ⁇ F508 strand (data not shown).
  • a minor band was seen (visible in lane 4) with electrophoretic mobility intermediate between that of CF164 and the target fragment. This band also increased with higher target concentration, suggesting that the target fragment contained a minor proportion of shorter PCR product, possibly due to primer dimer formation or alternative priming within the target.
  • this analysis reveals the quantity of target DNA needed to incorporate the labeled auxiliary oligonucleotide into the partially duplex form, prior to hybridization to the oligonucleotide probe array.
  • Array hybridization of labeled target molecules (prepared as described in Example 1) to oligonucleotide arrays tethered to glass slides (prepared as described in Example 1) was carried out in 3.3M tetramethylammonium chloride (TMAC) dissolved in 50mM Tris-HCl (pH 8.0), 2mM EDTA, 0.1 % (w/v) Na dodecyl sulfate and 10% (w/v) polyethylene glycol (Beattie et al, Molec. Biotechnol. 4:213-225, 1995; Beattie et al, Clin. Chem. 41 :700-706, 1995).
  • TMAC tetramethylammonium chloride
  • a humid environment such as a ' water bath
  • the normal target DNA preannealed with labeled CF 164 or with all four auxiliary oligonucleotides
  • the normal target DNA was hybridized to glass slides containing a variety of 9mer probes representing normal and mutant sequences in the CF target (displayed in Example 1 ).
  • the hybridization patterns are shown in Figure 4.
  • Hybridization signal intensities with the mutant probes varied from essentially undetectable for CF12M and CF13M to barely lower than for the corresponding wild-type probes (CF10M and CF1 1M), which is consistant with previous reports that mismatch discrimination depends on the type, location and number of mismatches between target and probe (Maskos & Southern, Nucl. Acids Res. 20: 1675- 1678, 1992; Mirzabekov, Trends Biotechnol. 12:27-32, 1994; Khrapko et al, FEB S ' Lett.
  • the hybridization of probes CF12W and CF12M with the wild-type target showed good signal with the wild-type probe and little or no signal with the mutant probe, as in the previous experiments.
  • the mutant probe (CF12M) gave slightly stronger ⁇ hybridization than the wild-type probe (CF12W), which produces one internal mismatch and one terminal mismatch with the normal target.
  • This hybridization intensity of the "mutant" probe (CF12M) with homyzygous mutant target would probably have been even stronger, except that a mistake in the design of the sequence of CF12M was made, under the incorrect assumption that the ⁇ F508 mutation is due to deletion of TTT (actually it is due to deletion of CTT).
  • Placement of oligonucleotide probe droplets onto glass slides was carried out manually, or with the aid of a Microlab 2200 fluid delivery robot (Hamilton, Reno, NV), as described previously (Beattie et al, Molec. Biotechnol. 4:213-225, 1995; Beattie et al, Clin. Chem. 41 :700-706, 1995).
  • Quantitation of 32 P-labeled target molecules bound across the hybridization array was carried out by autoradiography, followed by scanning of the X-ray films using a flat bed scanner. ⁇ 32 P-ATP (7,000 Ci/mmol, 100 ⁇ Ci/ ⁇ L) was obtained from ICN Radiochemicals (Irving, CA).
  • Microcon 3 filters were acquired from Amicon (Beverly, MA).
  • the oligonucleotides were synthesized at Genosys Biotechnologies (The Woodlands, TX) by means of the standard phosphoramidite procedure (Matteucci & Caruthers, J. Am. Chem. Soc. 103 :3185-91 , 1981 ), using a parallel synthesis strategy (Beattie & Frost, U.S. Patent No. 5, 175,209, 1992; Beattie et al, Appl. Biochem. Biotechnol. 10:510-521, 1988; Beattie & Hurst, In Innovation and Perspectives in Solid Phase Synthesis, Proc.
  • the "@” at the 3 '-end of probes denotes the amino modification used for attachment to glass slides.
  • the "P” at the 5 '-end of some probes denotes phosphorylation.
  • Hybridizations to oligonucleotide probes arrayed on glass slides were performed in 3.3M tetramethylammonium chloride (TMAC) plus 50mM Tris-HCl (pH 8.0), 2mM EDTA, 0.1 % (w/v) sodium dodecyl sulfate, 10% (w/v) polyethylene glycol, plus 20 ⁇ L (5 pmol) of the partially duplex target strand.
