EP1169474A1 - Ensemble d'oligonucl otides et ses m thodes d'utilisation - Google Patents

Ensemble d'oligonucl otides et ses m thodes d'utilisation

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Publication number
EP1169474A1
EP1169474A1 EP00901800A EP00901800A EP1169474A1 EP 1169474 A1 EP1169474 A1 EP 1169474A1 EP 00901800 A EP00901800 A EP 00901800A EP 00901800 A EP00901800 A EP 00901800A EP 1169474 A1 EP1169474 A1 EP 1169474A1
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EP
European Patent Office
Prior art keywords
complementary
oligonucleotides
targeting
oligonucleotide
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00901800A
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German (de)
English (en)
Inventor
Stephen Little
Neil James Gibson
David Mark Whitcombe
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AstraZeneca AB
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AstraZeneca AB
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Publication of EP1169474A1 publication Critical patent/EP1169474A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention is directed to an oligonucleotide array ('generic' array) consisting of a plurality of different oligonucleotides of predetermined sequence, attached to a solid surface at predetermined positionally distinct locations, characterised in that the oligonucleotides have substantially the same melting temperature (T m ).
  • the invention is also directed to probe and primer targeting polynucleotides which contain nucleotide sequences that that are complementary to different target nucleic acids from a test sample as well as to the oligonucleotides on the array.
  • the 'generic' array and targeting oligonucleotides prove useful in a number of hybridisation capture methods and assays such as, sequence identification by allele specific hybridisation, fingerprinting and genome typing, differential gene expression profile analysis, or in clinical diagnostic methods utilising for example, ARMS amplification primers.
  • the present invention is therefore also directed to methods or assays utilising such 'generic' arrays and, in particular, to the use of a multiplex amplification assay that utilises a plurality of ARMS primers possessing non-amplifiable tails in conjunction with a 'generic' array for detection amplification of variant nucleic acid sequences.
  • Microarray also termed hybridisation array, gene array or gene chip
  • hybridisation array gene array or gene chip
  • nucleic acid molecules attached to solid substrates at predefined locations in small areas and at high density are used, in conjunction with hybridisation reactions, for identifying and discriminating target nucleic acid sequences, has advanced rapidly in the past few years.
  • These chips or microarrays allow massive parallel data acquisition and are used, for example, in polymorphism detection, clinical mutation detection, expression monitoring, fingerprinting and sequencing. A variety of methods are currently available for making arrays of biological molecules.
  • WO 97/44134 also describes devices for delivery of small volumes of liquid (which may contain biological macromolecules) in a precise manner to produce microsized spots on a solid surface to generate a microarray.
  • International Publication No. WO 98/10858 also describes an apparatus for the automated synthesis of molecular arrays. Techniques exist for applying the oligonucleotides to the array at high density and for example, techniques exist for applying well in excess of 103 distinct polynucleotides per 1 cm 2 . Many of the advances in microarray technology concern increasing miniaturisation.
  • Oligonucleotide DNA arrays consisting of short oligonucleotides (e.g. typically 8mers to 20 mers) bound via their 3' termini to a solid surface such as glass or a silicon wafer have been proposed as tools for mutation detection or for the resequencing of genes (e.g. Chee et. al., Science, 1996, Vol. 274, pp 610 - 614; Drobyshev et. al., Gene, 1997, vol.188, pp 45-52).
  • the mechanism of analysing DNA sequences depends on the principles of allele specific hybridisation.
  • an oligonucleotide array is prepared with a set of overlapping oligonucleotide probes (e.g.
  • the target sequence is amplified using the polymerase chain reaction (PCR) and the products are then often transcribed into RNA which has incorporated therein a fluorescent label such as fluorescein-UTP.
  • PCR polymerase chain reaction
  • the labelled RNA is sheared into short fragments and then hybridised to the DNA array. The most stable hybrids form when the target sequence binds to its fully complementary sequence on the array.
  • Microarray technology also makes it possible to simultaneously study the expression of many thousands of genes in a single experiment. Differential expression profiles from, for example, normal versus diseased tissues or induced versus un-induced tissues can be obtained by hybridising the product of expressed mRNA to complementary nucleic acid at pre-defined locations on the array.
  • a time-course of expression of thousands of genes over several experiments from a single sample could be performed.
  • Analysis of gene expression in human tissue i.e. biopsy tissue
  • a comparison of levels of expression of various genes from patients with defined pathological disease conditions with normal patients enables an expression profile, characteristic of disease, to be created.
  • microarrays There are currently two approaches to analyse gene expression using microarrays. In the first approach, cDNA fragments, often generated by PCR, for each of the genes under study are attached to an array. Typically, mRNA isolated from the test samples (i.e. induced or un-induced) is reverse transcribed into cDNA with incorporation of a fluorescent label.
  • the cDNA is sheared and hybridised to the array.
  • the other test sample mRNA can be reverse transcribed with incorporation of a different fluorescent label to enable direct comparison of the expression level of each test gene on the same array (see WO 95/35505).
  • the second approach is similar to the first except that an oligonucleotide microarray is used. Because of the differences in hybridisation properties between short oligonucleotide probes, each gene must be represented by several oligonucleotides (typically 20 or more) on the chip. In addition, a partner control oligonucleotide identical to each oligonucleotide, except for one of the central nucleotides, is included on the array to serve as an internal control for hybridisation sensitivity.
  • each test gene must be represented by approximately 40 distinct oligonucleotides each at a different position on the array.
  • the advantage of oligonucleotide arrays over cDNA arrays concerns the shelf-life of the sample on the array.
  • a cDNA library prepared on an array is useable for weeks whereas, pre-prepared oligonucleotide arrays can be stored for considerably longer.
  • a solid support having immobilised thereon a plurality of oligonucleotides at pre-defined positionally distinct sites, characterised in that the sequence of each oligonucleotide that binds to its complementary sequence has substantially the same melting temperature (T .
  • a solid support having immobilised thereon a plurality of pre-selected oligonucleotides at pre-defined 5 positionally distinct sites, characterised in that the sequence of each oligonucleotide when bound to its complementary sequence has substantially the same melting temperature (T m ) as the other oligonucleotides on the support.
  • the oligonucleotides attached to the solid support are non- complementary with genomic DNA and non complementary with each other.
  • each single-stranded oligonucleotide immobilised on the solid support has, in increasing order of preference, a T m when bound to its complementary sequence, within 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, and 0.5°C of the average T m of all the oligonucleotides immobilised on the solid support.
  • solid support will possess melting temperatures within a range of 0 to 8°C of each other.
  • at least 90% of the oligonucleotides on the array have melting temperatures within 4°C, preferably within 2°C of each other.
  • the oligonucleotides immobilised on the solid support will possess melting temperatures within a range of 0 to 2°C of each other.
  • 25 number of oligonucleotides preferably less than 1-5%, more preferably less than 2% of the total number of oligonucleotides may fall outside these ranges or values.
  • T m The melting temperature (T m ) referred to herein, is defined as the temperature at which duplex DNA exists in a ratio of 50:50 in hybridised and dissociated form under equilibrium conditions.
  • the principal governing factors determining T m are sequence length
  • T m 81.5 - 16.6(log ⁇ 0 [Na+]) + 0.41(% G + C) - (600/N).
  • N chain length and [Na+] is the ionic strength of the hybridisation solution.
  • nucleotide can refer to nucleotides present in either DNA or RNA and thus includes nucleotides which incorporate adenine, cytosine, guanine, thymine and uracil as base, the sugar moiety being deoxyribose or ribose. It will be appreciated however that other modified bases capable of base pairing with one of the conventional bases, adenine, cytosine, guanine, thymine and uracil, may be used in the oligonucleotides, probes or primers employed in the present invention. Such modified bases include for example 8-azaguanine and hypoxanthine.
  • oligonucleotide as used herein is defined as a molecule comprised of two or more nucleotides (i.e. deoxyribonucleotides or ribonucleotides), preferably more than five. Its exact size will depend on many factors, such as the reaction temperature, salt concentration, the presence of denaturants such as formamide, and the degree of complementarity with the sequence to which the oligonucleotide is intended to hybridise.
