Methods of amplifying target nucleic acids in a sample

Abstract

A method of amplifying target nucleic acids in a sample uses a PCR amplification process in which the hybridizing step comprising hybridizing a part of any target nucleic acid in the sample to the corresponding first primer of the corresponding amplification site, hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the first primer on the amplification site can selectively bind to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pair.

Description

METHODS OF AMPLIFYING TARGET NUCLEIC ACIDS IN A SAMPLECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Application No.63 / 548, 695, titled SYSTEMS, APPARATUS AND METHODS FOR MULTIDIMENSIONAL MOVEMENT OF AT LEAST ONE COMPONENT, filed 01 February 2024, which is hereby incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] This disclosure relates to methods of amplifying target nucleic acids in a sample.BACKGROUND

[0003] Polymerase chain reaction (PCR) amplification is a widely used technique for amplifying nucleic acids. In a typical PCR amplification process, a target nucleic acid - in the presence of a primer and PCR reagents - can be subjected to repeated cycles of denaturation, annealing, and extension to generate amplification products corresponding to the target nucleic acid. PCR can generate large quantities of the nucleic acid, for use in numerous downstream techniques such as sequencing and diagnostics.

[0004] PCR amplification processes in which multiple target nucleic acids can be amplified simultaneously in a single vessel (“multiplex PCR”) have been developed, for example for diagnostic screening. However, these are often significantly limited in scope. For example, primer design is onerous owing to the need to work efficiently in the same reaction and there are limits in existing systems on the number of primers (typically 4-5). Improvements are needed. Further, improvements in handling the resultant amplified products are needed.SUMMARY OF THE DISCLOSURE

[0005] According to a first aspect of the present disclosure, there is provided a method of amplifying target nucleic acids in a sample. The method comprises providing a solid-phase amplification device comprising a plurality of amplification sites provided on a surface, each of the plurality of amplification sites comprising an individually controllable thermal device configured to control the temperature at a corresponding amplification site; immobilising a first primer corresponding to a target nucleic acid to each amplification site, wherein the first primer on each amplification site corresponds to a part of a different target nucleic acid such that each amplification site corresponds to a different one of a plurality of target nucleic acids; providing reagents for thermal amplification of each of the plurality of different target nucleic acids to the surface; providing the sample to the surface; and amplifying any of the plurality of target nucleic acids present in the sample at the corresponding amplification site using the corresponding first primer, the corresponding reagents and a polymerase chain reaction (PCR) amplification process to form amplification products corresponding to each of the plurality of target nucleic acids present in the sample. The PCR amplification process comprises hybridizing a part ofany target nucleic acid present in the sample to the corresponding first primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the first primer on the amplification site selective binds to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pairBRIEF DESCRIPTION OF THE DRAWINGS

[0006] The present disclosure will now be described in more detail with reference to the accompanying drawings, which are not intended to be limiting:

[0007] FIG. 1 illustrates a flowchart of a method according to the disclosure.

[0008] FIG. 2A depicts a top view of an amplification device used in a method according to the disclosure, FIG. 2B depicts schematic cross-sectional view of the amplification device in use through line A-A of FIG. 2A and Figs. 2C to 2F depict schematic cross-sectional views of a part of the amplification device in use through a part of line A-A of FIG. 2A.

[0009] FIG. 3 A depicts a top view of an amplification device used in a method according to the disclosure and FIGS. 3B and 3C depict schematic cross-sectional views of a part of the amplification device in use through a part of line A-A of FIG.3A.

[0010] FIG. 4 provides atop view of an amplification device which can be used in the methods and systems according to the disclosure.

[0011] FIG. 5 provides an expanded view of a part of the amplification device of FIG. 4.

[0012] FIG. 6 provides a schematic cross-sectional views of the amplification device through line A- A of FIG. 4.

[0013] FIG. 7 provides atop view of an amplification device which can be used in the methods and systems according to the disclosure.

[0014] FIG. 8 provides an expanded view of a part of the amplification device of FIG. 7.

[0015] FIG. 9 provides a schematic cross-sectional views of the amplification device through line A- A of FIG. 8.DETAILED DESCRIPTION

[0016] Despite the advantages offered by multiplex PCR, existing systems and methods still have drawbacks such as limited primers and amplification product handling functionality. Amplification product handling functionality often relies on manual processing by a technician.

