Classifications

• • 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 the use of a multiplex amplification method that utilises a plurality of ARMS primers possessing non-amplifiable tails in conjunction with a generic oligonucleotide array for detection amplification of variant nucleic acid sequences.
• the invention is of particular use in fingerprinting and genome typing, including single nucleotide polymorphism typing, and clinical diagnosis.
• PCR polymerase chain reaction
• a significant improvement on the above amplification and detection methods is the Amplification Refractory Mutation System (ARMSTM) as claimed in European Patent no. 0 332 435 and corresponding US Patent No. 5,595,890.
• ARMS is a simple, reliable and rapid PCR-based method for the detection of any single base changes or small deletions, such as mutations or polymorphisms.
• ARMS detection is particularly useful if the target nucleic acid is an allele in low copy number compared to the alternate allele, for example with somatic underrepresented sequences a single mutant sequence may only be present amongst a background of 10,000 or more wild type sequences.
• European Patent EP-B-0416817 discloses non-amplifiable tailed PCR primers which may be adapted for use in the method of the present invention. This disclosure does not exemplify multiplex PCR, using normal or ARMS primers, nor does it teach the added and surprising benefit of enhanced ARMS primer binding specificity observed by the present inventors with large multiplex formats. It also does not demonstrate use of capture oligonucleotides that have minimal secondary structure and/or substantially the same T m for facilitating and optimising product capture.
• a method for simultaneously detecting the presence or absence of a variant nucleotide at, or within, each of a plurality of different target sequence in a sample which method comprises:-
• each primer having a 3 '-end portion substantially complementary to a distinct target nucleic acid sequence which may be present in the sample and a 5 '-end portion complementary to one of a set of oligonucleotides immobilised onto a solid surface and optionally, an amplification blocking moiety interposed between said 3 '-end and 5 '-end portions, the terminal nucleotide of the 3' end portion being either complementary to a suspected variant nucleotide or to the corresponding normal nucleotide, whereby an extension product of the primer is synthesised when the said terminal nucleotide of the primer is complementary to the corresponding nucleotide in the target sequence, no extension product being synthesised when the said terminal nucleotide of the primer is not complementary to the corresponding nu
• the 5 '-end portion represents a single stranded tail portion useful for targeting the amplified products onto a solid support.
• the amplification primers are amplification refractory mutation system (ARMS) primers, as described in EP-B-0332435 and corresponding US Patent No. 5595890, and as described in EP-B-416817, and additionally described in Newton et al. (Nucleic Acids Research. 17(7):2503-2516, 1989).
• ARMS amplification refractory mutation system
• 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 polymo ⁇ hisms (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 and under appropriate conditions may be used to detect down to 0.01% mutant sequence in 99.99% wild-type background, or less.
• 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 non-target allele is not detectable.
• the ARMS primers will preferably possess destabilising mismatches inco ⁇ orated 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 nearer to the 3' terminus of the primer that a destabilising mismatch is inco ⁇ orated, the greater the effect on destabilisation (See also Newton et al. Nucleic Acids Research. 17:2503-2516, 1989).
• the tailed primer molecules are used in conjunction with another amplification primer that binds downstream of the target region of interest and lies anti-parallel to the tailed-primer so as to amplify the target region of interest according to the polymerase chain reaction.
• this second amplification primer also 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.
• Suitable labelling molecules are well known in the art.
• a suitable label can be inco ⁇ orated into the amplified product during its synthesis. A suitable amplification reaction can then be performed so as to generate an amplification product.
• nucleotide can refer to nucleotides present in either DNA or RNA and thus includes nucleotides which inco ⁇ orate adenine, cytosine, guanine, thymine and uracil as base, the sugar moiety being deoxyribose or ribose.
• modified bases capable of base pairing with one of the conventional bases, adenine, cytosine, guanine, thymine and uracil, may be used in the probes or primers employed in the present invention.
• 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.