  • capture oligonucleotides In operation, under any hybridisation conditions adopted, all of the oligonucleotides on the solid support (herein referred to as “capture oligonucleotides”) that are to be used for capture of a target sequence, exhibit approximately the same hybridisation stability with their cognate complementary sequence as the other pairs of oligonucleotide and complementary target sequence. This ensures that approximately equivalent amounts of target DNA are bound to the complementary oligonucleotide on the array at any particular time, facilitating quantitative analysis. As the T m of all the duplexed oligonucleotides will be substantially the same however, the optimum temperature for hybridisation can be adopted.
  • the optimum hybridisation temperature is generally carried out under conditions that are 5-10°C below the T m , with the hybridisation and subsequent washes carried out under stringent conditions.
  • the hybridisation temperature is controlled precisely, preferably to +2°C , more preferably to ⁇ 0.5°C or better, particularly when the hybridisable length of the capture oligonucleotides are small and there is a need to discriminate between two sequences that may only differ by a single nucleotide at one or other of the termini of the hybridisable sequence. It will be apparent that the capture oligonucleotides need not all be of the same length. Depending on their relative G-C content, two oligonucleotides of different lengths may nevertheless, have the same T m . Capture oligonucleotides of substantially the same length and G-C content are preferred however.
  • a solid support having immobilised thereon a plurality of pre-selected oligonucleotides at pre-defined sites, characterised in that the capture portion of all of the oligonucleotides are of substantially the same length and they all have substantially the same G-C content.
  • the length of each capture portion of the oligonucleotide immobilised on the solid support will be within or equal to 16, 12, 10, 8, 6, 5, 4, 3, 2, and 1 nucleotide(s) of the average length of all the capture portions of the oligonucleotides immobilised on the solid support.
  • the oligonucleotide capture portions will each be of a length that is within 0-8 nucleotides of each other.
  • each of the oligonucleotides immobilised on the solid support will have a G-C content within or equal to 25%, 20%, 15%, 10%, 10%, 5% and 2% of the average G-C content of all the immobilised oligonucleotides.
  • 9-10%) of all the oligonucleotides immobilised on the support will have a percentage G-C content within 10%) of the median value and the remainder will preferably be within 25 % of the median value. More preferably the percentage G-C content of the oligonucleotides immobilised on the solid support will be within 8% of each other.
  • the capture portion of all of the oligonucleotides are of the same length and have the same G-C content.
  • oligonucleotides on the solid support fall within the ranges for length and G-C content as defined above, it is envisaged that a small number of oligonucleotides preferably less than 1-5%, more preferably less than 2% of the total number of oligonucleotides may fall outside these ranges.
  • the presence of oligonucleotides on the solid support that possess capture sequences that fall outside the preferred sequence composition (i.e. length and G-C content) need not diminish the utility of the generic array particularly if these capture sequences are excluded from being used as capture molecules when the array is in use.
  • oligonucleotides immobilised on the solid support there at least 50 different oligonucleotides immobilised on the solid support, and in increasing order of preference, there are at least 100, 500, 1000, 5000, 10000 or more different oligonucleotides immobilised on the solid support. In the most preferred embodiment, there are between 50 and 500 different oligonucleotides immobilised on the solid support. In a further embodiment, the oligonucleotides are immobilised on the solid support at a density in the range of, in increasing order of preference, 1 to 1000 per cm 2 , 200 to 1000 per cm 2 , 200 to 500 per cm 2 , 1 to 200 per cm 2 , 1 to 50 per cm 2 , and 1 to 10 per cm 2 .
  • each distinct capture oligonucleotide is immobilised to the base of a well of a conventional microtitre plate.
  • the oligonucleotides at each pre-determined location on the solid support are unique, this is not essential. Duplicate, triplicate etc., representation of one, more or all capture oligonucleotides on the solid support (array) may be desired in order to detect replicate values.
  • the capture oligonucleotides attached to the solid support generally have a hybridisable sequence between 5 and 100 nucleotides in length.
  • the preferred length of hybridisable sequence is in the range of 10 - 50, more preferred is 20 -35, still more preferred is 12-30.
  • the hybridisable sequence is that portion of the capture oligonucleotide (capture portion) that is designed and available for hybrid formation with its complementary sequence.
  • Non-hybridisable sequence of the capture oligonucleotide might represent flanking or tether sequences. Tether sequences not only serve to anchor the oligonucleotide to the solid support but also serve to distance the hybridisable portion of the capture oligonucleotide from the solid support to alleviate steric interference.
  • each unique capture oligonucleotide on the array can either be designed manually, or can be designed using a computer program to generate random nucleotide sequences.
  • a suitable macro for randomly designing oligonucleotides is disclosed in example 2 herein. It is preferable that none of the capture oligonucleotides on the array (solid support) are capable of hybridising, under stringent hybridisation conditions adopted, to any of the sample target sequences. In this respect, it is preferred that none of the capture oligonucleotides are capable of hybridising with any part of the genome of the test organism under study, be this human, simian, bacterial, viral or the like.
  • all of the capture oligonucleotides on the array are artificial and lack complementarity to any known sequence from whatever origin. It is also preferred that none of the capture oligonucleotides cross-hybridise to any test sample nucleic acid. However, if one or more of the capture oligonucleotides on the array do bind to a test sample nucleic acid, this is not detrimental provided that it is known in advance so that any false positive result can be discounted.
  • the capture oligonucleotide molecules may be individually synthesised on a standard oligonucleotide synthesiser. These oligonucleotide (oligos) may then be attached to the substrate matrix by any of a variety of techniques known in the art such as by using photochemical reagents, such as disclosed in US Patent No. 4,542,102 and 4,713,326. US Patent No. 4,562,157 also describes a method of using photo-activatable cross-linking groups to immobilise pre-synthesised ligands on surfaces. Alternatively, the oligonucleotides can be synthesised directly onto the solid surface using photolithography techniques, such as disclosed in US Patent No.
  • Patents 8(5):503-508, 1998 When constructing an array with relatively few capture oligonucleotides, say 500 or less, or with long oligonucleotides say 30-mers or more it may be preferable to synthesise the oligos separately and affix them to the solid support later rather than in situ synthesis.
  • a method for the preparation of a generic oligonucleotide microarray of the invention comprising synthesising a plurality of different oligonucleotides and then affixing them to a solid support at a pre-defined location, wherein each oligonucleotide possesses substantially the same T m as the other oligonucleotides when annealed to its complementary sequence.
  • the oligonucleotides are synthesised on a standard oligonucleotide synthesiser such as an Applied Biosystems model 340A synthesiser.
  • a method for the preparation of a generic oligonucleotide microarray comprising directly synthesising onto a solid support at pre-defined positions a plurality of different oligonucleotides, each oligonucleotide possessing substantially the same T m as the other oligonucleotides when annealed to its complementary sequence.
  • said synthesis is by the photolithography technique as described in US Patent No. 5,143,854.
  • a tether/linker molecule to tether the capture oligonucleotides to the solid support.
  • Shchepinov et al. disclose the use of various amino group-containing phosphoramidite moieties to distance the capture oligonucleotide from the solid support and thus alleviate steric interference. They found that with a linker of at least 40 atoms in length they obtained up to 150-fold increased hybridisation yields.
  • the oligonucleotides are attached to the solid support via a tether molecule, such as disclosed in Shchepinov et al. (N.A.R. 25:1155-1161, 1997).
  • the novel array with its population of unique capture oligonucleotides is generally used in conjunction with targeting polynucleotide molecules (herein referred to as "targeting polynucleotides").
  • the targeting polynucleotides are comprised of two adjacent oligonucleotide sequences, optionally separated by a spacer molecule.
  • the first sequence of the targeting polynucleotide is complementary to one of the capture oligonucleotide sequences on the array.
  • the second sequence is complementary to or substantially complementary to the target sequence to be detected and can therefore act as a detection probe or as an amplification primer.
  • the targeting sequence (the second sequence) need not reflect (be precisely complementary to) the exact sequence of the target, the more closely it does reflect the exact sequence the better the binding during the annealing process.