[0017] According to a first aspect of the present disclosure, there is provided a method of amplifying target nucleic acids in a sample. The method comprises providing a solid-phase amplification device comprising a plurality of amplification sites provided on a surface, each of the plurality of amplification sites comprising an individually controllable thermal device configured to control the temperature at acorresponding amplification site; immobilising a first primer corresponding to a target nucleic acid to each amplification site, wherein the first primer on each amplification site corresponds to a part of a different target nucleic acid such that each amplification site corresponds to a different one of a plurality of target nucleic acids; providing reagents for thermal amplification of each of the plurality of different target nucleic acids to the surface; providing the sample to the surface; and amplifying any of the plurality of target nucleic acids present in the sample at the corresponding amplification site using the corresponding first primer, the corresponding reagents and a polymerase chain reaction (PCR) amplification process to form amplification products corresponding to each of the plurality of target nucleic acids present in the sample. The PCR amplification process hybridizing a part of any target nucleic acid present in the sample to the corresponding first primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the first primer on the amplification site selective binds to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pair.

[0018] The methods disclosed herein provide an improved PCR process and system in which the individual conditions for a particular primer and target nucleic acid pair can be provided locally to each pair on an amplification site. It will be appreciated that each primer and corresponding target nucleic acid pair will form a specific annealing or hybridization pair with a specific melting temperature (Tm) (and hence annealing temperature(Ta)). By provision of individually controllable thermal devices for each amplification site - and therefore associated with each specific primer - the thermal devices can be set to temperature specific to that primer and target nucleic acid. This in turn provides two key benefits. The first is that the process is not limited to primers which each have a similar annealing temperature. For example, where primers are included in a mixture in solution within a single vessel in a conventional manner, the vessel is only ever operating at a single temperature. If the primers do not have similar Ta, then some will not anneal (or at least, not efficiently). Selecting primers with similar Tais onerous and requires extensive investment in primer design and may not always be possible within a single vessel. The second key benefit is that the efficiency is improved. Each amplification site can be individually set to a specific set temperature using the associated thermal device which suits the specific primer / target nucleic acid pair. This in turn can improve binding kinetics and reduce incubation time, speeding up analysis.

[0019] It will be appreciated that target nucleic acids may not always be present. For example, in the context of diagnostics, it may be that an absence of a nucleic acid is indicative of a negative result for the screening. The provision of the plurality of amplification sites and the corresponding thermal devices will still improve this analysis by providing confidence and the ability to screen for multiple nucleic acids (and thus, conditions or species) simultaneously. Where at least one target nucleic acid is present, the methods will therefore comprise amplifying at least one of the plurality of target nucleic acids present in the sample at the corresponding amplification site using the corresponding first primer,the corresponding reagents and a PCR amplification process to form amplification products corresponding to the at least one target nucleic acid. The PCR amplification process will also comprise a hybridizing step comprising hybridizing a part of the target nucleic acid to the corresponding first primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the first primer on the amplification site can selectively bind to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pair. Where the plurality of target nucleic acids are present, the methods will therefore comprise amplifying the plurality of target nucleic acids present in the sample at the corresponding amplification site using the corresponding first primer, the corresponding reagents and a polymerase chain reaction (PCR) amplification process to form amplification products corresponding to the plurality of target nucleic acids. The PCR amplification process will also comprise a hybridizing step comprising hybridizing a part of each target nucleic acid to the corresponding first primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the first primer on the amplification site can selectively bind to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pair.Nucleic Acids and Amplification Products

[0020] PCR can be performed using a variety of nucleic acid templates or sequences as the target nucleic acid and can yield different types of amplified products, depending on the starting material, reagents and conditions. For example, the target nucleic acid can be a single -stranded or doublestranded nucleic acid, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The amplified products for double -stranded nucleic acids can be single or double -stranded nucleic acids. For RNA specifically, the PCR may comprise reverse transcription so as to generate complementary DNA (cDNA).

[0021] The PCR amplification process according to the methods disclosed herein produces an amplification product from the target nucleic acid. Each amplification product may be a copy of a corresponding target nucleic acid. For example, where the target nucleic acid is derived from denatured double stranded nucleic acids (i.e. the sample comprises double stranded nucleic acids), this may be a copy of the double stranded nucleic acids. Alternatively or additionally, this can be a single extended nucleic acid strand (comprising the primer and a new section corresponding to the remainder of the target nucleic acid not bound to the primer)- i.e. a single stranded copy of the specific target nucleic acid. The amplification product may be a copy of the target single stranded nucleic acid.