  • Hybridization was carried out for 3 hr at 25°C or at the temperature indicated, under saturated humidity conditions (within a water bath). After hybridization the slides were washed for 5 min by dipping into fresh hybridization buffer without PEG. Slides were air dried, wrapped in plastic film, and placed against X-ray film for autoradiography. Autoradiograms were converted to digital image using a flat bed scanner.
  • Example 4 it was shown that terminal mismatch discrimination using 9mer capture probes with the stacking oligomer preannealed to single-stranded PCR product was better when hybridization was carried out at 25°C than at 15°C.
  • the experiment of Fig. 8 (using target CF179) was repeated at 25, 30, 35, 40 and 45°C and the results are shown in Figure 9 A and 9B.
  • Fig. 9 A are shown the ⁇ hybridization dots after 3 hr hybridization at each temperature, followed by 5 min washing at the same temperature. The hybridization signal decreased with increasing temperature, while simultaneously, the mismatch discrimination increased and was very good at 45°C.
  • Figure 9B shows the results after continued washing for successive 1-hr periods at 30, 35, 40 and ending with 45°C. Because very little change was observed when the slides were washed at the lower temperatures (data not shown), only the results after the final 45 °C washing are shown. The major effect of extensive washing at the higher temperature was decreased hybridization signal; mismatch discrimination, as reflected by the relative intensity of different signals, was only minimally improved by extended washing. Apparently, in the stacking (tandem) hybridization system mismatch discrimination occurs primarily at the binding step (forward reaction), rather than during washing (dissociation reaction).
  • Figure 10 shows the results of an experiment similar to that of Fig. 8 (Example 6), except that the probe length was 8mer and 7mer (rather than 9mer) and the synthetic targets contained no internal mismatch with the probe.
  • Comparison of the upper image in Fig. 10 with the upper image in Fig. 8 gives a reasonable indication of the effect of probe length (9mer, 8mer, 7mer) at the 25°C hybridization temperature.
  • the probe ⁇ length decreased there was decreased hybridization signal but increased mismatch discrimination at the 5 '-end of the probe, adjacent to the stacking oligonucleotide.
  • synthetic templates 180-7G, 181-7G and 182-7G with the 7mer and 8mer probes there was likewise good mismatch discrimination, with the strongest hybridization occurring with the perfectly matched probe.
  • the 3 '-aminopropanol function used to tether the capture probes to the glass slides is indicated by "@ .”
  • Each of the three possible mismatches are represented, at positions 1, 2, 3, 4 and 5 from the 3 ' end of the stacking probe CF164 (equivalently, from the 5 '-end of the 7mer capture probe).
  • the synthetic target was preannealed with 5 ' - 32 P-labeled stacking probe CF164 as described in Example 5, then the mixture was hybridized at 25 °C with arrays of 7mer capture probes applied to microscope slides , washed, and analyzed by autoradiography as described in earlier examples. The data, shown in Fig.
  • a nucleic acid sequence amplification method such as the polymerase chain reaction (PCR), ligase chain reaction (LCR), etc.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Highly multiplexed PCR reactions can be used to prepare numerous specific fragments containing several thousand known polymorphic sites.
  • a large collection of target DNA fragments of high genetic complexity can be amplified from a tiny biological sample, and the tandem hybridization strategy of the disclosed invention can then be advantageously used to genotype the fragments, using the general strategy described below.
  • the genomic DNA is first extracted from the biological sample using standard procedures known to the ordinary artisan. Multiplex PCR is first carried out (using a mixture of PCR primers known to reproducibly amplify a multiplicity of specific genomic fragments) to prepare the desired genomic target sequences which contain the known DNA sequence polymorphisms. The products of individual PCR reactions (each containing from one to numerous primer pairs) may be mixed together to form a complex mixture of amplicons representing up to thousands of polymorphic markers. The PCR fragments can be made single-stranded prior to hybridization, to avoid the re-annealing of complementary target strands, which may compete with the hybridization of target strands to the arrayed capture probes.
  • Single-stranded PCR fragments may be generated by asymetric PCR, by Streptavidin affinity purification when one member of a pair of PCR primers is labeled with biotin, or by exonuclease treatment of duplex PCR fragments, during which half of the target strands are digested.