• the agent for polymerization of the nucleoside triphosphates may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
• Suitable enzymes for this pu ⁇ ose include, for example, E.coli DNA Polymerase I, Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptase, and other enzymes, including thermostable enzymes.
• thermostable enzyme refers to an enzyme which is resistant to heat and catalyzes (facilitates) combination of the nucleotides in the proper manner to form the primer extension.
• Any convenient template dependent polymerase may be used, however, a thermostable polymerase enzyme such as TaqTM, Taq GoldTM, or other suitable enzyme lacking 3' to 5' proof-reading activity is particularly preferred.
• 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 however, 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.
• nucleoside triphosphates 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). Further guidance may be found in “Laboratory protocols for mutation detection” edited by Ulf Landegren, published by the Oxford University Press, Oxford, 1996, ISBN 0 19 857795 8. on products which are complementary to each nucleic acid strand. Generally, all four nucleotide triphosphates are provided in the amplification reaction.
• an extension product of the primer and, if desired, an extension product of the companion amplification primer may be formed in the presence of only the appropriate corresponding nucleoside triphosphates and all four different nucleoside triphosphates would not be necessary.
• adenosine triphosphate is complementary to uridine triphosphate or thymidine triphosphate and guanosine triphosphate is complementary to cytidine triphosphate. It is appreciated that whilst thymidine triphosphate and guanosine triphosphate may base pair under certain circumstances they are not regarded as complementary for the pu ⁇ oses 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 pu ⁇ oses of this specification.
• cytosine triphosphate and uracil triphosphate 5 "Precise 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
• target sequence means the particular nucleic acid sequence present in the test sample to which the primer is intended to bind, which sequence contains a potential variant nucleotide, the presence or absence of which is to be detected.
• target sequence means the particular nucleic acid sequence present in the test sample to which the primer is intended to bind, which sequence contains a potential variant nucleotide, the presence or absence of which is to be detected.
• the p53 gene may contain up to or more than 100, for example 50 target sequences, each target sequence containing a single potential variant nucleotide. It will be appreciated also that there may exist any of three possible variants at any single nucleotide position. It will also be appreciated that the presence of a variant nucleotide at a particular site may be the result of a number of genetic polymo ⁇ hisms, for example, it may be a simple
• nucleotide substitution or, it may be a deletion, an addition, or an inversion, and the like. It will be appreciated therefore that whilst the method of the present invention is of particular interest in detecting the presence or absence of point mutations, the method is equally applicable to detecting the presence or absence of deletions, including deletions of more than one nucleotide as well as to detecting the presence or absence of substitutions of more than one nucleotide or inversions of more than one nucleotide. In this regard it is simply necessary to know the relevant nucleotides, especially the relevant terminal nucleotide, so that the necessary diagnostic primer(s) may be designed appropriately.
• target amplification sequence refers to the target sequence and sequence immediately adjacent thereto.
• target amplification sequence refers to the sequence that becomes amplified.
• extension product formed may be detected in any convenient form, for example in single or double stranded form.
• Amplification of the target sequence can be effected by multiple rounds of primer extension from the primer in order to amplify the target nucleic acid such as up to 5, up to 10, up to 15, up to 20, up to 30, up to 40, up to 50 or more times.
• each targeting oligonucleotide primer is accompanied by a companion primer which facilitates amplification of the target sequence inte ⁇ osed between the two primers according to amplification procedures such as polymerase chain reaction (PCR) or ligase chain reaction (LCR), well known in the art.
• This companion primer may itself have a non- amplifiable tail portion or it may contain a suitable label which can be used to detect the presence of the amplification product bound onto the solid support.
• the target binding region of the primer and the 5 '-end tail region are advantageously arranged such that the tail region remains single stranded, ie. uncopied.
• the tail region is non-amplifiable in the amplification products.
• This facet of primer design is disclosed in our European Patent No. 0 416 817 and corresponding US Patent No. 5525494.
• a blocking moiety is conveniently inte ⁇ osed between the tail portion (5'-end) and the target template binding sequence portion (3'-end).
