GB2495909A - Arrays comprising immobilized primer pair spots for amplifying target nucleic acids - Google Patents
Arrays comprising immobilized primer pair spots for amplifying target nucleic acids Download PDFInfo
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- GB2495909A GB2495909A GB1118030.4A GB201118030A GB2495909A GB 2495909 A GB2495909 A GB 2495909A GB 201118030 A GB201118030 A GB 201118030A GB 2495909 A GB2495909 A GB 2495909A
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Abstract
Arrays are disclosed comprising a substrate and a plurality of oligonucleotide primer spots immobilised thereon, wherein within each spot a population of primer pairs for specifically amplifying a target nucleic acid (NA) by PCR is immobilised upon the substrate such that the 3 ends of said primers can be extended, at least two of said oligonucleotide primer spots being adapted to amplify different target NAs. Methods of amplifying target NAs using the arrays are claimed (effectively utilising bridge amplification techniques). Methods of preparing oligonucleotide primer spots on an array are also claimed, wherein the methods may comprise steps of immobilising a population of a combined primer construct comprising first and second primers linked via a cleavable linkage, or wherein the methods comprise immobilising one of the primers and then covalently linking or recombining a proportion of the immobilised primers with the second primer of the primer pair.
Description
NUCLEIC ACID AMPLIFiCATION TECHNOLOGY
Field of the Invention
The invention relates to nucleic acid amplifIcation (e.g. PCR) technology and specifically to arrays suitable for carrying out multiplex PCR, methods for their manufacture and uses thereof
Background to the Invention
Polymerase chain reaction (PCR) is one of the most important inventions in modern biology, initially developed to amplify (i.e. increase copy number of) nucleic acid sequences in vitro. A number of different variations and adaptations of the basic technique have since emerged, one of which being multiplex PCR. Multiplex PCR employs multiple primer sets within a single PCR reaction vessel to produce amplicons specific to different nucleic acid sequences. However, resolutioniidentification of separate amplicons depends on e.g. their sequencing (which is labour intensive) or size separation (introducing assay design constraints and limiting the number of amplicons of the multiplex assay). In addition, the level of multiplex is limited by the possibility of different primer scts!arnplicons interacting and generating artcfacts that would not occur if the reactions were carried out singly, although emulsion PCR (emPCR) -where each droplet of a water-oil emulsion provides a separate "microreactor" for each amplicon -has gone some way to addressing this latter issue.
Microarrays are another important technology, used for highly parallel hybridisation based analysis e.g. gene expression measurements and comparative genomic hybridisation. Microarrays can be made by spotting synthetic oligonuclcotidcs (synthesised separately using conventional techniques) to a prepared surface. They can also be synthesised in situ on a chemically activated array surface by adding and reacting each nuclcotide phosphoramidite using physical separation (Southern), ink-jet technology (e.g. Agilent), or by using photoactivateable phosphoramidite derivatives and the excitation by photons using masks (Affymetrix) or controllable micro-mirrors (Nimblegen'Roche) to add specific bases to specific arcas. It has been shown that oligonucleotides can be cleaved from arrays using strong bases or UV that cleave the linkers attaching them to the solid surface. Ink jet printing of oligonucleotides uses standard phosphoramidite monomers as used in normal oligonucleotidcs synthesis.
Each spot of a standard microarray comprises a population of a single oligonucleotide probe that can anneal to complementary targets in a sample. A detection signal from such arrays is thus limited to the number of copies of target molecules present in the sample. In addition, the probes of the array must be designed to anneal directly with a target of interest and do not enable the detection, capture or identification of adjacent sequences.
It is an object of the present invention to provide a solution to these problems.
Summary of the Invention
The invention provides an array comprising a substrate and a plurality of oligonucleotide primer spots immobilised thereon, wherein: (a) within each spot a population of a pair of oligonuclcotidc primers that can prime the amplification of a specific target nucleic acid sequence by PCR is immobilised upon the substrate such that the 3' ends of said primers can be extended; and (b) at least two of said oligonucleotide primer spots are adapted to amplify different target nucleic acid sequences.