  • Labeled oligonucleotides, annealing to the target strands immediately adjacent to each polymorphic site, are next mixed with the single-stranded amplified fragments, to introduce the label into the target strands and to serve as the "stacking probes" in the hybridization analysis.
  • Each of these labeled stacking probes is designed to bind to a single, unique site in the entire collection of amplified target fragments. Additional labeled or unlabeled auxiliary oligonucleotides may be optionally annealed to the target strands on either side of the polymorphic site, to minimize secondary structure within the single- stranded target or to introduce additional label into target molecules.
  • a short “capture probe” is synthesized and tethered to the surface of the genosensor chip at a specific site.
  • the capture probes typically about 6-10 nucleotides in length, are designed to hybridize to the target strand in tandem with the labeled stacking probe, such that mismatched bases are at or near the junction between capture and stacking probes.
  • Each allele of a given sequence polymorphism is represented on the genosensor chip by an allele-specific capture probe.
  • two allele-specific capture probes are tethered to the hybridization substrate, each at a specific location in the array.
  • the capture probes may be tethered to glass surfaces via reaction of a 5'- terminal modification with the glass (such as described in Example 1) or via immobilization at the 3 '-end as described in Example 5.
  • oligonucleotide attachment methods cited herein, since many alternative immobilization methods have been described in the prior art which may equally serve in the auxiliary oligonucleotide/tandem hybridization strategy of the disclosed invention.
  • the practitioner is not restricted to immobilization of oligonucleotide probes to glass surfaces in order to implement the disclosed invention, since one skilled in the art may readily adapt any of the numerous attachment chemistries described in the prior art for linking synthethic DNA probes to solid supports, including plastics, silicon, gold, platinum, any silanized solid surface, polymer matrix, membranes, etc.
  • a cocktail of labeled stacking probes (and optionally, additional labeled or unlabeled auxiliary oligonucleotides) is mixed with the amplified DNA, then the mixture is hybridized to an array of immobilized short allele-specific capture probes under conditions described in Examples 2-9.
  • the resulting quantitative hybridization fingerprint reveals the allele status at each polymorphic site. For a given polymorphic site, the relative hybridization intensity at positions in the genosensor array containing the different allele-specific capture probes will reveal which alleles are present, and whether the sample is homozygous or heterozygous at the polymorphic site.
  • auxiliary oligonucleotides annealed to the nucleic acid target strand in ⁇ the region of the binding site of the surface-tethered capture probe, to minimize the formation of secondary or higher order structure (such as stem-loops or hairpins) which may make the target sequence unavailable for hybridization to the surface-tethered probe, and also to inhibit reannealing of complementary strands of a denatured duplex target fragment, which would likewise make the target sequence unavailable for hybridization to the capture probe.
  • This use of auxiliary oligonucleotides is therefore intended to facilitate the analysis of heat-denatured duplex DNA fragments by oligonucleotide hybridization, eliminating the need to physically isolate single- stranded target DNA prior to hybridization analysis.
  • auxiliary oligonucleotides to facilitate hybridization analysis of denatured duplex DNA is illustrated schematically in Figure 13.
  • two auxiliary oligonucleotides complementary to the target sequence on both sides of the hybridization site of the capture probe, are preannealed in molar excess to the heat-denatured DNA target.
  • the presence of a molar excess of these auxiliary oligonucleotides will impede the reannealing of complementary analyte strands, as well as minimizing secondary structure within the target strand as discussed above.
  • flanking auxiliary oligonucleotides may be labeled, and one or both of them may serve as "stacking probes," hybridizing in tandem with the capture probe.
  • the flanking auxiliary oligonucleotides may alternatively be added to the heat-denatured target DNA at about the time of initiation of the hybridization reaction to the surface-tethered capture probe.
  • the length of auxiliary oligonucleotides flanking the capture probe may vary over a wide range, depending on the genetic ' complexity of the DNA analyte. For analysis of a single denatured PCR products or a mixture of several PCR products (up to about ten), the flanking auxiliary oligonucleotides may be 7mer or longer.
  • flanking auxiliary oligonucleotides may be of length about lOmer to about 50mer but preferably of length selected to ensure binding to a unique site within a target sequence of a given genetic complexity, as disclosed previously.