[0022] Accordingly, “target nucleic acid” (or “template nucleic acid”) in the sample means a nucleic acid chain or strand which is the target of the amplification process or a PCR cycle. This may be onechain or strand of a double stranded nucleic acid which is the ultimate target of the amplification process, for example, and the ultimate product. At first (e.g. in the first cycle or where unreacted in the first cycle), this may be the original target nucleic acid derived from the sample. As the cycles continue, the target or template nucleic acid may be original target nucleic acid or may be the amplification product of a previous cycle, or a nucleic acid formed by denaturing of the amplification product of a previous cycle.

[0023] Each amplification site and first primer will provide at least one corresponding amplification product, which may be immobilised on the amplification site or may be, in some embodiments, be released into the solution.

[0024] As used herein, the term “nucleic acid” (or NA) refers to a plurality of nucleotides, for example any oligomeric or polymeric form of nucleotides, including, but not limited to, DNA and RNA in either single-stranded or double-stranded form and synthetic analogues such as locked nucleic acids (LNA), peptide nucleic acids (PNA). An oligonucleotide may have nucleotide generally comprising a sugar moiety (e.g. a ribose or deoxyribose), a phosphate group, and a nucleobase. The nucleobase (or nitrogenous base) may be selected from the group consisting of adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), but may also include base analogues or chemically modified bases (e.g., 5- methylcytosine). The phosphate groups link the sugar moieties to form the backbone of the polymer or oligomer. The oligonucleotide may have contiguous bases but alternatively the bases may be interspersed with linker moieties. Each nucleic acid may have a 5'-terminus and a 3'-terminus, with the one or more nucleotides between the 5'- and 3 '-termini.PCR Overview

[0025] The methods disclosed herein comprise amplifying any of the plurality of target nucleic acids present in the sample at the corresponding amplification site using a PCR amplification process. A PCR amplification process according to the method can comprises a plurality of PCR cycles comprising the steps of (i) denaturing / release to form target nucleic acid(s) or template nucleic acid(s); (ii) hybridizing or “annealing” the target or template nucleic acid(s) to corresponding primers to form respective bound pairs; and (iii) extension of the primers while bound to the target or template nucleic acid(s). For the first cycle of the PCR amplification process, denaturing may only be carried out if the sample provided includes or may include double -stranded nucleic acids, where denaturing a doublestranded nucleic acid would lead to two target nucleic acids. Accordingly, for double stranded nucleic acids, the method comprises a plurality of PCR cycles comprising the steps of (i) denaturing / release of any double stranded nucleic acids in the sample to form a plurality of target nucleic acids. For single stranded-nucleic acids, the first cycle does not need an initial denature step and instead the single stranded-nucleic acid may form the target nucleic acid. So it may only be for the second cycle onwards that the PCR cycles comprise a plurality of steps of (i) denaturing / release; (ii) hybridizing or “annealing”; and (iii) extension. Denature or release in respect of the second cycle onwards may referto the amplification product of the previous cycle: that is, the target nucleic acid bound to the extended nucleic acid strand.

[0026] With each cycle of the PCR amplification process, the temperature is varied for each of the stages. That is, the temperature is changed depending on whether (i) denaturing / release; (ii) hybridizing or “annealing”; and (iii) extension is occurring. In conventional systems and methods, including those carrying out multiplex PCR, the temperature of the system is varied through the system as a whole, such that the whole system is held at the same temperature for each of these steps. The methods of the present disclosure allow for the varying of these temperatures on a site-by-site basis so that the control of each individual PCR amplification process occurring on each amplification site can be controlled, in some cases for each of these steps.