• a preferred blocking moiety is a hexethylene glycol (HEG) monomer.
• the primer tail comprises material such as 2'-O-alkyl RNA which will not permit polymerase mediated replication by enzymes lacking reverse transcriptase activity and thus will block the enzyme preventing it from creating a double-strand of the tail region.
• An alternative way of preventing the tail region (the 5' portion of the primer) from acting as template for the polymerase enzyme is to link the 3' portion (target binding region) and the 5' portion (tail region) of the primer 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 can then be utilised for capture of the amplification product onto a complementary capture oligonucleotide attached to a solid support.
• the more preferred means of blocking the polymerisation agent is to inco ⁇ orate a blocking 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 pu ⁇ ose.
• 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 phosphormo ⁇ holidates, or other residues known er 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.
• EP-B-416817 describe tailed primers with blocking moieties, interposed between the tail and the target binding portion of the primer, that can be inco ⁇ orated into the polynucleotides of this invention.
• the blocking moiety should be easily inco ⁇ orated by solid phase oligonucleotide chemistry and should also form a substrate for further extension in the same chemistry.
• suitable blocking moieties include hexethylene glycol (HEG) and tetraethylene glycol (TEG) phosphoramidites.
• the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
• the primer is an oligodeoxyribonucleotide.
• the 3' portion of the tailed ARMS primer represents the target binding sequence. This sequence must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
• This sequence can be of any length, depending on the complexity of the target sequence, although it will typically contain between 8 and 60 nucleotides in length such as 10-40 nucleotides, 15-30 nucleotides, particularly 20- 30, 17-22, 16-23 or 15-24 nucleotides capable of hybridisation to the target nucleotide sequence, although they may contain more or fewer such nucleotides.
• Each of the above ranges is a separate and independent embodiment of the invention. Primers having only short sequences capable of hybridisation to the target nucleotide sequence generally require lower temperatures to form sufficiently stable hybrid complexes with the template.
• the 3' end target binding portion of the tailed ARMS primer 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. It may be advantageous to inco ⁇ orate nucleotide mismatches into the target binding portion of the ARMS 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.
• a major source of artefactual products is probably allowing the reaction temperature to fall too low, thus permitting too low a stringency, for example by removing the reaction mixture from the heat cycling means, even briefly for example to add the agent for polymerisation (eg. Taq polymerase) especially in the first reaction cycle.
• the agent for polymerisation eg. Taq polymerase
• a primer may give rise to difficulty in this regard if it is G/C rich as a whole or particularly if it is G/C rich at its relevant, normally, 3' end.
• the precise nature of the base pairing in the region of the 3 1 end of the primer when in use may be the cause of an artefactual result.
• any one or more of the 10, for example 6 nucleotides adjacent to the terminal mismatch may be altered to introduce further mismatching.
• one mismatch in addition to the terminal mismatch may be necessary, positioned for example, 1, 2 or 3 bases from the terminal mismatch.
• the third nucleotide from the 3 1 terminal nucleotide is altered to generate a mismatch in use.
• diagnostic primer may thus be determined by straightforward experimentation based on the above criteria, such experimentation being well within the ability of the skilled molecular biologist.
• One feature of the present method is the su ⁇ rising finding that the incidence of artefactual results, generating false positives, is reduced when a multiplex ARMS assay is performed.
• the capture hybridisation is to be effected in the presence of all the test sample nucleic acid, i.e. without separation of amplified products from unreacted primers and sample 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.
• the target binding region of the primer hybridises to template nucleic acid from a sample. The region is of any convenient design available to the person of ordinary skill and is limited only by practical considerations.
• Template binding can be effected at any desired stringency, that is to say under appropriate hybridisation stringency conditions the template binding region of the primer may hybridise to the template region (if present in the template) to the exclusion of other regions.
• template binding may be effected at reduced stringency to extend the primer on any convenient number of related template sequences, such as for example human leukocyte antigen (HLA) genes, or other conserved genes, particularly bacterial or ribosomal RNA genes.