  • the use of labeled stacking probes on both sides of the capture probe is conceptually related to the use of a single longer stacking probe (of length sufficient to ensure binding to the target sequence at a unique location), except that the longer stacking probe is divided into two sections, contiguously stacking to both sides of the capture probe.
  • the total length of contiguously stacked duplex DNA formed at the analysis site will be 21mer, similar to that formed using a 7mer capture probe plus a single 14mer stacking probe.
  • the inventors have achieved excellent mismatch discrimination using 7mer and 8mer capture probes, equal or better than achieved using a single stacking probe, and in this embodiment of the invention, best mismatch discrimination was achieved when the mismatch was located near the center of the capture probe.
  • the latter technique may be employed whether there is a single stacking probe or flanking stacking/auxiliary probes, and the length of the "complementary auxiliary oligonucleotide" is preferably at least equal to the combined length of stacking/auxiliary probes and capture probe.
  • the "complementary auxiliary oligonucleotide" is preferably at least 34 bases long, the length of contiguously stacked duplex region formed by capture and capture probes .
  • mRNA is extracted from the biological sample, converted to DNA using reverse transcriptase, then PCR or other amplification strategies can be used to prepare fragments whose relative abundance reflect the relative abundance of specific mRNA species in the original mRNA sample: Sequence-targeted multiplex PCR can be used to amplify one or more known sequences within each transcript; arbitrary sequence PCR can be used to prepare fragments representing a random subset of sequences within the mRNA population; or oligo(dT) primers can be used in combination with sequence-targeted or arbitrary sequence primers, to amplify 3 ' -untranslated regions of the mRNA population.
  • single-stranded PCR fragments may be generated prior to hybridization to the genosensor array.
  • preannealing of PCR fragments with a molar excess of multiple auxiliary oligonucleotides to prevent reannealing of the target in the region of analysis can be employed to enable direct analysis of duplex PCR fragments without isolation of single strands.
  • Each gene transcript is represented in the genosensor array by a short "capture probe” which is designed to hybridize to the corresponding target strand (derived from the relevant gene transcript) in tandem with the corresponding labeled “stacking probe.”
  • the capture probes and stacking probes can be designed to analyze a specific subset of genes, or a complete set of potential transcripts of an organism, depending on the purpose of the analysis and the extent to which sequence information is available for the organism of interest. This approach can also be used for mixed populations of organisms, provided that sequence information is known for the genes and species of interest.
  • nucleic acid analyses described in Examples 1-4 and 10-12 represent special circumstances, under which the quantity of nucleic acid available from a small biological sample is insufficient for direct genosensor analysis, and amplification of target strands by PCR or other target enhancement methods is therefore required. In many types of nucleic acid analysis, however, a sufficient quantity of nucleic acid will be available for direct analysis using the tandem hybridization strategy of the disclosed invention, without the need to perform a costly and time consuming initial step of DNA amplification.
  • Avoidance of PCR amplification also eliminates the occurrence of a variety of artifacts (eg., self-priming/primer dimer, random priming, amplification of contaminant sequences, and other causes of irreproducible or misleading results), which are well known problems in PCR applications.
  • Examples of nucleic acid sequence analyses that may be performed directly using the tandem hybridization strategy of the disclosed invention, without employing DNA amplification, include DNA marker analysis, genotyping, mutational screening, microbial identification and gene expression/mRNA profiling using nucleic acids extracted from cultured organisms, tissue biopsies, or biological samples collected from plentiful agricultural stocks or natural ecosystems.
  • the critical consideration which enables direct application of the disclosed tandem hybridization approach without DNA amplification is the appropriate design of the labeled stacking probes, so that the hybridization is specifically targeted to unique sites within nucleic acid analytes of high genetic complexity.
  • a nucleic acid sample such as total genomic DNA or bulk mRNA or cDNA derived therefrom
  • a collection of labeled "stacking probes" is synthesized, of sufficient length (typically about 10-30 bases, the exact length depending on the genetic complexity of the nucleic acid analyte) to ensure that each stacking probe anneals to a unique position within the entire nucleic acid sample.
  • the labeled stacking probes are added to the nucleic acid sample, and the mixture is hybridized with the array of short capture probes (typically about 6-10 bases in length), each designed to bind to a specific target sequence, in tandem with the appropriate stacking probe.