In preferred embodiments, the minimum distance between the edges of any two adjacent spots is at least equal to the length of the longest target nucleic acid sequence that can be amplified by said adjacent spots and/or the array is configured to reduce stcric interference between the substrate and any 3' overhang of template nucleic acid.
Also provided is a first method of preparing an oligonucleotide primer spot on an array according to the invention, said method comprising the steps of: (a) immobilising a population of a combined primer construct on the substrate via its 5' end, where said construct comprises a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR, wherein the 3' end of the first of said primer pair is covalently linked to the 5' end of the second of said primer pair, and where in a proportion of said population the linkage between the pair of primers is selectively clcavable; and (b) selectively cleaving the linkage between the pair of primers in said proportion of said population.
In preferred embodiments, said immobilising step comprises synthesising said population via a single, separate synthesis reaction and attaching the resultant population on the substrate via the 5' end or it comprises synthesising said population in situ using a single synthesis reaction.
The invention also provides a second method of preparing an oligonuclcotidc primer spot on an array according to the invention, said method comprising the steps of: (a) immobilising on a substrate via its 5' end a population of the first ofa pair of oligonuclcotidc primers that can prime the amplification of a specific target nucleic acid sequence by PCR; and b) recombining a proportion of said population with a nucleic acid construct such that the 3' ends of said proportion then consist of the second of said pair of oligonucleotide primers.
The invention additionally provides a third method of preparing an oligonucleotide primer spot on an array according to the invention, said method comprising the steps of: (a) immobilising on a substrate via its 5' end a population of the first of a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR; and (b) covalently linking to a proportion of the 3' ends of said population the second of said pair of oligonucleotide primers.
In preferred embodiments of said second/third methods, said immobilising step comprises immobilising a pre-formed population on the substrate or synthesising said population in situ using a single synthesis reaction.
In preferred embodiments of said third method, said linking step comprises reversibly blocking a proportion of the 3' ends of the population of immobilised primer, extending the unblocked 3' ends using in situ synthesis, and unblocking the blocked 3' ends.
Alternatively, said linking step comprises annealing a bridging oligonucleotide to a proportion of the 3' ends of the population of immobilised primer, wherein the 3' end of said bridging oligonucleotide can anneal to the first of the pair of oligonuelcotide primers and the 5' end of said bridging oligonucleotide can anneal to the second of the pair of oligonucleotide primers, and: (a) extending the 3' ends of said proportion using a polymcrase that uses the bridging oligonucleotide as a template; or (b) annealing the second of the pair of oligonuclcotide primers to the 5' end of the bridging oligonuclcotidc primer and ligating said second primer to the 3' ends of said proportion.
In a further preferred embodiment of said third method, the 5' end of the first of the pair of primers is immobilised on the substrate via a construct that has at its proximal end a sequence that can anneal to the 3' end of the immobilised primer and has at its distal end a sequence that can anneal to the second of the pair of primers, and said linking step comprises the steps of: (a) annealing a proportion of the 3' ends of the population of the immobilised primer to the proximal end of said construct; and (i) extending the 3' ends of said proportion using a polymerase that uses the distal end of said construct as a template; or (ii) annealing the second of the pair of primers to the distal end of said construct and ligating said second primer to the 3' ends of said proportion.
The invention further provides a method of ampliling a specific target nucleic acid sequence from within a sample, said method comprising the steps of: (a) contacting an array according to the invention with said sample; (b) enabling a strand of the target nucleic acid sequence to anneal to one of the pair ofoligonucleotide primers that can prime the amplification of said specific target nucleic acid sequence by PCR and that are immobilised in a single oligonncleotide primer spot of the array; and (c) enabling extension of the 3' end of said primer, using said strand as a template, to an extent such that the extension product comprises a sequence that can anneal to the other primer of the pair of oligonucleotide primers in that primer spot.