[0027] The number of PCR cycles in total may be from at least 5 cycles, such as at least 10 cycles. This may be from 5 to 100 cycles, such as from 5 to 50 cycles, or 20 to 40 cycles or 25 to 35 cycles. Where the sample comprises single stranded nucleic acids, this may include the first cycle which may not include a denaturing step. The conditions (e.g. temperature, duration of each step) in each PCR cycle may vary such that each cycle may comprise the same process but not the same precise conditions.PCR Denaturing / Release

[0028] As set out above, PCR amplification processes comprise a step of denaturing / release. The PCR amplification process of the methods may therefore comprise heating each amplification site to a target denature temperature at which a double stranded nucleic acid is denatured to form two nucleic acids (i.e. target or template nucleic acids). These can then be used in subsequent hybridization and extension steps. It will be appreciated for the second and subsequent cycles that the denaturing is a post-extension denature step such that the double stranded nucleic acid is one formed by the previous PCR cycle, such that it will comprise a target or template nucleic acid strand and an extended nucleic acid strand (formed of the primer and an extended portion). Denaturing in this case may cause release of the target or template nucleic acid strand and the extended nucleic acid strand - comprising the primer - may remain immobilised on the surface by virtue of the immobilisation of the primer.

[0029] This step may be present in the first cycle of the PCR amplification process or it may be that the first cycle of the PCR amplification process comprises a different initial denaturing step or no denaturing step. For example, where the sample comprises double stranded nucleic acids (or may comprise double stranded nucleic acids), an initial denaturing step may be carried out if the sample provided includes or may include double -stranded nucleic acids, where the initial denaturing step a double-stranded nucleic acid would lead to two target nucleic acids. The method may therefore, for the first cycle, comprise heating each amplification site to a target initial denature temperature. This may be for an extended duration and / or at a higher temperature compared to the other denaturing steps.

[0030] By heating each amplification site to a target denature temperature or initial target denature temperature, it is meant that the source of the heat (e.g. the individually controllable thermal) devices are set to maintain a specific temperature or specific temperature range. This may be that eachamplification site is at this temperature, for example that the centre of the amplification site as defined by the centre of the thermal device is at this target denature temperature.

[0031] In the denature step and / or initial denaturing step, where present, the heating may be using the thermal devices. That is, heating the sample to a denature temperature comprises controlling the individually controllable thermal devices to heat the sample to a target denature temperature. The thermal devices may be used together to heat the sample across the surface to a single temperature during the denature steps (including the initial denature step). For example, each may be set to the same target denaturing temperature. Alternatively, it may be that the thermal heater of each amplification site is set to a target temperature which is different for each amplification site and corresponds to a temperature at which the double stranded nucleic acids comprising the target nucleic acid will denature. This may increase the likelihood of interaction between the target nucleic acid species and the corresponding primer on the amplification site of interest. This is because double stranded nucleic acids having a denaturing temperature above the set denature temperature of a specific amplification will not denature at that site and, therefore, the risk of erroneous binding or other interference which may be caused by single stranded nucleic acids can be reduced.

[0032] The denaturing steps may be carried out at a temperature of greater than 80 °C, such as greater than 85 °C or greater than 90 °C. It will be appreciated that the denaturing temperature should not be so high as to degrade the various components used in PCR. Accordingly, the temperature range may be from 80 to 110 °C, such as 90 to 100 °C, such as 94 to 98 °C.

[0033] The duration of each the denaturing step may be at least 5 seconds, such as at least 30 seconds or at least 1 minute. For example, this may be from 5 seconds to 10 minutes, for example from 10 seconds to 2 minutes, from 15 seconds to 60 seconds. For an initial denaturing step, this may be at least 5 seconds, such as at least 30 seconds or at least 1 minute. For example, from 5 seconds to 10 minutes, for example from 1 minutes to 5 minutes)

[0034] An exemplary denaturing step may be from 94°C to 98°C for a duration of from 15 to 60 seconds. An exemplary initial denaturing step may be from 94°C to 98°C for a duration of from 1 to 5 minutes.PCR hybridization

[0035] PCR amplification processes include hybridisation of the target or template nucleic acid to the primer. As set out above, one drawback of existing multiplex PCR systems and methods is that these cannot account for different primer and corresponding target nucleic acid pair Taacross the various pairs. This either means that the selection of primers is very limited or the PCR amplification process is inefficient and / or inaccurate.

[0036] The methods disclosed herein comprise a PCR amplification process comprising a hybridizing step comprising hybridizing a part of any target nucleic acid in the sample to the corresponding first primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site isdifferent and corresponds to a temperature at which the first primer on the amplification site can selectively bind to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pair. By provision of individually controllable thermal devices for each amplification site - and therefore associated with each specific primer - the thermal devices can be set to temperature specific to that primer and target nucleic acid. This avoids these drawbacks.