• HLA human leukocyte antigen
• Part of the invention lies in the ability of the primers to specifically distinguish sequences that differ only in the variant nucleotide.
• the method of the invention utilises at least 20, at least 40, at least 50, at least 60, at least 80, at least 100, at least 200, at least 500, or more distinct primers.
• the preferred range is between about 50 and 200 primers.
• a suitable format for diagnostic pu ⁇ oses is to utilise suitably adapted microtitre plates (such as 96-well plate) each well possessing a targeting oligonucleotide capable of capturing one of the ARMS primer tails.
• the amplification reaction solution is treated to remove those primers that have not participated in primer extension or target template amplification.
• This may conveniently be performed using techniques well known in the art such as column chromatography using a column substrate suitable to discriminate between oligonucleotides of the particular primer length adopted from predominantly double-stranded target amplified products, or alternatively, by ethanol precipitation or gel filtration.
• the solid surface with oligonucleotides immobilised thereon at pre-determined positions is more commonly known in the art as an oligonucleotide microarray.
• 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 polymo ⁇ hism detection, clinical mutation detection, expression monitoring, finge ⁇ rinting and sequencing.
• oligonucleotide DNA arrays consisting of short oligonucleotides (e.g.
• solid support is well known in the art. Essentially, 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. Suitable examples of existing laboratory materials that can be utilised are glass microscope slides and microtitre (such as 96-well) plates.
• 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.
• each of the 5 '-end tail sequences of the ARMS primers duplexed to their capture oligonucleotides on the microarray possess substantially the same T m (for example, within 15°C of the median value, preferably within 8°C of the median value, more preferably within 4°C of the median value), such that under any hybridisation conditions adopted, all of the oligonucleotides on the solid support (herein referred to as "capture oligonucleotides”) exhibit approximately the same hybridisation stability with their cognate complementary sequence as the other pairs of oligonucleotide and complementary target sequence.
• the optimum temperature for hybridisation can be adopted.
• the capture oligonucleotides immobilised on the microarray are designed so as to have minimal secondary structure (such as hai ⁇ in loop structure) that might impede hybridisation.
• 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.
• 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 .
• 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.
• the oligonucleotides immobilised on the solid support will possess melting temperatures within a range of 0 to 8°C of each other.
• At least 90% of the oligonucleotides immobilised on the solid support will possess melting temperatures within 4°C, more preferably 2°C, of each other. In an even more preferred embodiment the oligonucleotides immobilised on the solid support will possess melting temperatures within a range of 0 to 2°C of each other. In the most preferred embodiment, 90-100%) of all the oligonucleotides immobilised on the solid support will possess the same melting temperature, and the remaining oligonucleotides will preferably possess melting temperatures within a range of 0 to 2°C of this mode value.
• oligonucleotides on the solid support fall within the ranges or values for melting temperature 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 or values.
• the length of each capture portion of the oligonucleotide immobilised on the solid support will be within or equal tol6, 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.
• 95-100%> 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.
• all of the 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 l-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
• 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 and G-C content.
• the theoretical and experimental procedure for determimng the T m is disclosed in Molecular Cloning-A Laboratory Manual, Second Edition, J Sambrook et al., Cold Spring Harbor, Chapter 11 section 46 and 55. In essence, for oligonucleotides shorter than 18 nucleotides, the T m of the hybrid is estimated by multiplying the number of A + T residues in the hybrid by 2°C and the number of G + C residues by 4°C and adding the two together.
• T m 81.5 - 16.6(log 10 [Na+]) + 0.41 (% G + C) - (600/N).
• N chain length and [Na+] is the ionic strength of the hybridisation solution.
• the preferred length of hybridisable sequence is in the range of 10 - 50, more preferred is 12-20.
• 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.
• 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.
• the oligonucleotides can be synthesised directly onto the solid surface using photolithography techniques, such as disclosed in US Patent No. 5,143,854, or other methods such as disclosed in US Patent No. 5,700,637, or International Publication No's: WO 95/35505, WO 97/44134 or WO 98/10858.