In preferred embodiments, said method comprises the subsequent step of removing said strand from the extension product, preferably further comprises the subsequent step of enabling the extension product to orientate such that it anneals to the other primer of said pair of oligonucleotide primers, and preferably comprises the subsequent step of enabling extension of the 3' end of said other primer using the first extension product as a template.
In preferred embodiments, at least one other specific target nucleic acid sequence from within the sample is simultaneously amplified at a different oligonucleotide primer spot.
Also within the scope of the invention is an array substantially as described herein, a method of preparing an oligonucleotide primer spot on an array substantially as described herein, and a method of amplifying a specific target nucleic acid sequence from within a sample substantially as described herein.
Brief Description of the Drawings
The invention will be described with reference to the accompanying drawings, in which: Figure 1 shows a target nucleic acid sequence; Figure 2 shows an array spot with primers a and b' immobilised upon the array via their 5' ends; Figure 3 shows how the spot of Figure 2 can be used to ampli' the sequence of Figure 1 using a template strand from within a sample; Figure 4 shows an embodiment of the arrangement of primers a and b' within an array spot; Figure 5 shows an embodiment of a method of preparing an oligonucleotide primer spot on an array according to thc invention; and Figures 6 and 7 illustrate elements of embodiments of a method of preparing an oligonucleotide primer spot on an array according to the invention.
Detailed description of Preferred Embodiment(s)
The invention relates to an array comprising a substrate. The substrate typically comprises a solid surface or matrix (such as a glass slide or silicon thin-film cell), such as those found in conventional so-called "microarrays", but could also be e.g. a collection of beads or optical fibres. Inimobilised upon (e.g. covalently attached to) the substrate is a plurality of oligonueleotide primer spots (i.e. discrete, separate regions) and within each spot there is a population of a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by Polymerase Chain Reaction (PCR); that population is immobilised upon the substrate such that the 3' ends of the primers can be extended (i.e. for each primer the 3'-hydroxyl group at its 3' end is free to participate in a condensation reaction). By amplification it is intended to mean "increase the copy number of'. The target nucleic acid sequence can be a double-stranded nucleic acid or either of the strands of a double-stranded nucleic acid; it can also be a single stranded molecule and!or its complimentary sequence. Though the oligonueleotide primer pair is defined by its ability to prime the amplification of a specific target nucleic acid sequence by PCR it is to be understood that this does not limit the use of the array to amplification of a target sequence by PCR (i.e. other amplification methods such as Recombinase Polymerase Amplification (RPA) and Multiple Displacement Amplification (MDA) are also contemplated).
The oligonucleotide primers can be any nucleic acid polymers (e.g. DNA or RNA or mixtures thereof), typically 5 to 200 nucleotide rcsiducs in length (more typically 10 to residues, and more typically 15-50 residues), that are configured such that they are capable of specifically priming amplification of a target nucleic acid e.g. each is complimentary to separate 3' ends of the (e.g. sense and anti-sense) strands of a double-stranded target sequence, or complimentary to the 3' end of a single stranded target sequence and to the 3' end of a sequence that is complimentary to that target sequence. At least two (preferably at least 10, more preferably at least 50, more prcferably at least 100, more preferably at least 500, more preferably at least 1000, more preferably at least 10000, most preferably at least 100000) of the oligonucleotide primer spots of the array are adapted (via the specific oligonucleotide primer pairs within them) to amplif' different target nucleic acid sequences. In preferred embodiments, no oligonucleotide primers are present within eacb spot other than the population of a pair of oligonuclcotidc primers that can prime the amplification of a specific target nucleic acid sequence by PCR.