  • the capture probes may be designed to bind to the target on either the 5 '-side or 3 '-side of the tandemly hybridizing "stacking probe.”
  • the labeled stacking probes are typically preannealed to the nucleic acid target strands, but alternatively, may be added to the analyte at the beginning of hybridization to the oligonucleotide array, or even after application of analyte to the array.
  • the hybridization is carried out under conditions such that significant binding of the nucleic acid analyte to the oligonucleotide array will occur only if the capture probe and stacking probe hybridize in tandem with the target strand.
  • a 7mer capture probe will not form a stable duplex structure with isolated complementary sequences within a nucleic acid sample, even though the 7mer will likely have numerous complements within a sample of high genetic complexity, but the short capture probe will hybridize specifically with the nucleic acid target at the site uniquely placed into register for contiguous stacking hybridization, via annealing of a longer stacking probe.
  • the experimental strategy described above may be used with flat surface genosensor arrays if sufficient time is allowed for hybridization, considering the very low concentration of individual short sequences (combined length of tandemly hybridizing capture and stacking probes) within a nucleic acid sample of high genetic complexity.
  • a preferred hybridization substrate, however, for direct nucleic acid analysis using the disclosed tandem hybridization method is the flowthrough genosensor chip (Beattie et al, Clin. Chem. 41 :700- 706, 1995; Doktycz & Beattie, In Automated Technologies for Genome Characterization, Beugelsdiik, Ed., J. Wiley & Sons, Inc., pp.
  • nucleic acid analyte is flowed through a porous silicon or microchannel glass chip, in which the capture probes are tethered within patches of densely packed channels connecting the two faces of the chip.
  • the flowthrough configuration is particularly preferred for analysis of heat-denatured dilute solutions of nucleic acids, and offers improved sensitivity and dynamic range, compared with the flat surface genosensor configuration.
  • hybridization substrates of high effective surface area may likewise serve to facilitate analysis of complex nucleic acid mixtures using the disclosed tandem hybridization strategy, including rigid fritted materials, rigid or flexible membrane materials, layers of matted fibrous materials, woven fabric materials, micromachined silicon structures, porous plastics, polymer gel matrices, etc.
  • the sample is preferably fragmented by sonication, microwave treatment or by enzymatic or chemical degradation, mixed with the appropriate set of labeled stacking probes, then hybridized with the allele-specific capture probes arrayed across the genosensor chip.
  • the relative intensity of hybridization signals at each position across the chip reveals the allelic status at each site, and the procedure can be used to simultaneously analyze thousands of known DNA sequence variations.
  • mRNA is extracted from the biological sample, optionally converted to cDNA, heat-denatured and mixed with the desired set of gene-specific labeled stacking probes (typically of length 10-30 bases), then hybridized with the appropriate set of arrayed capture probes (typically of length 6- 10 bases) specifically designed to hybridize to the target strand in tandem with the labeled probes.
  • desired set of gene-specific labeled stacking probes typically of length 10-30 bases
  • arrayed capture probes typically of length 6- 10 bases
  • the labeled stacking probe is designed to anneal to sequences present in all target molecules, such as the poly(A) tail in mRNA or repetitive sequence elements in genomic DNA, then a single labeled stacking probe can be preannealed to the target nucleic acid, and the mixture hybridized to an array of surface-tethered capture probes which uniquely represent each sequence analyzed.
  • a universal labeled stacking probe comprising oligo(dT) (for direct analysis of poly(A) + mRNA) or oligo(dA) (for analysis of reverse-transcribed cDNA) plus 1 -3 redundant residues (mixture of all 4 bases or universal base analog) extending into the expressed sequence of mRNA or cDNA can be preannealed with the mRNA or cDNA, and the mixture hybridized to an array of transcript- specific capture probes designed to bind to the target in tandem with the universal labeled probe.
  • the capture probes are preferably longer (typically about 10- 15 bases) than when the stacking probe is gene-specific, to ensure that the capture probe binds to a unique transcript.
  • the required site-specificity of the hybridization analysis must be achieved using capture probes of length sufficient to enable each capture probe to hybridize uniquely to a single target sequence " among all expressed sequences present in the sample.
  • the relative hybridization intensity at each position in the array will then reflect the relative abundance of expressed sequences in the sample.