• Schena et al. (TIBTECH 16(7):301-306, 1998) reviews the recent advances in microarray technology including the various means of constructing these arrays.
• 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.
• An advantage of using non-amplifiable single-stranded tailed primers in the method of the invention is that the target 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.
• the oligonucleotide microarray suitable for capturing the tailed ARMS primers used in the multiplex amplification method of the present invention is another aspect of the invention.
• the multiplex tailed ARMS primers amplification with oligonucleotide microarray capture method of the invention is 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 finge ⁇ rinting, diagnostic identification, genotyping of organisms (including single nucleotide polymo ⁇ hism detection) and environmental monitoring. A particular use is in the detection of inherited disease or acquired disease, such as tumours. 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.
• allelic variation requires a mutation discrimination technique, optionally an amplification reaction and a signal generation system.
• Table 1 lists a number of conventional signal generation systems that may be employed to detect the tailed ARMS products generated according to the invention.
• Fluorescence Fluorescence intensity, Fluorescence resonance energy transfer (FRET), Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998.
• Other Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetric, Hybridisation protection assay, Mass spectrometry.
• One way of detectably labelling the amplified products is to inco ⁇ orate a suitable label into the tailed ARMS primers and/or the companion amplification primer, or to inco ⁇ orate a label such as fluorescein-dUTP into the amplified products.
• a label such as fluorescein-dUTP into the amplified products.
• any number of conventional detectable markers such as radioisotopes, fluorescent labels, chemiluminescent compounds, labelled binding proteins, magnetic labels, spectroscopic markers and linked enzymes might be used.
• the labelling technique employed is a matter of personal choice for the person skilled in the art.
• 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. If the tailed ARMS primers are labelled, the unextended ARMS primers can be separated from extended primers prior to the capture step using conventional techniques such as column chromatography, ethanol precipitation or gel filtration.
• a ligand binding to the minor groove such as Hoechst 33258 fluorescent dye or the use of 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 and minor groove binders 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.
• 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). 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.
• 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. This results in the microarray oligonucleotides fluorescing as a result of the binding of the appropriate product. In this format a fluorescent signal is produced by separating two species bound to the array surface.
• This format 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 inco ⁇ orated, 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.
• 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 tailed ARMS primers or products 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.
• 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. At 1M NaCl, G:C base pairs are more stable than A:T base pairs. Similarly, 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.
• 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.
• 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).
• kit for detecting the presence or absence of one or more variant nucleotides in one or more nucleic acids contained in a sample comprises:- (i) a plurality of primers, one primer for each potential variant nucleotide of a target sequence to be detected, each primer having a 3 '-end portion substantially complementary to a distinct target nucleic acid sequence which may be present in the sample and a 5 '-end portion complementary to one of a set of oligonucleotides immobilised onto a solid surface and optionally, an amplification blocking moiety inte ⁇ osed between said 3 '-end and 5 '-end portions, the terminal nucleotide of the 3' end portion being either complementary to a suspected variant nucleotide or to the corresponding normal nucleotide, whereby an extension product of the primer is synthesised when the said terminal nucleotide of the primer is complementary to the corresponding nucleotide in the target sequence,
• the kit also contains one or more of the four different nucleoside triphosphates and or an agent for polymerisation of the nucleoside triphosphates and/or instructions for use.
• 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 or a polymo ⁇ hism 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 solid support is a microtitre plate with each capture oligonucleotide immobilised in a distinct well of the plate.
• Figure la Records the absorbance values (OD405nm) of the captured amplification products for Experiment 1 (23-plex). Signals generated from the 10%) mutant template are shown as bars and those from 100% wild-type template are shown as diamonds.
• Figure lb Records the ratio of signals from mutant and wild-type templates (ODm/ODw) from Experiment 1.
• Figure 2a Records the absorbance values (OD405nm) of the captured amplification products for Experiment 2 (84-plex). Signals generated from the 10%> mutant template are shown as bars and those from 100%o wild-type template are shown as diamonds.