As an example of the application of the array of the invention, we can consider that a specific spot is adapted to ampli' sequence X (comprising sequences a and b at its 5' and 3' ends, respectively), and complimentary sequence X' (with complimentary sequences a' and b' at its 3' and 5' ends, respectively) (Figure 1). That spot therefore comprises within it a population of primers a and b' (though Figure 2 shows only a single copy of each primer for clarity) that are immobilised upon the array substrate such that their 3' ends can be extended. Figure 3 shows how the spot can begin to amplify sequence XX using a template strand from within a sample, shown as a'-X'-b', that anneals via a! to immobilised primer a. The template strand can thus act as template for extension of the 3' end of immobilised primer a (e.g. using a suitable polymerase activity e.g. a DNA polymerase).
Once the original template strand is dissociated from the immobilised extension product that extension product can orientate such that its sequence at b can anneal to immobilised
S
primer b'. In this way the extension product can itself act as a template for extension of the 3' cnd of immobilised primer b'. Immobilised extension products can act as templates for non-extended, immobilised primers in the spot so exponential (or near exponential) amplification of a target sequence can occur within the spot with subsequent amplification cycles. Each spot of the array can be considered as a separate "microreactor" although spots can share a liquid phase (containing the other, general reagents needed for the amplification e.g. polymcrase activity, free nucleotides etc.) and reaction conditions.
Conditions permitting amplification within every spot of the array can be selected by one of ordinary skill in the art (e.g. extension time would need to be long enough to permit full extension of thc longest target sequence).
Thc array of the invention thus provides the possibility of large-scale, simultaneous amplification of different target sequences in the same vessel (e.g. multiplex PCR). By immobilising the separate reactions on an array the inventors are able to readily idcnti the target sequences that are being amplified by their position on the array; this has numerous applications such as detection of specific sequences from within a sample (useful for e.g. diagnostic purposes, monitoring expression profiles) and further characterisation of the immobilised, amplified sequence (e.g. sequencing thereof, useful for e.g. detection of allelie variants). Furthermore, immobilisation of discrete spots prevents interaction between different primer pair/template units, removing the artefacts that are commonly seen in conventional multiplex PCR systems. The use of a specific primer pair within a spot potentially enables exponential amplification of target sequences and thus increases the sensitivity & signal: noise ratio for any particular assay. The use of a primer pair, in comparison to a single probe as per e.g. a conventional microarray, facilitates the detection, capture and characterisation of generalised target sequences (e.g. a particular gene transcript, for which there might be several splice variants).
In preferred embodiments of the array of the invention, the minimum distance between the edges of any two adjacent spots is at least equal to the length of the longest target nucleic acid sequence that can be amplified (or that is expected to be amplified) by said adjacent spots, more preferably at least equal to the sum of the lengths of the target nucleic acid sequences that can be amplified by said adjacent spots. In this way the chances that the extension products of one spot can interfere with amplification within the adjacent spot are minimised.
Tn preferred embodiments the array of the invention is configured to reduce steric interference between the substrate and any 3' overhang of template nucleic acid (where a 3' overhang is any sequence that is 3' to the region that can anneal to one of the pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid within the template). In this way the annealing between template nucleic acid and primer can be maximised, especially for templates originating from a sampLe. For example: -the substrate could be at least partially porous to nucleic acid; and/or -any particular oligonucicotide primer could be raised from the substrate surface via a linker at its 5' end; and/or -any particular oligonucleotide primer could be immobilised upon a location of the substrate that is raised relative to a neighbouring location (see e.g. Figure 4).
The invention also provides a method of amplifying a specific target nucleic acid sequence from within a sample, said method initially comprising contacting an array of the invention with said sample and enabling a strand of the target nucleic acid sequence (e.g. one of the sense or anti-sense strands of a double-stranded target) to anneal to one of the pair of oligonucleotide primers that can prime the amplification of said specific target nucleic acid sequence by PCR and that are immobilised in a single oligonucleotide primer spot of the array. Where a double-stranded target is present this step might involve denaturing that target (e.g. using an appropriate elevation of temperature) or using a recombinase to align complementary sequences (as used within RPA). The method then comprises enabling extension of the 3' end of said one primer (typically with a polymerase enzyme) -using the strand of the target nucleic acid sequence as a temphite -to an extent such that the extension product comprises a sequence that can anneal to the other primer of the pair of oligonueleotide primers in that primer spot.