  • the universal labeled probe could contain a sequence (or mixture of closely related "consensus sequences") at the beginning or end of a repetitive sequence element such as SINE (including Alu), LINE, or "short tandem repeat” ("microsatellite”) sequence, plus a short redundant sequence (string of mixed bases or universal base, as in the polA/mRNA embodiment) to position the labeled probe at the junction of each member of the repetitive sequence element in genomic DNA.
  • SINE including Alu
  • LINE LINE
  • microsatellite short tandem repeat
  • string of mixed bases or universal base, as in the polA/mRNA embodiment to position the labeled probe at the junction of each member of the repetitive sequence element in genomic DNA.
  • the array of surface-tethered capture probes would then correspond to DNA sequence polymorphisms flanking the repetitive sequence elements. As in Example 14, the capture probes would need to be long enough to hybridize to a unique site within the genomic DNA.
  • the approach described above may be similarly used to analyze mitochondrial or chloroplast DNA in eukaryotic organisms, where stretches of conserved sequence are flanked by highly variable sequence in a high copy number extranuclear organelle.
  • the universal labeled tandem probes will be annealed to the conserved sequence and the allele-specific short capture probes will hybridize in tandem with the variable sequence adjacent to the universal stacking probes.
  • the tandem hybridization method can also be used to "unambiguously detect and identify bacterial, viral or other microbial species or strains on the basis of known, unique features of nucleic acid sequences.
  • a target amplification method such as multiplex PCR can first be used to prepare a collection of genomic regions known to contain unique sequences. If sufficient biological material is available, such as cultured cells or large clinical sample, the nucleic acids can be directly analyzed to identify the species or strain, without using PCR.
  • flat surface oligonucleotide arrays may be used for the tandem hybridization strategy with DNA targets of high genetic complexity, provided sufficient hybridization time is allowed, however a flowthrough hybridization substrate is preferable when the concentration of target sequence complementary to the surface- tethered capture probes is very low.
  • the analysis can be targeted to the well known highly variable regions of 16S ribosomal RNA genes, present in multiple copies per cell.
  • PCR primers targeted to conserved regions of bacterial 16S rRNA genes can be used to amplify 16S rRNA gene fragments from DNA extracted from essentially any bacteria.
  • the amplification step may be unnecessary; DNA or RNA extracted from the culture may be analyzed directly.
  • Labeled stacking probes of length sufficient to insure specific hybridization to the conserved 16S rRNA or rRNA gene sequence flanking the variable 16S rRNA or rRNA gene sequence, are mixed with the extracted DNA or RNA, then the mixture is hybridized to an array of species- or strain-specific capture probes, hybridizing to the target ' strands in tandem with the corresponding labeled stacking probes.
  • the analysis is preferably simplified by using a small number of "universal" or "group-specific" labeled stacking probes, designed to hybridize to conserved regions of 16S rRNA genes, immediately adjacent to the hypervariable regions to which the species- or strain- specific capture probes would be targeted.
  • the tandem hybridization strategy can also be used in viral nucleic acid analysis, for example, in the detection and typing of human papilloma virus from a tissue specimen.
  • multiplex PCR is first used to specifically amplify one or more fragments from any of the known HPV genotypes.
  • HPV type-specific combinations of labeled stacking probes and surface-tethered capture probes, designed such that each HPV genotype will bind to a specific site within the genosensor array, are then used to reveal the HPV genotype in a single hybridization assay.
  • the tandem hybridization approach of the disclosed invention may be used with "universal" oligonucleotide arrays containing all sequences of a given oligomer length, for example an array of all 4,096 hexamer (6mer) capture probes, all 16,384 heptamer (7mer) capture probes, all 65,536 octamer (8mer) capture probes, etc.
  • the "universal" array of capture probes to be used in the tandem hybridization strategy may also comprise a mixture of oligonucleotide lengths, selected from the sets of all sequences of each given length, to minimize the effects of base composition on duplex stability and thus enable any of the capture probes to function under a single hybridization condition.
  • a modified 7mer array may contain some 8mers and 9mers in which the A-rich, T-rich or AT-rich sequences contain one or more additional nucleotides added (onto the end opposite from that which will abut with the stacking probe) to increase the stability of their dupleces (formed with complementary target sequences) closer to that of the GC-rich 7mer capture probes within the "universal" array.