  • polynucleotide as used herein is used to define a molecule composed often or more deoxyribonucleotides or ribonucleotides, preferably more than 25.
  • a polynucleotide 5 molecule may be made up of two or more oligonucleotide molecules.
  • adenosine triphosphate is complementary to uridine triphosphate or thymidine triphosphate and guanosine triphosphate is complementary to cytidine triphosphate. It is appreciated that whilst
  • 10 thymidine triphosphate and guanosine triphosphate may base pair under certain circumstances they are not regarded as complementary for the purposes of this specification. It will also be appreciated that whilst cytosine triphosphate and adenosine triphosphate may base pair under certain circumstances they are not regarded as complementary for the purposes of this specification. The same applies to cytosine triphosphate and uracil triphosphate.
  • Precision complementarity or "perfectly matched” as used herein, is in reference to the duplex that the poly- or oligonucleotide strands make with one another to form a double stranded structure such that every nucleotide in each strand undergoes Watson-Crick base pairing with a nucleotide on the other strand.
  • the term also encompasses the pairing of nucleoside analogues, such as deoxinosine, nucleotides with 2-aminopurine bases, and the
  • substantially complementary refers to poly- or oligonucleotide molecules (or strands) that, under suitable hybridisation conditions (i.e. with reduced stringency), have sufficient complementarity to specifically anneal together, i.e. to the
  • the number of mismatch nucleotides does not exceed 20%, more preferably 15%, and still more preferably
  • the targeting polynucleotide molecules may be individually synthesised on a standard oligonucleotide synthesiser.
  • the 5 ' portion is preferably blocked from acting as template for the polymerase enzyme.
  • This blocking can be effected by linking the 3' portion and the 5' portion of the polynucleotide in the opposite sense to one another, the 3' end being generally in the 5' -> 3' sense and the 5' end of the polynucleotide being in the 3' -» 5' sense, with linkage via their 5' termini.
  • the presence of the nucleotide sequence of the 5' portion in the opposite orientation prevents the polymerase enzyme from making a fully double stranded amplification product.
  • the 5' portion of the polynucleotide thus becomes a single stranded tail on the amplification product.
  • This single stranded tail (the 5' end portion) can then be utilised for capture of the amplification product onto the complementary capture oligonucleotide attached to a solid support.
  • the more preferred means of blocking the polymerisation agent is to incorporate a blocking moiety (as the spacer moiety) between the 5' portion and 3' portion of the targeting polynucleotide.
  • blocking moiety means any moiety which when linked, for example covalently linked, between the 3' portion and 5' portion of the polynucleotide is effective to inhibit and preferably prevent, more preferably completely prevent amplification (which term includes any detectable copying) beyond the polymerisation blocking moiety, thus leaving the amplification product with a single stranded tail which is the 5' portion of the polynucleotide.
  • blocking moieties may be envisaged for this purpose.
  • the polymerisation blocking moiety may comprise a bead, for example a polystyrene, glass or polyacrylamide bead or the polymerisation blocking moiety may comprise a transition metal such as for example iron, chromium, cobalt or nickel (for example in the form of a transition metal complex with the oligonucleotide tail and the target binding nucleotide moiety) or an element capable of substituting phosphorus such as for example arsenic, antimony or bismuth linked between the oligonucleotide tail and the target binding nucleotide moiety.
  • a transition metal such as for example iron, chromium, cobalt or nickel (for example in the form of a transition metal complex with the oligonucleotide tail and the target binding nucleotide moiety) or an element capable of substituting phosphorus such as for example arsenic, antimony or bismuth linked between the oligonucleotide tail and the target binding nucleotide moiety.
  • the blocking moiety might similarly involve substitution of the usual phosphate linking groups, for example where oxygen is replaced, leading to inter alia phosphorodithioates, phosphorothioates, methylphosphonates, phosphoramidates such as phosphormorpholidates, or other residues known per se.
  • Alternative blocking moieties include any 3'-deoxynucleotide not recognised by restriction endonucleases and seco nucleotides which have no 2'-3' bond in the sugar ring and are also not recognised by restriction endonucleases. Newton et al. (Nucleic Acids Research. 21(5):1155-1162, 1993) and EP-B-416817, describe tailed primers with blocking moieties, interposed between the tail and the target binding portion of the primer, that can be incorporated into the polynucleotides of this invention.
  • the spacer comprises a non-amplifiable blocking moiety such as hexethylene glycol (HEG) monomer, alone or combined with further nucleotides, more preferably alone.
  • HOG hexethylene glycol
  • the spacer could comprise material such as 2'-O-alkyl RNA which will not permit replication of a complementary strand by DNA polymerase enzymes that lack a reverse transcriptase function.
  • the targeting polynucleotides of the invention it is desirable that none of the targeting polynucleotides are capable of hybridising to each other. Naturally, it is also desirable that none of the tails (the 5' portions of the targeting polynucleotides) nor spacer moieties are capable of binding to any nucleic acid in the nucleic acid sample. If the capture hybridisation is to be effected in the presence of all the test sample nucleic acid, i.e. without separation of targeting polynucleotide bound nucleic acid from unbound nucleic acid, it is also desirable that none of the capture oligonucleotides on the solid support are capable of binding to any target nucleic acid in the original test sample.
  • each polynucleotide comprising a unique 3 ' portion substantially complementary to a unique target nucleic acid sequence which may be present in a sample, a 5' portion complementary to one of a group of pre-selected oligonucleotides that each possess substantially the same melting temperature (T m ) and are attached at pre-defined positions to a solid support, and optionally a spacer moiety interposed between said 3' portion and said 5' portion.
  • T m melting temperature
  • each of the unique 3' portions is precisely complementary with its cognate target sequence. It is to be expected however, that not all the target sequences, complementary to each and every unique 3' portion, will be present in a test sample.
  • each of the unique 5' portions from within the population of polynucleotides has substantially the same G-C content and substantially the same length as each of the other 5' end portions.
  • each of the unique 5' portions from within the population of polynucleotides has the same G-C content and length as each of the other 5' end portions.
  • the 3' portion of the targeting polynucleotide represents the target binding sequence.
  • This sequence can be of any length although it will preferably be between 8 and 60 nucleotides in length, more preferably between 12 and 35 nucleotides in length.
  • the target binding portion of the targeting polynucleotide sequence need not possess precise complementarity to the target sequence however, it must have sufficient complementarity (be substantially complementary) to bind specifically to the target sequence, that is to say under appropriate hybridisation stringency conditions the target binding region of the primer will hybridise to the target region (if present in the sample) to the exclusion of other regions.
  • nucleotide mismatches are incorporated into the target binding primer in order to assist in destabilising primer binding to incorrect target sequences.
  • the presence of certain mismatches need not however, prevent primer binding to the desired target template sequence.
  • the target binding portion of the targeting polynucleotides has precise complementarity to its target sequence.
  • target nucleotide sequence or “target nucleic acid” or “target sequence” as used herein means a nucleotide or nucleic acid sequence comprising the sequence to be detected by probe or amplified by primer.
  • a sample may contain as many as 60, for example 50, separate potential variant sequences.
  • Each variant sequence is a potential target sequence for probe hybridisation or primer amplification according to the invention disclosed herein.
  • each targeting oligonucleotide primer is accompanied by a companion primer which facilitates amplification of the target sequence interposed between the two primers according to amplification procedures such as polymerase chain reaction (PCR) or ligase chain reaction (LCR), well known in the art.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the spacer moiety interposed between the two hybridisable elements can serve to prevent steric interference of the two hybrid elements, these two elements being the hybrid duplex molecule consisting of the oligonucleotide targeting portion (the 3 ' portion) and its complementary sequence from the test sample, and the hybrid duplex consisting of the capture oligonucleotide on the array and the complementary sequence on the targeting oligonucleotide (the 5' portion).
  • the spacer moiety might consist of straight chain or branched alkyl groups, polyglycol residues of any desired number of repeating unit, or modified nucleotides such as 2'-deoxyribose or l 'napthalene-2'-deoxyribose may be interposed between the first and second portions of the targeting oligonucleotides so as to provide spatial distance between the capture hybrid and the target hybrid.