In preferred embodiments the method comprises the subsequent step of removing the template strand from the extension product. This can be done using e.g. an appropriate elevation of temperature and/or denaturing agent (e.g. formamide or a single strand binding protein). Preferably the method comprises the further subsequent step of enabling the extension product to orientate such that it anneals to the other primer of the pair of oligonucleotidc primers in that primer spot (via the sequence within the extension product that can anneal to the other primer of said pair of oligonucleotide primers -as described above). Preferably the method then involves enabling extension of the 3' end of said other primer using the first extension product as a template (preferably to an extent such that the second extension product comprises a sequence that can anneal to the first primer of the pair ofoligonucleotide primers in that primer spot). The duplex so-formed between extension products can be denatured (e.g. using an appropriate elevation of temperature andlor denaturing agent (e.g. formamide)) and such an extension-product-primingicxtcnsionidenaturation cycle can then be repeated (e.g. at least once, at least twice, at least 5 times, at least 15 times or at least 30 times).
In preferred embodiments of the above method(s), at least one other specific target nucleic acid sequence from within the sample is simultaneously amplified at a different oligonucleotide primer spot. Preferably at least 10, more preferably at least 50, niore preferably at least 100, more preferably at least 500, more preferably at least 1000, more preferably at least 10000, most preferably at least 100000 different target nucleic acid sequences are simultaneously amplified.
The detection of amplification of a target sequence can be carried out using conventional techniques, such as via the localisation of an intercalating agent or a major-or minor-groove binding dye to a particular spot (through reversible interaction with duplexes formed between immobiliscd cxtension products), and this can be quantified and followed over the course of the amplification (e.g. repeated rounds of PCR).
Methods of preparing the array of the invention The invention also provides methods of preparing an array as described above, and specifically methods of preparing an oligonucleotide primer spot on an array as described above. For example, separate, single synthesis reactions can be used to produce each of the primers of a primer pair, where a single synthesis reaction is one where the entire length of the primer is produced via sequential addition of bases (e.g. in conventional oligonucleotide synthesis, nucleotides are sequentially coupled to a growing oligonucleotide chain using the phosphoramidite method; the chain is grown whilst immobilised on a substrate and then released from that substrate when the chain is complete). The primers so produced can then be attached (e.g. "spotted") onto an array via their 5' ends using conventional methods e.g. linkage between an amine group at the primer 5' terminus and epoxy-silane on the array substrate. The attachment is preferably covalent.
However, in a first preferred aspect, a method is provided that comprises the initial step of immobilising a population of a combined primer construct on the substrate via its 5' end.
The combined primer construct comprises a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR, wherein the 3' end of the first of said primer pair is covalently linked to the 5' end of the second of said primer pair, and where in a proportion of said population the linkage between the pair of primers is selectively eleavable. This embodiment further comprises the step of selectively cleaving the linkage between the pair of primers in said proportion of said population.
Figure 5 illustrates this, where a population (two individuals) of a combined primer construct is attached to the array via the 5' end of the construct as a whole; the construct has been formed by linking the 3' end of primer a to the 5' end of primer b'. In a proportion (50%) of the affixed eonstmcts the linkage (a) is selectively cleavable i.e. under specific conditions linkage a can be cleaved without cleaving linkage J3 and without cleaving any other part of the combined primer construct. This aspect of the invention significantly reduces the costs associated with preparing an array with multiple spots because, for each spot, only one primer synthesis is required instead of two (and a single cleavage step can potentially be used to subsequently treat all spots).
Tn preferred embodiments of this aspect of the invention said proportion is 20-80%, more preferably 30-70%, more preferably 40-60%, and most preferably about 50%. Such ratios increase the chances of exponential amplification at the respective spot.