  • the "universal" array of capture probes may in addition be edited to remove sequences that are judged to be problematic or uninformative, such as repetitive sequences.
  • a universal array of capture probes is that a single array may be mass-produced and used for a large variety of nucleic acid analytes, together with labeled stacking probes designed for each type of assay.
  • a universal array of short capture probes may be used with a mixture of gene-specific labeled stacking probes (each designed to represent a known, unique coding sequence or open reading frame) to obtain a gene expression (transcriptional) profile using bulk mRNA or cDNA, whereby the quantitative hybridization pattern across the array reflects the relative abundance of each expressed sequence, thus the relative activity of different genes.
  • a universal capture probe array may be used with the universal labeled stacking probe mixture (as described in Example 14) which binds to the poly A tail of each mRNA (or to the polyT tail of the corresponding reverse transcribed cDNA.
  • a universal array of capture probes may be used for simultaneous analysis of numerous mutations or DNA sequence polymorphisms, using a mixture of labeled stacking probes, each designed to anneal to a target nucleic acid at a unique site, adjacent to the site of a known mutation or polymorphism.
  • allelic status for each mutational site or polymorphism will be revealed from the quantitative hybridization pattern.
  • a universal capture probe array may be used in combination with a universal stacking probe comprised of repetitive sequences (eg, SINE, STRP, etc.), to simultaneously analyze numerous DNA sequence polymorphisms flanking repetitive sequences in genomic DNA, as described in Example 14.
  • each of the stacking probes used herein are of longer length (than those employed by Mirzabekov), designed to hybridize at a single unique position within a nucleic acid analyte of high genetic complexity.
  • stacking probes used herein are added to the nucleic acid analyte prior to hybridization to the array, whereas those employed by Mirzabekov are added in one or more additional cycles of hybridization, following the initial binding of target strand to the array of capture probes.
  • long stacking probes (as disclosed herein) is expected to minimize the effects of base composition on duplex stability, improving the ability of all short capture probes within a univeral array to hybridize with a complementary sequence within the target nucleic acid, since the major duplex stability in the stacked configuration is derived from the stability of the longer stacking probe.
  • the tandem hybridization approach of the disclosed invention may be used to analyze the most ubiquitous type of DNA sequence polymorphism, STRPs, also known as “microsatellites” and “variable number tandem repeats” (VNTRs).
  • STRPs DNA sequence polymorphism
  • VNTRs variable number tandem repeats
  • FIG 14A illustrates schematically the use of allele-specific labeled stacking probes for STRP analysis.
  • An array of short (preferably 7mer-9mer) capture probes is used, representing the nonrepetitive (unique) sequences known to be flanking each of the STRP markers to be analyzed.
  • the univeral array of capture probes as disclosed in Example 16, may be used.
  • a labeled stacking probe For each STRP allele of the set of STRP markers to be analyzed, a labeled stacking probe is used, which contains a nonrepetitive (unique) sequence at one end (functioning to position the stacking probe at the opposite side of the STRP marker from the likewise "positioned" capture probe), plus a specific number of short tandem repeat elements.
  • binding of label corresponding to a given STRP allele
  • binding of label corresponding to a given STRP allele
  • each STRP marker is represented by a capture probe tethered to a specific location on the array, bearing a nonrepetitive sequence at one end of the marker, and each STRP allele is represented by a labeled stacking probe bearing a nonrepetitive sequence at the other end of the marker, plus a specific number of short tandem repeat units.
  • a capture probe bearing four repeat units would not stack contiguously with the stacking probe bearing four repeat units, thus there would be little or no binding of label to this array position (with the target bearing 10 repeat units), however, the label would be bound at a position in the array containing a capture probe bearing six repeat units, and if the target contained 8 repeat units, the label would bind (due to contiguous stacking) to the array position containing the capture probe with four repeat units, hybridized in tandem with a stacking probe bearing four repeat units.
  • each STRP marker allele is represented at a different position within the array of capture probes.
  • each marker analyzed will have a labeled stacking probe containing a nonrepetitive sequence flanking one side of the marker, and different alleles are detected from the position within the array (among the capture probes bearing variable number repeat units for that marker) to which label is bound.
  • arrays of surface-tethered capture probes are employed, wherein the quantitative hybridization pattern across the array reveals the desired sequence information about the nucleic acid analyte.