  • the capture hybrid refers to the duplex molecule formed by annealing the capture oligonucleotide to the 5' portion (the single stranded "tail") of the targeting polynucleotide. This targeting polynucleotide may or may not already have bound its target sequence.
  • the target hybrid refers to the duplex molecule formed by annealing of the target binding portion (the 3' end) of the targeting polynucleotide to its target sequence.
  • An advantage of the invention is that the target product, either probe-target hybrid or amplified product, has a single-stranded portion which can be hybridised without denaturation to the solid support containing the immobilised pre-selected capture oligonucleotide sequences.
  • Current array technology requires the target nucleic acids to be denatured or rendered single-stranded some other way, prior to capture on the array.
  • microarray and targeting polynucleotides of the invention are useful in any setting where it is desirable to identify the presence of one or more specific nucleic acid sequences from a population of sequences. Examples of uses are in de novo or re-sequencing methods, gene expression studies, fingerprinting, diagnostic identification, genotyping of organisms and environmental monitoring. Any population of nucleic acids represents a suitable test sample.
  • Sources of test sample nucleic acid include human cells such as circulating blood, buccal epithelial cells, cultured cells and tumour cells. Other mammalian tissues and cultured cells are also suitable sources of template nucleic acids.
  • viruses, bacteriophage, bacteria, fungi and other micro-organisms can be the source of nucleic acid for analysis.
  • the DNA may be genomic or it may be cloned in plasmids, bacteriophage, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) or other vectors.
  • RNA may be isolated directly from the relevant cells or it may be produced by in vitro priming from a suitable RNA promoter or by in vitro transcription.
  • the present invention may be used for the detection of variation in genomic DNA whether human, animal or other. It finds particular use in the analysis of inherited or acquired diseases or disorders. A particular use is in the detection of inherited disease. It will be appreciated that the target nucleic acid is directly or indirectly linked to the sequence or region of interest for analysis.
  • a method for identifying the presence or absence of one or more test nucleic acid sequences in a sample comprising the following steps: i) contacting a nucleic acid containing sample with a plurality of single stranded targeting polynucleotide molecules under suitable hybridisation conditions to ensure hybrid formation between the targeting nucleotide portion of the targeting polynucleotide molecule and its complementary target nucleic acid sequence in the sample, each of said targeting polynucleotide molecules possessing, in addition to the targeting nucleotide portion, a unique single-stranded oligonucleotide tail sequence complementary to a unique capture oligonucleotide attached to a solid support; ii) contacting the population of hybrid molecules produced in step (i) to a solid support having attached thereon at pre-defined locations unique capture oligonucleotides, each capture oligonucleotide being complementary to one or other of the oligonucleotide
  • a method for identifying the presence or absence of one or more test nucleic acid sequences in a sample comprising: i) contacting a nucleic acid containing sample with a plurality of single stranded targeting polynucleotide molecules under suitable hybridisation conditions to ensure hybrid formation between the targeting nucleotide portion of the targeting polynucleotide molecule and its complementary target nucleic acid sequence in the sample, each of said targeting polynucleotide molecules possessing, in addition to the targeting nucleotide portion, a unique single-stranded oligonucleotide tail sequence complementary to a unique capture oligonucleotide sequence attached to a solid support, characterised in that substantially all of the oligonucleotides the capture sequence of each oligonucleotide and its complementary sequences on the tail possess substantially the same T m ; ii) contacting the population of hybrid molecules produced in step (i) to a solid support having attached
  • the hybrid molecules produced in step (i) are separated from the unhybridised targeting molecules prior to step (ii). This may be conveniently done by, for example, ethanol precipitation, column chromatography or gel filtration.
  • the targeting polynucleotide binds to the target nucleic acid in the sample ("the wet reaction"). If the targeting polynucleotide is serving as a probe, for example as in an allele specific hybridisation, the hybrid molecule is available for capture via the unhybridised single stranded tail portion of the targeting polynucleotide which is complementary to a capture oligonucleotide attached at pre-defined position on the solid support. In a preferred embodiment the targeting polynucleotide possesses a detectable label. If the targeting polynucleotide is serving as an amplification primer, an amplification reaction must be carried out on the polynucleotide-bound target nucleic acid sample.
  • the targeting polynucleotide is serving as an amplification primer in an amplification system such as the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the target binding region (3' end) and the tail region (5' end) are advantageously arranged such that the tail region is non-amplifiable in the PCR amplification reaction but remains single stranded, thus ensuring that the amplified product has at least one single stranded tail complementary to a capture oligonucleotide on the solid.
  • This facet of primer design is described in European Patent No. 0 416 817 and corresponding US Patent No. 5525494.
  • This second oligonucleotide primer may also have a non-amplifiable single stranded tail, possibly identical to the capture portion of the targeting polynucleotide so as to facilitate capture on the solid support.
  • This second primer might also be suitably labelled to facilitate detection of the captured amplification product on the solid support.
  • a suitable label (such as a labelled nucleoside tri-phosphate) might be incorporated into the amplified product during the amplification process.
  • Any convenient template dependent polymerase may be used, this is preferably a thermostable polymerase enzyme such as TaqTM, more preferably Taq GoldTM.
  • any convenient nucleoside triphosphates for conventional base pairing may be used. If required these may be modified for fluorescence. As these may affect polymerisation rates, for best results, the fluorescently labelled dNTPs are admixed with an excess of wild-type dNTPs, for example in an admixture of between about 1 :3 and 1:20. Further details of convenient polymerases, nucleoside triphosphates, other PCR reagents, primer design, instruments and consumables are given in "PCR” by C.R. Newton and A. Graham (The Introduction to Biotechniques series, Second Edition 1997, ISBN 1 85996 011 1, Bios Scientific Publishers Limited, Oxford).
  • the targeting polynucleotide molecules are amplification primers, such as those disclosed in EP-B-416817.
  • the targeting amplification primer molecules are used in conjunction with another amplification primer to amplify the target region of interest.
  • the amplification primers are amplification refractory mutation system (ARMS) primers, as described in EP-B-0332435 and corresponding US Patent No. 5595890, and additionally described in Newton et al. (Nucleic Acids Research.
  • ARMS is a technique suitable for detecting the presence or absence of variant nucleotides at a particular loci. It is particularly useful therefore, in diagnostic detection of mutated nucleic acid indicative of tumour phenotype, or in the detection of single nucleotide polymorphisms (SNPs). ARMS is a particularly useful technique where the target sequence is present in low copy number or there is a need to discriminate between two or more alleles, as for example in mutation detection. ARMS mutation detection enables the sensitive detection of specific alleles in the presence of an excess of alternate alleles. In this way somatic mutations can be detected in a background of wild type DNA.
  • ARMS can readily be used to detect 1% mutant sequence in a 99% wild-type background.
  • ARMS uses primers that allow amplification in an allele specific manner. Allele specificity is provided by the complementarity of the 3'- terminal base of a primer with its' respective allele. Amplification is inhibited when the 3'- terminal base of the primer is mismatched. This specificity is maintained when Taq DNA polymerase or other suitable enzyme lacking 3' to 5' proof-reading activity (such as Klenow) is used.
  • An ARMS test is specific when the yield of product from the target allele exceeds the threshold of detection of the system in use and the yield of product from the nontarget allele is not detectable.
  • the ARMS primers will preferably possess destabilising mismatches incorporated close to the 3 '-terminal nucleotide that discriminates between the different alleles, to enhance specific binding and template amplification from the desired allele target sequence.
  • the second amplification primer for amplifying any particular target sequence possess an oligonucleotide tail identical to that of the first targeting primer.
  • the second amplification primer possesses a detectable label, such as a fluorophor or radioisotope to enable the eventual detection of the amplified product on the microarray.
  • either or both primer molecules possess a detectable label.
  • Suitable labelling molecules are well known in the art.
  • a suitable label can be incorporated into the amplified product during its synthesis. A suitable amplification reaction can then be performed so as to generate an amplification product.