In preferred embodiments said immobilising step comprises synthesising said population via a single, separate synthesis reaction and attaching the resultant population on the substrate via the 5' end. Alternatively, said immobilising step comprises synthesising said population in situ using a single synthesis reaction (i.e. a synthesis reaction that builds the primer from its 5' end whilst attached to the substrate of the array); this particular embodiment is preferred because it removes the need for the attachment step.
Tn either case, a mixed population of the combined primer construct -where a proportion has a selectively cleavable linkage -can be formed e.g. by having a mixture of at least two different nueleotide monomers present during the step of adding (to the growing construct) a monomer at a site between the two primers of the construct, wherein one of those different nucleotide monomers provides the resultant construct comprising it with a site that is selectively cleavable.
For example, said monomer m[ght give rise to a backbone region providing for selective cleavage (e.g. a ribonuclcotide, for DNA primers (and vice-versa), or modified sugar-phosphate moiety (e.g. PNA, LNA or Morpholino)).
A1temativey, the nature of the base of the monomer might cnaNc selective base cxcision, to be followed by cleavage of the abasic site. For example, an RNA specific base (e.g. uracil. inosine) could be used for DNA primers. Else one could use other bases that can be targets for base excision (e.g. 5-Nitroindole, 7-deaza-7-nitro-dATP, 7-deaza-7-nitro-dGTP, 5-hydroxy-dCTP, and 5-hydroxy-dUTP, or oxidised, alkylated or deaminated bases), including those that recruit nucleotide excision repair machinery by forming a dimer with an adjacent base. As a variation of this, an abasic site mimic (e.g. tetrahydrofliran) could be used to direct abasic-site cleavage. In particularly preferred embodimcnts, a single enzyme activity can selectively remove the chosen base and cleave the abasic site. Hence for DNA primers it is particularly preferred that the monomer comprises the uracil base; uracil can be removed using an enzyme with Uracil-DNA Glycosylase activity and an enzyme with AP-lyase activity, though preferably an enzyme with both activities is used instead (e.g. Tma Endonuclease III).
A further option is to use a monomer that gives rise to a specific sequence (comprising it) that can bc used to direct cleavage. For example, that sequence could be a restriction enzyme site or could form a hairpin (which can subsequently be cleaved). Alternatively, the sequence could recruit a specific oligonucleotide probe to direct cleavage e.g. of double-stranded and/or mismatched nucleic acid.
In a second preferred aspect, a method is provided that comprises the steps of immobilising on a substrate via its 5' end a population of the first of a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR, and then recombining (e.g. using a recombinase) a proportion of said population with a nucleic acid construct such that the 3' ends of said proportion then consist of the second of said pair of oligonucleotide primers. In preferred embodiments said construct is double stranded nucleic acid. In preferred embodiments of this aspect of the invention said proportion is 20-80%, more preferably 30-70%, more preferably 40- 60%, and most preferably about 50%. Such proportions could be controlled e.g. by using an excess amount of the nucleic acid construct and utilising a relatively inefficient rccombination activity. Such preferred proportions increase the chances of exponential amplification at the respective spot.
In a third preferred aspect, a method is provided that comprises the steps of immobilising on a substrate via its 5' end a population of the first of a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR, and then covalently linking to the 3' ends of a proportion of said population the second of said pair ofoligonucleotide primers. In preferred embodiments of this aspect of the invention said proportion is 20-80%, more preferably 30-70%, more preferably 40-60%, and most preferably about 50%.
Tn preferred embodiments the immobilising steps as referred to above comprise immobilising a pre-formed population (i.e. a population synthesised via a single, separate synthesis reaction) on the substrate or, more preferably, synthesising said population in situ using a single synthesis reaction (where no attachment step is needed).
In preferred embodiments of the third aspect, the linking step comprises reversibly blocking a proportion of the 3' ends of the population of immobilised primer, extending the unblocked 3t ends using in situ synthesis (to the extent that the whole of the second of said pair of oligonucleotidc primers is formed 3' to the unblocked end), and unblocking the blocked 3' ends.