[0037] This may be a hybridizing step of one cycle of the PCR amplification process, for example the first, or it may be that each hybridizing step will occur in this way. For the subsequent PCR cycles (i.e. the second and subsequent), the correspond hybridizing step may comprise hybridizing a part of any target nucleic acid (which may be initial target nucleic acid or the amplification product of a previous cycle, or template or target nucleic acids formed by denature of the amplification product of a previous cycle) in the sample to the corresponding first primer of the corresponding amplification site (or in some cases to templates formed by the previous cycles); and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the first primer (or templates formed by the previous cycles) on the amplification site can selectively bind to a corresponding part of the target nucleic acid so that the first primer (or templates formed by the previous cycles) and the part of the target nucleic acid can hybridize to form a bound pair.

[0038] By heating each amplification site to a target temperature (or target hybridization temperature), it is meant that each individually controllable thermal device is set to maintain a specific temperature or specific temperature range. This may be that each amplification site is at this temperature, for example that the centre of the amplification site as defined by the centre of the thermal device is at this target temperature. The target temperature is therefore a temperature at which the thermal device is set - i.e. is seeking to achieve on the amplification site. Accordingly, hybridizing may comprise individually controlling each of the thermal devices so that the temperature amplification site is different and corresponds to a temperature at which the first primer on the amplification site can selectively bind to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pair. “At each amplification site” means that the temperature of the environment surrounding the primers located on each site, and in some embodiments the temperature of the primer itself is controlled so as to be at a temperature at which the corresponding primer and nucleic acid will hybridize or anneal.

[0039] The target temperature (or target hybridization temperature) may be at least 3 °C or at least 5 °C below the melting temperature (Tm) (i.e. Tm- 3, or Tm- 5) for the corresponding primer and nucleic acid pair. Accordingly, individually actuating each of the individually actuatable thermal devices so as to raise the temperature of the primer on each site to a target temperature at which the primer and the corresponding nucleic acid pair can anneal is a temperature of at least 3 °C less than the melting point of the corresponding pair. For example, this may be a temperature of from 3 to 15 °C below the Tm,such as from 4 to 12 °C below the Tm, such as from 5 to 10 °C below the TmTmfor a bound pair of primer and nucleic acid is the temperature at which 50% of the primer and nucleic acid are bound (in duplex). This may be the Ta.

[0040] There are a number of different methods which can be used to calculate the annealing temperature (Ta) of a bound pair. However, it will be appreciated that no one particular method need be employed to determine a temperature at which the primer set and the corresponding nucleic acid pair can anneal. Instead, this could be determined through experimental testing, for example to determine when optimal PCR occurs for a particular bound pair. This may use a melting curve to determine the Tmand deriving the appropriate target temperature based on Tm, as set out above.

[0041] The target temperature on each site may be a range or value of from 40°C to 65°C, for example from 50 °C to 65 °C. This may be for a duration of at least 1 second, such as at least 5 seconds, or at least 10 seconds. For example, this may be from 1 second to 2 minutes, for example from 5 seconds to 60 seconds, from 15 seconds to 60 seconds.

[0042] Although the hybridization has been discussed with respect to a target nucleic acid and the primer, it will be appreciated that other hybridization may occur during the PCR amplification process. At least some of the nucleic acid molecules are elongated and released (see below). This is referred to as ‘interfacial amplification’ as nucleic acid in solution will repeatedly hybridise to attached primers. There is also a further mechanism in which the immobilized copies (i.e. extended nucleic acid strands) formed also hybridise to other primers and form additional copies on the same amplification site. This second process is called ‘surface amplification’. Accordingly, in such a circumstance, the “target nucleic acid” is immobilised on the surface.PCR extension

[0043] The PCR amplification process comprises an extension step. This may comprise heating each amplification site to a target extension temperature at which an extension on the first primer of the bound pair can occur to form a corresponding immobilised extended nucleic acid strand. This creates the copies of the target nucleic acid using a polymerase which can be provided on the surface (for example, provided to each amplification site as a reagent). Example polymerases include DNA polymerase, for example Taq polymerase or another suitable variant with high fidelity and / or reduced error rates (e.g., Pfii, Phusion™, or Q5™). For PCR of RNA, an example polymerase which can be use is reverse transcriptase (RT). The polymerase acts to add nucleotides to the 3'-end of the primers of each bound pair, creating a new strand on the primer which is complementary to the target or template nucleic acid.