  • the target nucleic acids is preferably labelled in order to be able to discriminate targeting polynucleotides bound to the capture oligonucleotides on the array that have target nucleic acid attached from those that do not.
  • the nucleic acid are degraded to form fragments, degradation is preferably random using for example sonication or shearing, to generate average lengths of target nucleic acid around the lengths of the complementary sequences on the targeting oligonucleotides. These fragments can then be labelled.
  • any number of conventional detectable markers such as radioisotopes, fluorescent labels, chemi luminescent compounds, labelled binding proteins, magnetic labels, spectroscopic markers and linked enzymes might be used.
  • One particular example well known in the art is end-labelling with 3 2 P.
  • Fluorescent labels are preferred because they are less hazardous than radiolabels, they provide a strong signal with low background and various different fluorophors capable of absorbing light at different wavelengths and/or giving off different colour signals exist to enable comparative analysis in the same analysis. For example, fluorescein gives off a green colour, rhodamine gives off a red colour and both together give off a yellow colour.
  • target bound targeting oligonucleotides are not separated from unbound targeting oligonucleotides (following step (i) of the method disclosed herein), and the target nucleic acid is not specifically labelled, other means of discriminating between those captured targeting oligonucleotides that have bound their cognate target sequence from the test sample from those that have not bound their test sample will be required. Suitable means for doing this include the use of intercalating agents (i.e. dyes such as ethidium bromide) that become incorporated into duplex nucleic acid or the use of labelled binding proteins or antibodies or other reagents that recognise helix formation (such as the target nucleic acid/targeting oligonucleotide hybrid), see for example US Patent No.
  • intercalating agents i.e. dyes such as ethidium bromide
  • a ligand binding to the minor groove such as Hoechst 33258 fluorescent dye or the use of fluorescently labelled ligands which recognise the minor groove of DNA in a sequence specific manner, see for example, "Recognition of the Four Watson- Crick Base Pairs in the DNA Minor Groove by Synthetic Ligands.” S. White, J. W. Szewczyk, J. M. Turner, E. E. Baird and P. B. Dervan, Nature, 391, 468 (1998). Convenient intercalators will be apparent to the person skilled in the art (cf Higuchi et al. BioTechnology. 10:413-417, 1992).
  • capture of the hybrid detection product by the oligonucleotide on the solid support is detected using one or more minor groove binding probes.
  • Further systems include two-component systems where a signal is created or abolished when the two components are brought into close proximity with one another. Alternatively, a signal is created or abolished when the two components are separated following binding of the target binding region.
  • Both elements of the two component system may be provided on the same or different molecules. By way of example the elements are placed on different molecules, target specific binding displaces one of the molecules into solution leading to a detectable signal.
  • One of the components may be attached to the capture oligonucleotide, or the solid support itself.
  • the array could consist of fluorescein labelled oligonucleotides of, for example 20 residues in length.
  • a set of short quencher oligonucleotides (say 10 residues in length) complementary to the array and labelled with DABCYL (or methyl red) could be added to the array.
  • the short complementary DABCYL oligonucleotides bind to the corresponding 'address' on the array and the fluorescence of the fluorescein labels is quenched.
  • the sample is then purified so that unextended primers or unbound probes are separated from extended or bound products.
  • the bound or amplified products are then added to the microarray and the tail portions which are fully complementary to the oligonucleotides on the microarray bind to the microarray with displacement of the quencher oligonucleotides.
  • microarray oligonucleotides fluorescing as a result of the binding of the appropriate product (see Figure 1).
  • this format a fluorescent signal is produced by separating two species bound to the array surface.
  • One advantage of this format is that it permits quality control of the array.
  • the fluorescent array When the fluorescent array is manufactured it can be scanned in a fluorescent scanner and any defects such as a probe oligonucleotide which has failed to attach to the surface will be detected as a non or weakly fluorescent spot on the array. Efficient quenching when the quencher oligonucleotides are added can also be monitored before the test products are added.
  • Convenient two-component systems may be based on the use of energy transfer, for example between a fluorophore and a quencher.
  • the detection system comprises a fluorophore/quencher pair.
  • Convenient and preferred attachment points for energy transfer partners may be determined by routine experimentation.
  • a number of convenient fluorophore/quencher pairs are detailed in the literature (for example Glazer et al, Current Opinion in Biotechnology. 8:94-102, 1997,) and in catalogues such as those from Molecular Probes, Glen and Applied Biosystems (ABI). Any fluorescent molecule is suitable for signalling provided it may be detected on the instrumentation available.
  • the quencher must be able to quench the dye in question and this may be via a Fluorescence Resonance Energy Transfer (FRET) mechanism involving a second, receptor fluorophore, or more preferably via a collisional mechanism involving a non- fluorogenic quencher such as DABCYL, which is a "Universal" quencher of fluorescence or methyl red.
  • FRET Fluorescence Resonance Energy Transfer
  • the selected fluorophores and quenchers are incorporated, most conveniently via phosphoramidite chemistry, into the capture oligonucleotides and/or targeting polynucleotides and/or second primer required for example, when undertaking PCR-based amplification.
  • FAM a fluorescein dye with an excitation optimum at ⁇ 490nm, is a convenient donor.
  • the oligonucleotides on the support are detectably labelled.
  • a further embodiment consists of a fluorescently labelled DNA array where the array oligonucleotides are labelled with a fluorophore which either does not fluoresce at the irradiation frequency or is only weakly fluorescent at this frequency.
  • the ARMS primers are labelled with a fluorophore which is substantially fluorescent at the irradiation frequency and which forms an energy transfer pair with the fluorophore label on the DNA array.
  • the array fluorophore at the specific binding sites increase substantially in its fluorescent brightness and this may be detected by scanning the array. This is an example of increasing the fluorescent signal on the array by bringing two species close together by hybridisation.
  • the oligonucleotide microarrays and targeting polynucleotides of the invention can be used for large scale hybridisation assays in numerous applications, including genetic and physical mapping of genomes, gene expression studies, sequencing, fingerprinting and genotype mapping, genetic diagnosis and environmental monitoring.
  • the microarray, targeting polynucleotides and method of the invention are particularly suitable for differential gene expression studies.
  • RNA can be isolated from a cell or cell population and labelled, for example by attaching a fluorescent molecule to isolated RNA or by end labelling using T4 polynucleotide kinase.
  • mRNA can be reverse transcribed into cDNA and a suitable label incorporated during cDNA synthesis. Fragmentation of these labelled molecules can then precede hybridisation to the targeting polynucleotides prior to capture on the generic array of the invention.
  • different fluorescent labels for example Cy3(green) or Cy5(red)-labelled deoxyuridine triphosphate
  • test samples i.e. induced versus un-induced
  • each target gene must be represented by several oligonucleotides (typically 20 or more) on the chip.
  • a partner control oligonucleotide identical to each oligonucleotide, except for one of the central nucleotides, is included on the array to serve as an internal control for hybridisation sensitivity.
  • each test gene must be represented by at least 40 distinct oligonucleotides each at a different position on the array.
  • a suitable sample might be a tissue sample, for example a biopsy or bodily fluid, such as blood, sample, or a cell sample, for example epithelial or buccal cells, or a cultured cell or cell line such as a mammalian cell or cell line, or a bacterial or yeast cell. Alternatively, it may be from a whole organism, such as Arabidopsis thaliana. Any sample containing one or a plurality of different genes is suitable.
  • a method for quantifying the expression level of a gene comprising: (i) converting mRNA from a test sample into cDNA;
  • step (iii) contacting the cDNA with a plurality of targeting polynucleotide molecules under suitable conditions to allow hybridisation between substantially complementary sequences to occur, each polynucleotide molecule comprising a unique 3 ' portion substantially complementary to a unique region of the cDNA, a 5' tail portion complementary to one of a group of pre-selected oligonucleotides that are attached at pre-defined positions to a solid support, and optionally a spacer moiety interposed between said 3' and 5' portions; (iv) contacting the components from step (iii) with a substrate on which is immobilised at pre-determined positions a plurality of capture oligonucleotide sequences each complementary to one or other of the 5' tail portions of the targeting polynucleotides so as to allow the tailed cDNA/targeting polynucleotide duplex molecules to bind to their complementary capture oligonucleotide on the substrate; and
  • step (v) detecting the amount of bound cDNA/targeting polynucleotide duplex at each position on the substrate.