A combination of the preceding two embodiments is particularly preferred as it results in the need for just one synthesis reaction and no attachment step (reducing production cost).
In an alternative embodiment of the third aspect, the linking step comprises annealing a bridging oligonucleotide to a proportion of the 3' ends of the population of immobilised primer, wherein the 3' end of said bridging oligonucleotide can anneal to the first of the pair of oligonucleotide primers and the Send of said bridging oligonucleotide can anneal to the second of the pair of oligonucleotide primers. Figure 6 shows how a bridging oligonucleotide 3'-a'-b-S' can be used to extend immobilised primer a to feature primer b' at its 3' end. The method then comprises eithcr cxtcnding the 3' ends of said proportion using a polymerase that uses the bridging oligonucleotide as a template or annealing the second of the pair ofoligonucleotide primers to the 5' end of thc bridging o1igonuceotide primer and ligating said second primer to the 3' ends of said proportion.
In yet a further alternative embodiment of the third aspect, the 5' end of the first of the pair ofprimers is immobiliscd on the substrate via a construct that has at its proximal end (proximal to the first primer) a sequence that can anneal to the 3' end of the immobilised primer and has at its distal end (distal to the first primer) a sequence that can anneal to the second of the pair of primers. Figure 7(i) shows, as an example, primer a immobilised via a construct that has b at its distal end and z' at its proximal end (where z' can anneal to sequence z, where z is the 3' end of primer a). The method comprises the step of annealing a proportion of the 3' ends of the population of the immobilised primer to the proximal end of the construct, forming a hairpin structure. Figure 7(H) shows the hairpin that can be formed using the structure shown in Figure 7(i). The method then comprises extending the 3' ends of said proportion using a polymcrasc that uses the distal cnd of the construct (sequence b in Figure 7(il)) as a template or annealing the second of the pair of primers to the distal end of the construct and ligating the second primer to the 3' ends of said proportion.
Claims (1)
- <claim-text>Claims 1. An array comprising a substrate and a plurality of oligonucleotidc primer spots immobilised thereon, wherein: (a) within each spot a population of a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR is immobilised upon the substrate such that the 3' ends of said primers can be extended; and (b) at least two of said oligonuelcotidc primer spots arc adapted to amplify different target nucleic acid sequences.</claim-text> <claim-text>2. An array according to claim 1 wherein the minimum distance between the edges of any two adjacent spots is at least equal to the length of the longest target nucleic acid sequence that can be amplified by said adjacent spots.</claim-text> <claim-text>3. An array according to claim I or claim 2 that is configured to reduce steric interference between the substrate and any 3' overhang of template nucleic acid.</claim-text> <claim-text>4. A method of preparing an oligonucleotide primer spot on an array according to any one of the preceding claims, said method comprising the steps of: (a) immobilising a population of a combined primer construct on the substrate via its 5' end, where said construct comprises a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR, wherein the 3' end of the first of said primer pair is covalently linked to the 5' end of the second of said primer pair, and where in a proportion of said population the linkage between the pair of primers is se1ectivcy clcavable; and (b) selectively cleaving the linkage between the pair of primers in said proportion of said population.</claim-text> <claim-text>5. A method according to claim 4 wherein said immobilising step comprises synthesising said population via a single, separate synthesis reaction and attaching the resultant population on the substrate via the 5' end.</claim-text> <claim-text>6. A method according to claim 4 wherein said immobilising step comprises synthesising said population in situ using a single synthesis reaction.</claim-text> <claim-text>7. A method of preparing an oligonucleotide primer spot on an array according to any one of claims I to 3, said method comprising the steps of: (a) immobilising on a substrate via its 5 end a population of the first of a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR; and (b) recombining a proportion of said population with a nucleic acid construct such that the 3' ends of said proportion then consist of the second of said pair of oligonucleotide primers.