[0044] The extension step is carried out under heating. Heating each amplification site may comprise individually controlling each of the thermal devices so that a target extension temperature for each amplification site is different and corresponds to a temperature at which the first primer of the bound pair on each amplification site can be extended to form the corresponding extended nucleic acid strand.The target extension temperature may be being between the target denature temperature and the target hybridization temperature.

[0045] In the final cycle of the PCR amplification process, the extension step may be an extended extension step, which has a duration longer than that of the other extension steps. The set temperature may be the same.

[0046] By heating each amplification site to a target extension temperature, it is meant that the source of the heat (e.g. the individually controllable thermal) devices are set to maintain a specific temperature or specific temperature range. This may be that each amplification site is at this temperature, for example that the centre of the amplification site as defined by the centre of the thermal device is at this target temperature .

[0047] Individual control of the extension on each amplification site can be advantageous. First, as with the other steps, this allows for setting of conditions optimised for a particular primer / target nucleic acid, since the optimal temperature conditions will differ. Second, this can be used to selective stop and start extension on each amplification site. For example, the amplification may be monitored and once sufficient extension on a specific amplification site (for example, as detected by a sensing element) has occurred, the method may comprise reducing the temperature of the corresponding site while at least one other amplification site remains at the target temperature. This may prevent or reduce unwanted side reactions and / or may be used to speed up the cycle process by beginning the next step (e.g. cooling) on particular amplification sites as soon as the extension step is complete for that site.

[0048] The extension steps may be carried out at a temperature of from 60 °C to 80 °C, such as from 65 °C to 75 °C, such as 68 °C to 72 °C. This may be for a duration of at least 1 second, such as at least 5 seconds, or at least 10 seconds. For example, this may be from 1 second to 2 minutes, for example from 5 seconds to 60 seconds, from 15 seconds to 60 seconds. Where present, the extended extension step may be for a duration of at least 30 seconds, such as at least 1 minute or at least 5 minutes. For example, this may be from 30 seconds to 20 minutes, for example from 1 minute to 15 minutes.

[0049] An exemplary extension step may be from 68 °C to 72 °C for a duration of from 15 to 60 seconds. An exemplary extended extension step may be from 68 °C to 72 °C for a duration of from 1 minute to 15 minutes.Release

[0050] The methods disclosed herein can result in a bound amplification product on the amplification site in the form of a bound target nucleic acid and extended nucleic acid strand. After the PCR amplification process has been completed - i.e. after the desired number of cycles have been completed - there may be a further step of releasing at least a part of this bound amplification product from the amplification site, for example for analysis or further use.

[0051] In one embodiment, the method may further comprise a release step comprising applying a stimulus to at least one amplification site such that a bound target nucleic acid and extended nucleicacid strand on the amplification site are released together from the amplification site. For example, each primer may be embolised on (i.e.) bound to the site by a cleavable linker, such as an electro-active linker (cleavable by the application of voltage or current) or a heat -cleavable linker (cleavable by the application of heat). Application of the stimulus can cleave this linkage, releasing the bound pair. Similarly, the method may also or alternatively comprise a release step comprising applying a stimulus to at least one amplification site such that an immobilised extended nucleic acid strand is released from the amplification site. Alternatively or additionally, the method may further comprise a post-extension denature step (i.e. a release step) comprising heating each amplification site to a target denature temperature at which the target nucleic acid is released from the immobilised extended nucleic acid strand.

[0052] These release steps can be selective release steps. In particular, the method may comprise selectively releasing the amplification products on one amplification site, for example while the amplification products on other amplification sites are retained thereon or after they have been removed from the surface. The selective release (and recovery) may be so as to purify the amplification product - i.e. remove the product from a mixture with at least one other species, but optionally it may be to isolate the amplification product from any other species (such as other amplification products, reagents, sample). The method may further comprise recovering the released amplification product separately to the other of the plurality of amplification products. The method may then further comprise analysing the amplification product.