  • unhybridised targeting polynucleotides are removed from the reaction mixture after step (iii).
  • the sub-set of targeting polynucleotides directed to a specific gene all possess the same 5' tail portion sequence so that all targeting polynucleotide/cDNA duplex molecules formed can be captured at the same location (by the same oligonucleotide) on the support (array).
  • the method is used to determine expression levels of various genes in a test sample, each gene capable of being detected by a different sub-set of targeting polynucleotides and addressed to distinct positions on the support.
  • the cDNA generated in step (i) is detectably labelled.
  • the gene or each gene to be detected, as represented by cDNA molecules produced in step (i) is detected by between 5 and 80 distinct polynucleotides that bind at distinct parts of the gene.
  • a method for identifying the differential expression of each of a plurality of genes in a first cell type with respect to expression of the same genes in a second cell type comprising:
  • each polynucleotide molecule comprising a unique 3' portion substantially complementary to a unique target nucleic acid sequence which may be present in a sample, a 5' tail portion complementary to one of a group of pre-selected oligonucleotides that each possess substantially the same melting temperature (T m ) and are attached at pre-defined positions to a solid support, and optionally a spacer moiety interposed between said 3 ' portion and said 5 ' portion, (iv) detectably hybridising the hybridised products from step (iii) to a solid surface on which is immobilised at pre-determined positions a plurality of oligonucleotide sequences complementary to one or other of the 5' tail portions of the targeting polynucleotides; and (v) examining the solid support by fluorescence under fluorescence excitation conditions to detect
  • a method for identifying the differential expression of each of a plurality of genes in a first cell type with respect to expression of the same genes in a second cell type comprising: (i) isolating mRNA from each cell type and converting said mRNA into cDNA with incorporation of a different fluorescent label into the newly synthesised cDNA for each cell type; (ii) optionally, fragmenting said newly synthesised cDNA into appropriate length nucleic acid fragments; (iii) contacting said labelled nucleic acid with a plurality of targeting polynucleotide molecules under suitable conditions to effect hybridisation between substantially complementary sequences, each polynucleotide molecule comprising a unique 3' portion substantially complementary to a unique target nucleic acid sequence which may be present in a sample, a 5' tail portion complementary to one of a group of pre-selected oligonucleotides that each possess substantially the same melting temperature (T m ) and
  • each target gene is detected by a plurality of targeting polynucleotides that bind at distinct parts of the target gene.
  • Each range being a separate and independent embodiment of the invention.
  • each of the targeting polynucleotide for a particular gene possesses an identical single stranded oligonucleotide tail portion capable of capturing the target:polynucleotide hybrid molecule onto a solid support at a pre-defined position.
  • sequence location 200-230 of gene X isolated from normal tissue sample is captured at position A whereas sequence location 200-230 of gene X isolated from tumour tissue is captured at position B.
  • sequence location 350-380 of gene X isolated from normal tissue sample can either also be captured at location A (to pool all the results for one gene from one cell type at one site) or at its own unique location C.
  • sequence location 350-380 of gene X isolated from tumour tissue can either be captured at position B or position D.
  • the nucleotide sequence of each of the capture portions of the targeting polynucleotides and complementary sequences on the capture oligonucleotides on the solid support have substantially the same ⁇ m .
  • hybridisation conditions are adopted to ensure maximum annealing of the targeting polynucleotides to their target sequences. This may be effected by pooling together those targeting polynucleotides whose target binding sequence possesses on binding to its target approximately the same T m as the others in the pool. More preferably, the target binding portion of each targeting polynucleotide in a particular pool is of the same nucleotide length and has the same G-C content as the others in the pool. According to a preferred embodiment therefore, each pool of targeting polynucleotides is hybridised to a portion of the test nucleic acid sample under optimum conditions for hybrid formation with their respective target sequences. The hybridisation reactions from each pool of targeting polynucleotides are then pooled together. Following this, capture of the target bound sequences onto the solid support is effected using hybridisation conditions adapted according to the particular T m of the capture duplexes.
  • hybridisation conditions chosen are designed to be as close as possible to the T m of the duplexes.
  • concentration of salt in the hybridisation solution used is particularly significant.
  • G:C base pairs are more stable than A:T base pairs.
  • double stranded oligonucleotides with a higher G-C content have a higher T m than those of the same length but with a higher A-T content. If slight differences, i.e. single nucleotide differences, amongst the target nucleic acids need to be distinguished, establishing optimum hybridisation conditions is important, particularly, when the hybridisable length of the oligonucleotides is small ( ⁇ approximately 30-mers).
  • Tetramethylammoniumchloride is particularly suitable when used at concentrations of between 2M and 5.5M. A preferred concentration range being 3M - 4M. Compared to the presence of 1M NaCl in the hybridisation solution, use of up to 5M TMAC1 can enhance hybridisation specificity by up to 40-fold.
  • a preferred means of ensuring maximum hybrid formation despite there being a range of T m due to target sequence composition is to divide the population of targeting polynucleotides into groups according to their optimum T m , and then to undertake separate hybridisation reactions using sub-groups of pooled targeting polynucleotides that are grouped according to the T m of the targeting polynucleotide :target sequence hybrid portion.
  • Each hybridisation can then be carried out under optimum hybridisation conditions for the particular group of targeting polynucleotides. In this manner, optimum hybridisation conditions can be adopted which will ensure approximately equivalent duplex formation. This may be of particular importance if quantitative analysis is required.
  • the products from the different hybridisation reactions can then be pooled ready for capture hybridisation to the solid support (Reaction B, see below).
  • An example of a suitable hybridisation solution involving oligonucleotides of between 15 and 50 nucleotides is: 3M TMAC1, 0.01M sodium phosphate (pH 6.8), ImM EDTA(pH 7.6), 0.5% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA and 0.1% dried skimmed milk (i.e. MarvelTM).
  • hybridisation A involves hybrid formation between the targeting polynucleotides and the target nucleic acid in the test sample ("the wet reaction").
  • B involves hybrid formation between the targeting polynucleotides and the capture oligonucleotides bound to the solid support.
  • the hybridisations can be carried out in either order or together, although it is preferred that the wet reaction A is carried out prior to reaction B. With differential expression studies it is preferred that hybridisation A be carried out under conditions that ensure maximal hybridisation of the targeting polynucleotides to target sequence.
  • This invention is concerned with a novel microarray of universal use comprising a plurality of oligonucleotides, each possessing substantially the same T m , attached at predefined positions to a solid support.
  • solid support is well known in the art.
  • any conceivable solid substrate may be employed in the invention.
  • a suitable substrate is a material having a rigid or semi-rigid surface, generally insoluble in a solvent of interest such as water.
  • Specific suitable substrates are glass, plastics, polymers, polysaccharides, resins, metal, silica or silica-based material, nylon or nitrocellulose filters, and the like.
  • the solid support may comprise a single sheet of a suitable material such as glass, silicon or plastic so that the pre-selected oligonucleotides are positioned at pre-defined sites based on each oligonucleotide having a distinct and distinguishable set of x,y co-ordinates.
  • the solid support may comprise a set of beads of a suitable material such as glass or plastic so that the pre-selected oligonucleotides are positioned at pre-defined sites based on each oligonucleotide residing on a distinct bead.
  • the substrate and/or its surface will be flat glass or single-crystal silicon.
  • the surface of the substrate will preferably contain reactive groups such as carboxyl, amino, hydroxyl, or the like.
  • a polycationic polymer such as polylysine is particularly useful.
  • the surface is non-fluorescent at the wavelength that the analysis is to be performed.
  • the surface of the substrate is also preferably provided with a layer of cross- linking groups to assist attachment of the oligonucleotides to the support. These cross-linking groups will preferably be of sufficient length to permit the oligonucleotides attached to interact freely with their binding partners in solution.