• Figure 2b Records the ratio of signals from mutant and wild-type templates (ODm/ODw) from Experiment 2.
• Figure 3a Records the absorbance values (OD405nm) of the captured amplification products for Experiment 3 (23-plex). Signals generated from the 10%) mutant template are shown as bars and those from 100% wild-type template are shown as diamonds.
• Figure 3b Records the ratio of signals from mutant and wild-type templates (ODm/ODw) from Experiment 3.
• Figure 4a Records the absorbance values (OD405nm) of the captured amplification products for Experiment 4 (84-plex). Signals generated from the 10%o mutant template are shown as bars and those from 100%> wild-type template are shown as diamonds.
• Figure 4b Records the ratio of signals from mutant and wild-type templates (ODm/ODw) from Experiment 4.
• ARMS primers were designed for the specific detection of some of the mutations in exons 5-8 of the p53 tumour suppresser gene (Table 2 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 2). The 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.
• * - denotes the mutant codon for which a tailed ARMS primer was prepared.
• the following represents the specific tailed ARMS primer used to detect p53 175 CAC mutation (* denotes the HEG group): 5 '-GCTTTATGTCCACAGATTTC* ATACACAGCACATGACGGAGGTTGTGAGCCA-3 ' SEQ ID No. 1 represents the 5' tail portion; SEQ ID No. 2 represents the 3' targeting portion
• the specific reverse primer used with the above ARMS primer is: 5'-ACCCGGAGGGCCACTGACAAC-3' (SEQ ID No. 3)
• Two templates, one containing the 175 CAC p53 mutation and the other containing the 132 CAG mutation 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). Reaction conditions: Each ARMS primer was present at 50 nM concentration, the reverse primers 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 KC1, 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.
• 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, O.lmg/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.
• PBS phosphate buffered saline
• Admixtures were prepared between template containing p53 mutant codon 175 CAC and wild-type template with the mutant sequence present at 10% of the total.
• Three separate amplifications (using the multiplex of 19 tailed ARMS primers, two reverse primers and 2 control template primers) were carried out on: (a) the admixture; (b) wild-type template; and, (c) no-template control.
• the amplification products were then each added to a separate array consisting of a microtitre dish with 17 capture oligos for a subset of the primers plus 2 capture oligo for the exon 5&6 control reaction immobilised thereon.
• Figures la shows the ODm and ODw values with the relevant no-template control values subtracted for the mutant (10% 175 CAC) and 100%> wild-type templates amplified with a multiplex of 19 mutant primers, 2 reverse primers and 2 control primers for different exons of the p53 gene plus two reverse primers.
• the signals generated from the mutant template are shown as bars and those from the WT as diamonds. All signals are background corrected.
• the control primer generates signal from both templates whereas the 175 CAC primer only generates signal from the mutant template.
• a further two primers produce signal non-specifically from both templates (132 CAG and 220 TGT).
• Figure lb shows the ratio of signals from the mutant and WT templates (ODm/ODw).
• the primer specific for the mutation (175 CAC) produces a significant signal from the mutant template only with the ratio being approximately 50.
• the control signals and inappropriate priming signals are similar on both mutant and WT templates and produce ratios of approximately 1.
• Experiment 1 was repeated except that the 175 CAC mutant template was substituted for by the 132 CAG mutant template.
• efficient target amplification was demonstrated using just 8 key capture oligonucleotides.
• Figures 3 a shows the ODm and ODw values with the relevant no-template control values subtracted for the mutant (10% 132 CAG) and 100%> wild-type templates amplified with a multiplex of 19 mutant primers, 2 reverse primers and 2 control primers for different exons of the p53 gene.
• the 132 CAG primer primes and extends off WT template in the context of the 23-plex.
• the signal from the mutant template is greater than that from the WT template, the priming of this primer on the WT template results in an ODm/ODw ratio of only 7 (Figure 3b).

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