</claim-text> <claim-text>8. A method of preparing an oligonucleotide primer spot on an array according to any one of claims 1 to 3, said method comprising the steps of: (a) irnmobilising on a substrate via its 5' end a population ofthe first of a pair of oligonucleotide primers that can prime the amplification of a specific target nucleic acid sequence by PCR; and (b) covalently linking to a proportion of the 3' ends of said population the second of said pair of oligonucleotide primers.</claim-text> <claim-text>9. A method according to claim 7 or S wherein said immobilising step comprises immobilising a pre-formcd population on the substrate or synthesising said population in situ using a single synthesis reaction.</claim-text> <claim-text>10. A method according to claim 8 or claim 9 wherein said linking step comprises reversibly blocking a proportion ofthe 3' ends of the population of immobilised primer, extending the unblocked 3 ends using in situ synthesis, and unblocking the blocked 3' ends.</claim-text> <claim-text>11. A method according to claim 8 or claim 9 wherein said linking step comprises annealing a bridging oligonucleotide to a proportion of the 3' ends of the population of immobilised primer, wherein the 3' end of said bridging oligonucleotide can anneal to the first of thc pair ofoligonucleotide primers and the 5' end of said bridging oligonucleotide can anneal to the second of the pair of oligonucleotide primers, and: (a) extending the 3' ends of said proportion using a polymerase that uses the bridging oligonucleotide as a template; or (b) annealing the second of the pair of oligonucleotide primers to the 5' end of the bridging oligonucleotide primer and ligating said second primer to the 3' ends of said proportion.</claim-text> <claim-text>U. A method according to claimS or claim 9 wherein the 5' end of the first of the pair of primers is immobilised on the substrate via a construct that has at its proximal end a sequence that can anneal to the 3' end of the immobilised primer and has at its distal end a sequence that can anneal to the second of the pair of primers, said linking step comprising the steps of: (a) annealing a proportion of the 3' ends of the population of the immobilised primer to the proximal end of said construct; and (i) extending the 3' ends of said proportion using a polymerase that uses the distal end of said construct as a template; or (ii) annealing the second of the pair of primers to the distal end of said construct and ligating said second primer to the 3' ends of said proportion.</claim-text> <claim-text>13. A method of ampIi'ing a specific target nucleic acid sequence from within a sample, said method comprising the steps of: (a) contacting an array according to any one of claims 1 to 3 with said sample; (b) enabling a strand of the target nucleic acid sequence to anneal to one of the pair ofoligonuckotidc primers that can prime the amplification of said specific target nucicic acid sequence by PCR and that are immobilised in a single oligonucleotide primer spot of the array; and (c) enabling extension of the 3' end of said primer, using said strand as a template, to an extent such that the extension product comprises a sequence that can anneal to the other primer of the pair of oligonucleotide primers in that primer spot.</claim-text> <claim-text>14. A method according to claim 13 comprising the subsequent step of removing said strand from the extension product.</claim-text> <claim-text>15. A method according to claim 14 comprising the subsequent step of enabling the extension product to orientate such that it anneals to the other primer of said pair of oligonucleotide primers.</claim-text> <claim-text>16. A method according to claim 15 comprising the subsequent step of enabling extension of the 3' end of said other primer using the first extension product as a template.</claim-text> <claim-text>17. A method according to any one of claims 13 to 16 wherein at least one other specific target nucleic acid sequence from within the sample is simultaneously amplified at a different oligonucleotide primer spot.</claim-text> <claim-text>18. An array substantially as described herein.</claim-text> <claim-text>19. A method of preparing an oligonueleotide primer spot on an array substantially as described herein.</claim-text> <claim-text>20. A method of amplifying a specific target nucleic acid sequence from within a sample substantially as described herein.</claim-text>
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| EP3034617A1 (en) * | 2014-12-16 | 2016-06-22 | Marcos Isamat Riviere | Method for the simultaneous identification of multiple same-genome fragments or multiple fragments from different genomes using a PCR-technique |
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