  • Crosslinking groups may be selected from any suitable class of compounds, for example, aryl acetylenes, ethylene glycol oligomers containing 2-1 monomer units, diamines, diacids, amino acids, and the like.
  • the cross-linking groups may be attached by a variety of methods which are readily apparent to the person skilled in the art. For example, by esterification or amidation reactions of an activated ester of the linking group with a reactive hydroxyl or amine group on the free end of the cross-linking group.
  • the detection of specific interactions may be performed by detecting the positions where the labelled target sequences are attached to the array.
  • Radiolabelled probes can be detected using conventional autoradiography techniques. Use of scanning autoradiography with a digitised scanner and suitable software for analysing the results is preferred.
  • the label is a fluorescent label
  • the apparatus described e.g in International Publication No. WO 90/15070, US Patent No. 5, 143,854 or US Patent No. 5,744,305 may be advantageously applied. Indeed, most array formats use fluorescent readouts to detect labelled capture: target duplex formation. Laser confocal fluorescence microscopy is another technique routinely in use (M.J.Kozal et al., Nature Medicine. 2:753-759, 1996).
  • Mass spectrometry may also be used to detect oligonucleotides bound to a DNA array (Little DP et al, Analytical Chemistry. 69(22) :4540-4546, 1997). Whatever the reporter system used, sophisticated gadgetry and software may be required in order to inte ⁇ ret large numbers of readouts into meaningful data (such as described, for example, in US Patent No. 5,800,992 or International Publication No. WO 90/04652).
  • the microarray can be treated to remove the bound sequences in preparation for reuse of the microarray by exposure to a second or subsequent set of target sequences.
  • the hybrid duplexes are disrupted and the solid support matrix treated in order to remove all traces of the original target.
  • the matrix may be treated with various detergents or solvents to which the substrate, the oligonucleotides and the linkages to the substrate are inert. This treatment may involve an elevated temperature treatment, treatment with organic or inorganic solvents, modifications in pH, and other means for disrupting specific interactions.
  • Examples of methods that could be used are: (1) Washing the array with 50 mM sodium hydroxide to disrupt base pairing by high pH. (2) Washing the array with pure water and at high temperature (e.g. > 80°C) to disrupt base pairing by high stringency. (3) Addition of oligonucleotide sequences complementary to the tail sequences (and identical to the chip sequences) to disrupt base pairing by exchange with the sequences in free solution. Other methods for disrupting duplex formation are well known in the art (see for example Sambrook et al. ibid).
  • the microarray of the invention is not a custom chip, but rather a generic chip which interacts with specific custom targeting oligonucleotides, once the microarray has been cleaned, it can be reused in any appropriate procedure and is not limited to reuse in the particular procedure used before.
  • the discriminatory ability lies with the "wet reaction" involving the target nucleic acids with the custom targeting polynucleotides that co-operate with the generic microarray of the invention.
  • kit for detecting the presence or absence of one or more target nucleic acid sequences contained in a sample which kit comprises :-
  • each polynucleotide comprising a unique 3' portion substantially complementary to a unique target nucleic acid sequence which may be present in a sample, a 5' portion complementary to one of a group of pre-selected oligonucleotides that each possess substantially the same melting temperature (T m ) and are attached at pre-defined positions to a solid support, and optionally a spacer moiety inte ⁇ osed between said 3' portion and said 5' portion; and (ii) a solid support having immobilised thereon a plurality of pre-selected oligonucleotides at pre-defined positionally distinct sites, characterised in that the composition of each of the oligonucleotides is such that they all have substantially the same melting temperature (T m ) when annealed to their complementary sequence, each capture oligonucleotide having a sequence complementary to a 5' portion of one of the polynucleotides in (i).
  • the kit also contains some or all four different nucleoside triphosphates and/or an agent for polymerisation of the nucleoside triphosphates and/or instructions for use.
  • the targeting portion of the polynucleotides may be acting as hybridisation probes or amplification detection primers.
  • the kit comprises a set of at least two primers for each target sequence, the terminal nucleotide of at least one primer being complementary to a suspected variant nucleotide associated with a known genetic disorder and at least one of the other primers being a companion primer as described hereinbefore.
  • the kit may therefore comprise sets of oligonucleotide primers, each set targeting different alleles at a specific loci.
  • the polynucleotide molecules referred to in (i) are ARMS primers with non-amplifiable tails as described hereinbefore.
  • the solid support is a microscope slide or microtitre plate, such as a 96-well plate.
  • kits may also comprise control DNA and control primers or probes, and the like.
  • Figure 1 Illustrates the use of quenching oligonucleotides for measuring primer binding.
  • Figure 2a Results from Experiment 1, 84- ARMS primer multiplex on 1% p53 codon 175 CAC admixture template and wild-type; OD 405nm mutant (ODm) and wild-type (ODw) plot for the 11 test ARMS primers and one control primer.
  • Figure 2b Results from Experiment 1, (ODm/ODw)-l ratio of absorbance (405nm) on p53 wild-type and mutant admixture for l%o 175 CAC.
  • Figure 3a Results from Experiment 1, 84- ARMS primer multiplex on 5% p53 codon 175 CAC admixture template and wild-type; OD 405nm mutant (ODm) and wild-type (ODw) plot for the 11 test ARMS primers and one control primer.
  • Figure 3b Results from Experiment 1, (ODm/ODw)-l ratio of absorbance (405nm) on p53 wild-type and mutant admixture for 5% 175 CAC.
  • Figure 4a Results from Experiment 2, 84-ARMS primer multiplex on 1% p53 codon 175 CAC admixture template and wild-type; OD 405nm mutant (ODm) and wild-type (ODw) plot for the 17 test ARMS primers and two control primer.
  • 80 ARMS primers were designed for the specific detection of some of the mutations in exons 5-8 of the p53 tumour suppresser gene (Table 3 lists the 80 codon positions and specific mutations for which ARMS primers were designed and prepared). Uniquely identifying non-amplifiable tails (with T m s in the range of 53°C to 58°C) with hexaethylene glycol links between the primer and the tail sequences were added to 19 of these ARMS primers (marked * in Table 3). The 80 ARMS primers were then multiplexed together with 2 reverse primers designed to give PCR products with the ARMS primers specific for mutations in exons 5&6 and 8 respectively. Tailed primer sets which act as control primers for the detection of p53 exons 5&6 and exon 8 sequence were also included. Table 3.
  • * - denotes the mutant codon for which a tailed ARMS primer was prepared.
  • Templates containing mutations in the p53 gene were prepared by primer directed mutagenesis (see Higachi et al. NAR. 16:7350-7367, 1988). This gave templates of 500 - 800 bp containing the mutation of interest. Wild type templates were prepared by PCR amplification of wild-type DNA to give the corresponding 500 - 800 bp fragments.
  • Oligonucleotides of precise complementary sequence to the 19 tail sequences were synthesised with 3' biotin moieties. These capture sequences were bound to the wells of a streptavidin coated microtitre plate (one capture sequence per well).
  • Each ARMS primer was present at 50 nM concentration, the reverse primers were present at 500mM concentration and wild type dNTPs at 50 ⁇ M each. Fluorescein-dUTP was also included at 0.5 ⁇ M.
  • the buffer was 50 mM KCl, 10 mM tris, 1.2 mM MgCl 2 at pH 8.3. 4 Units of AmpliTaq GoldTM were used per amplification. 10 5 copies of template were added. Cycling conditions were 94°C for 20 minutes then 35 cycles of (94°C for 1 minute, 60°C for 1 minute). In each experiment, three parallel experiments were run using (a) wild type template, (b) mutant/wild-type admixture template and (c) a no- template control.
  • PCR products were divided between the capture wells of the microtitre plate. Hybridisation between the PCR products and the capture oligos took place overnight at 55°C in 3M TMAC, 1M Tris (pH 7.5), 0.5M EDTA, 0.01% Triton-X-100, 0.1mg/ml herring sperm DNA . Unbound products were then washed off (2 washes in phosphate buffered saline (PBS)). The PCR products were detected by ELISA detection of inco ⁇ orated fluorescein-dUTP using an anti-fluorescein-alkaline phosphatase antibody- enzyme conjugate. Colour development was by addition ofp-nitrophenyl phosphate and the OD 405 was determined after 30 minutes.