  • the stacking hybridization approach of the disclosed invention is equally applicable to "bead technology" in which different capture probe sequences are tethered to microspheres which are distinguishable by any measurable (detectable) unique physical or chemical property associated with each bead, such as size, shape, mass, spectral profile, chemical reactivity, electronic properties, etc.
  • FIGS 15A and 15B A specific example of this approach is illustrated in Figures 15A and 15B, wherein the FlowMetrix system of Luminex Corp., invloving flow cytometry with spectral analysis of color-coded latex microspheres (McDade and Fulton, Medical Device & Diagnostic Industry, April 1997) is employed to enable multiplex analysis of numerous hybridization reactions.
  • This Example is intended to illustrate only one possible combination of stacking hybridization with bead technology. It will be evident to one skilled in the art that as indicated above, other physical or chemical properties of the beads (besides luminescence spectral properties used in the FlowMetrix system) may be advantageously employed to distinguish and quantitate the binding of analyte nucleic acid to specific capture probes tethered to distinguishable beads.
  • Figure 15 A shows the basic approach to genotyping, mutational analysis and gene expression profiling.
  • the nucleic acid analyte is annealed with a labeled stacking probe, of sequence and length designed to bind to a unique position within the analyte nucleic acid.
  • Each short capture probe sequence is immobilized to a specific color-coded bead, and upon hybridization with the stacking probe/target strand, the label will bind to the bead only when the stacking probe is hybridized to the target sequence in tandem with the capture probe.
  • allele-specific capture probes are hybridized with genomic DNA (or mixture of PCR products) preannealed with a mixture of stacking probes (binding to the target DNA adjacent to a set of polymorphic or mutation-bearing sites).
  • genomic DNA or mixture of PCR products
  • stacking probes binding to the target DNA adjacent to a set of polymorphic or mutation-bearing sites.
  • the quantity of label associated with each color-coded bead (quantitatively determined using flow cytometry with spectral analysis of individual beads streaming past the detector window) will reveal the allele status at each marker or mutational site analyzed.
  • the relative level of label (from stacking probes) bound to each color-coded bead will provide a gene expression (transcriptional) profile.
  • the stacking probe must be labeled with a tag that is distinguishable from the spectral properties of color-coded beads.
  • the capture probes tethered to color-coded beads are of sequence and length necessary to bind uniquely to the desired regions of the analyte nucleic acid.
  • the mixture of bead-tethered capture probes are mixed with the analyte nucleic acid along with short labeled sequence-specific oligonucleotide probes, each designed to hybridize in tandem with a specific capture probe.
  • each expressed sequence is represented by a specific capture probe tethered to a color- coded bead, plus a labeled probe which hybridizes in tandem with the capture probe.
  • each polymorphic marker is represented by a sequence specific longer capture probe designed to stack contiguously with a shorter labeled probe (when hybridized with the DNA analyte) or with a number of allele-specific labeled probes.
  • each label from the shorter labeled probes
  • the level of each label (from the shorter labeled probes) bound to each color-coded bead will then reveal the allelic status at each polymorphic or mutation-bearing site.
  • a high degree of multiplexing is provided by the use of color-coded beads in the FlowMetrix system, for example thousands of different color codes can be distinguished using several fluorescent dyes mixed together in defined ratios at different levels, providing a large number of distinct spectral profiles.
  • the tandem hybridization approach can be used with bead technology as long as the labels associated with the stacking probes are distinguishable from those of the "coded" beads, and a wide variety of physical or chemical properties may be incorporated into the microspheres to enable alternative bead-identifying detection schemes.

Abstract

La présente invention concerne une nouvelle technique d'analyse de l'ADN génomique et des séquences exprimées, qui utilise des oligonucléotides auxiliaires, préalablement annelés avec l'acide nucléique monocaténaire cible pour former une molécule cible partiellement duplex. L'invention présente plusieurs avantages dans l'analyse de séquences d'acide nucléique par hybridation avec des matrices de 'génocapteurs' ou puces à ADN. L'invention concerne également un procédé permettant d'analyser et de comparer directement des modèles d'expression génique au niveau de la transcription dans différents échantillons cellulaires.
PCT/US1999/025693 1998-11-02 1999-11-02 Analyse d'acides nucleiques par hybridation en tandem ciblee sur des sequences WO2000026412A1 (fr)

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