[0053] By selective release, it is meant that one specific amplification product (whether this is a single nucleic acid strand (i.e. the target nucleic acid), immobilised extended nucleic acid strand or the bound pair) is / are released from the surface. This may be that a specific amplification product is / are released from the corresponding amplification site(s) to which it is / they are bound, while the other amplification products are retained on their respective amplification sites. In this way, the method can separate the amplification products for further use. In some cases, it may be that all of the different amplification products on one amplification site are released from the corresponding amplification site(s) to which it they are bound, while the other amplification products on other amplification sites are retained on their respective amplification sites. In this way, the method can separate the amplification products for further use. It will be appreciated that where one amplification product (i.e. one type) is retained across plural amplification sites, the amplification products may be selectively released from the plural amplification sites on which it is retained, for example simultaneously.

[0054] Selective release may be triggered by an external stimulus, or may be caused by the amplification device.

[0055] The stimulus (or “release stimulus”) is an input which either directly or indirectly causes the release or detachment from the amplification site. The stimulus may accordingly be an input which directly acts on the bonds or interaction between the bound pair, such as an electrical input (e.g. current or voltage), light energy, thermal energy. Alternatively or additionally, it may be indirect, for examplethe stimulus may cause a change in the environmental conditions directly on and adjacent the amplification site (such as a change in temperature or change in pH), which in turn causes the detachment (or “dissociation”).

[0056] The stimulus may be provided by the amplification device. For example, this may be provided by a modification element configured provide an electrical input (e.g. current or voltage), light energy, thermal energy to a specific amplification site. Each amplification site may comprise a corresponding modification element. This may be an electrode, for example. In some embodiments, this may be the individually controllable thermal device. Alternatively or in addition, it may be a force generated by the device (such as an electric field) which can cause debinding.

[0057] Where the individually controllable thermal device is used to provide the stimulus, care must be taken not to interfere with the PCR amplification process, damage the resultant amplification products or to cause unintended detachment during the PCR amplification process.

[0058] Prior to applying the release stimulus, the method may comprise applying a first stimulus to the amplification site, the first stimulus having a lower intensity than the release stimulus (for example, where the release is a post-extension denature step (i.e. a release step) comprising heating each amplification site to a target denature temperature at which the target nucleic acid is released from the immobilised extended nucleic acid strand) such that it can cause debinding of any species not bound to the surface of the amplification site with the same strength. It will be appreciated that the PCR amplification process may result in imperfect matches which may be less strongly bound due to imperfected base pair interactions. Further, there can under certain circumstances be non-specific binding between species. This binding will be non-specific and results in a weaker interaction between the two components in question. The first stimulus can be selected to remove these other and non- specifically bound components which are then removed before selective release and / or analysis of the amplification products. In some embodiments, the amplification of the first stimulus may occur prior the denaturing step of at least one cycle of the PCR amplification process, for example from the second cycle onwards. This may be each cycle.

[0059] Where the type of stimulus used as the first and as the release stimulus is the same, the first stimulus may therefore be less than the release stimulus. For example, if the stimulus is application of a voltage or current, the first stimulus may provide a lower voltage or current than the release stimulus. This may take the form of a continuous increase in the stimulus applied to the amplification site(s) in question, for example ramping up from or through the first stimulus sand to the debinding stimulus but separately (or only) collecting the species portion released at the release stimulus. By “lower intensity” it is meant that the force is lower. Variation of the force may be based on the strength or intensity (e.g. as measured by the input voltage (V / m or mV / m)) of the field applied to generate the force, such as the electric field. The strength of the electric field may vary dependent on the species being moved.Primers

[0060] The method comprises immobilising a (different) first primer to each amplification site. By primer it may be singular or may be plural, such that immobilising a first primer may comprise immobilising a plurality of first primer molecules to each amplification site. Primers are single chain nucleic acids, for example an oligonucleotide, having a structure (sequence) corresponding to or complimentary to the target nucleic acid (e.g. formed of DNA nucleotide bases (also known as dNTPs) A, T, C and G, such that it can anneal to or hybridize to a part of the target nucleic acid. Primers used in the methods of the present disclosure may have a length of from 10 nucleotides to about 40 nucleotides, for example from 10 to 35 nucleotides.

[0061] A primer generally comprises a phosphodiester backbone and a sequence of ribonucleotide or deoxyribonucleotide bases configured to hybridize with the complementary region of a target nucleic acid. “Primer” as used herein may be a forward primer or reverse primer.