  • ODm mutant template
  • ODw wild-type template
  • no-template control the no-template control ODs were subtracted from ODm and ODw to give background corrected values.
  • ODm, ODw values and the (ODm/ODw -1) ratio were then plotted.
  • Admixtures were prepared between template containing p53 mutant codon 175 CAC and wild-type template with the mutant sequence present at 1% and 5% of the total.
  • SEQ ID No.l represents the 5' tail portion; SEQ ID No. 2 represents the 3' targeting portion.
  • the * denotes the HEG group.
  • the ODm and ODw values with the relevant no-template control values subtracted are shown in figures 2a and 3a.
  • the figures show that the primer for 175 CAC gives a signal on the mutant template (bars) which is higher than the one obtained with wild-type template (diamonds).
  • the size of the signal is dependant on the amount of mutant template present in the substrate and is therefore greater for the 5% mutant template than for the 1% mutant template.
  • the ability of the primer for 175 CAC to differentially detect low levels of mutant sequence can be also seen by calculating the (ODm ODw-1) ratios (see Figures 2b and 3b). From these values it can be clearly seen that only the primer for 175 CAC demonstrates a significantly higher OD on the mutant template than on the wild-type template.
  • An admixture was prepared between template containing p53 mutant codon 175 CAC at 10% in a wild type background.
  • three separate amplifications were carried out on: (a) the admixture; (b) wild-type template; and, (c) no-template control.
  • the products of the amplifications were then each added to a separate array consisting of 17 capture oligos for the primers plus 2 capture oligos for exon 5, 6 and exon 8 control reactions.
  • the ODm and ODw values with the relevant no-template control values subtracted are shown in Figure 4a.
  • Example 1 demonstrates the use of ARMS for the sensitive detection of under- represented sequences in combination with the use of non-amplifiable tails and oligonucleotide arrays to provide a method for the large scale multiplex analysis of polymo ⁇ hisms in gene sequences.
  • ARMS in solution phase permits the more sensitive detection of gene variation than can be achieved with solid phase allele specific oligonucleotide (ASO) hybridisation.
  • non-amplifiable tails and oligonucleotide arrays presents a more generic and widely applicable technique then can be achieved with ASO arrays which must be individually designed on a target to target basis. In this way a maximally efficient mutation detection system is produced because each component used in the process is suited to the process it is required to carry out. Mutation detection can be carried out in solution phase using ARMS and DNA arrays used for separating complex mixtures of oligonucleotide sequences.
  • non-amplifiable tails and oligonucleotide arrays presents a simpler hybridisation technique then can be achieved with ASO arrays.
  • ASO arrays all of the probe to targets hybridisations require to be carried out under the same conditions including the same temperature and buffer. These conditions may not be ideal for many of the probe to target hybridisations which require to be performed.
  • generic arrays and non-amplifiable tails a single set of unified hybridisation conditions can be pre-selected which permit all probe to target hybridisations to be carried out under the same optimal conditions because all probe to target hybridisations can be selected to occur between sequences of substantially the same T m and GC content.
  • non-amplifiable tails and oligonucleotide arrays also presents a more cost effective way of screening for mutations than can be achieved with ASO arrays.
  • manufacture of the array used in the screening process is cheaper than is the case with specific ASO arrays because: 1.
  • the generic array will likely require far fewer capture sequences for the analysis of each variant in the gene of interest than is possible with a specific ASO array and therefore, is far simpler and cheaper to manufacture. 2.
  • Once the array has been designed it may be used for the analysis of any gene target. The costs of designing and developing new arrays for new gene targets is avoided.
  • Oligonucleotide sequences of substantially the same T m can conveniently be generated by use of a spreadsheet computer program inco ⁇ orating a random number generator.
  • a spreadsheet computer program inco ⁇ orating a random number generator.
  • the program sorts the sequences in order of increasing T m . In this way oligos of substantially the same T m can be selected as candidate sequences for use as tails. It should be understood that the use of this T m algorithm is illustrative only and that any convenient algorithm could be used. Macro:
  • T m [2* (#A or T) + 4*(#G or C)].
  • cDNA is prepared from mRNA purified from two types of tissue representing normal and altered cells.
  • the altered cells may have been treated differently to the normal cells prior to mRNA purification.
  • Examples of 'different treatment' include starving the cells of a metabolite or metabolites, stimulating them with a specific metabolite such as a growth factor or treating them with a drug or hormone.
  • different cell conditions might exist already, i.e. normal Ns tumour.
  • reverse transcription from an oligo-dT primer is carried out with inco ⁇ oration of fluorescent labels into the cD ⁇ A prepared.
  • one label such as Cy-3 is used to label the cD ⁇ A from the normal tissue and another label such as Cy-5 is used to label the cD ⁇ A from the treated tissue.
  • the cD ⁇ A population is optionally fragmented by sonication or mechanical shearing (i.e. by passage through a 19G needle).
  • Targeting polynucleotides are added to the cD ⁇ A mixture and hybridisation permitted to occur, for example, under the following hybridisation condition: labelled cD ⁇ A is resuspended in 10 ml of 3.5 x SSC containing 4mg of poly dA D ⁇ A, 2.5 mg E. coli tR ⁇ A, 4mg human Cotl D ⁇ A and 1ml 10 10% SDS. Hybridisation is carried out at 62°C for 3hours.
  • each cD ⁇ A is targeted by a number of distinct targeting polynucleotides each capable of hybridising to a different region of the cD ⁇ A, but all possessing the same tail sequence to facilitate capture at the same pre-determined location on the microarray.
  • each cD ⁇ A is targeted by 20 or more distinct targeting polynucleotides.
  • the targeting polynucleotides comprise two principal domains, the first domain is complementary to a distinct region of one of the cDNAs in the sample mixture and the second domain (tail portion) is complementary to one of the capture oligonucleotides on the DNA array.
  • the targeting polynucleotides may be preferable to group the targeting polynucleotides into pools of substantially the same T m . In this way, separate sample.-targeting polynucleotide hybridisations can be carried out under the optimum hybridisation conditions for each pool. All the pooled reaction products can then be mixed prior to the capture hybridisation. Once hybridisation between the cDNA and the targeting polynucleotides is substantially complete, the mixture is added to the surface of the oligonucleotide microarray under suitable conditions to allow hybridisation between the targeting polynucleotide tail portion to occur.
  • Hybridisation is carried out at 62°C for 1- 3hours in a suitable volume of hybridisation solution such aslOml of 6x SSC, 0.1%SDS and 0.25% dried skimmed milk (MarvelTM) in a suitable enclosed vessel.
  • a proprietary hybridisation apparatus such as model HB-1 (Techne Ltd) provides reproducible conditions for the experiment.
  • the microarray is subjected to a stringency wash (such as in 2xSSC, 0.2%SDS, then 0.2xSSC) and the array surface is subjected to fluorescence. Fluorescent output from the two dyes is captured and stored as separate channels.
  • the intensity of the two data sets is normalised by reference to a common housekeeping gene whose expression is considered to be invariant in all tissues. There are many such genes but one example is GAPDH.

Abstract

La présente invention concerne un ensemble d'oligonucléotides (ou ensemble «générique») constitué d'une pluralité d'oligonucléotides différents de séquence prédéterminée, liés à une surface solide à des emplacements distincts prédéfinis. Cet ensemble se caractérise notamment par le fait que les oligonucléotides présentent sensiblement une température de fusion identique (Tm). Cette invention concerne enfin des méthodes d'utilisation dudit ensemble «générique».
EP00901800A 1999-02-11 2000-02-08 Ensemble d'oligonucl otides et ses m thodes d'utilisation Withdrawn EP1169474A1 (fr)

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GBGB9902970.4A GB9902970D0 (en) 1999-02-11 1999-02-11 Novel matrix
GB9902970 1999-02-11
PCT/GB2000/000357 WO2000047767A1 (fr) 1999-02-11 2000-02-08 Ensemble d'oligonucléotides et ses méthodes d'utilisation

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