[0062] In these SP-PCR methods where the or each primer is tethered or bound to the amplification site. The or each primer (for example, a plurality of individual primer molecules or a plurality of different types of primers) can be located on a surface providing the amplification site. This can be achieved in any suitable manner, such as by covalently or non-covalently immobilizing the primer(s) to the surface. The or each primer may be bound by their 5 '-terminus so as to form 5 ’-bound primers. This enables the free 3'-end to undergo extension (with polymerase). In other embodiments, this may be bound by the 3 ’-end. The primer may further comprise functional groups such as amines, thiols, or other reactive moieties (e.g., aldehydes or maleimides) to facilitate covalent attachment to the surface (e.g. at the 5 ’-terminus). For example, an amine -modified 5' end can react with an NHS-ester-modified surface, forming a stable amide (linker) bond. The surface defining the amplification site may be functionalised or otherwise provided so at to bond to the or each primer(s) provided thereon. A linker molecule may also be provided between the surface and the primer.

[0063] It will be appreciated that in the methods disclosed herein, only one of the primers for a PCR amplification of a double stranded nucleic acid is bound to the surface, and the other can be present in solution or both primers can be bound to the surface (corresponds to bridge amplification).

[0064] Where the sample comprises double stranded nucleic acids, the methods may further comprise immobilising a second primer to each amplification site, wherein the first primer on each amplification site corresponds to a first target nucleic acid of one double stranded nucleic acid and the second primer on each amplification site corresponds to a second target nucleic acid of the same double stranded nucleic acid. The first primer and the second primer may correspond to forward and reverse primers for the double stranded nucleic acid. That is, one may correspond to one target nucleic acid of the double stranded nucleic and one may correspond to the other. Accordingly, amplification of the double stranded nucleic acid may occur on one amplification site.

[0065] In such a case, there may advantageously be multiple processes for amplification. As set out above, during PCR, at least some of the nucleic acid molecules are elongated and released (see below). This is referred to as ‘interfacial amplification’ as nucleic acid in solution will repeatedly hybridise toattached primers. There is also a further mechanism in which the immobilized copies (i.e. extended nucleic acid strands) formed also hybridise to other primers and form additional copies on the same amplification site. This second process is called ‘surface amplification’. Accordingly, in such a circumstance, the “target nucleic acid” is immobilised on the surface.

[0066] Accordingly, in some embodiments, the first primer and second primers are adjacent on the amplification surface.

[0067] Where there is a second primer, the hybridizing step of the PCR amplification process further comprises hybridizing a part of the second target nucleic acid to the corresponding second primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the second primer on the amplification site can selectively bind to a corresponding part of the second target nucleic acid so that the second primer and the part of the second target nucleic acid can hybridize to form a bound pair. The other embodiments set out for the first primer apply to the second primer.

[0068] Where there are first and second primers, it will be appreciated that the Tmand Tamay vary as this will depend on the region of overlap between each primer and the corresponding target nucleic acid, and hence the target hybridisation temperature for each may differ. In such a case, either a composite target temperature at which both may hybridize may be used or, in other embodiments, the method may comprise individually controlling each of the thermal devices so that a first target temperature is used, followed by a second target temperature. The same heating configurations for the first primer apply equally to the second primer.

[0069] By “first” primer, it is meant a first primer is of that amplification site. This may be a singular primer or may be plural molecules or species of one type of primer. In the broadest aspect, the structures of each first primer of each of the plurality of sites are different. Any amplification site may further include additional primers, such as a second primer. The first and second primers have different structures and correspond to different nucleic acids (i.e. have different sequences).

[0070] In some embodiments, immobilising a first primer corresponding to a target nucleic acid to each amplification site comprises synthesising the first primer on each amplification site. That is, the first primer - and indeed any primer - may be synthesised on the corresponding amplification site (i.e. in situ). For example, this may be achieved using a solid-phase synthesis based on the phosphoramidite method. This may be achieved by tethering or attaching a base oligonucleotide to the surface and connecting oligonucleotides fragments on site, for example through annealing and ligation or polymerase reactions, for example. In some embodiments, the in situ synthesis of the primer(s) on each amplification site may be controlled by the thermal control, and this may be carried out by the corresponding thermal device. For example, WO 2019 / 145713 Al, the content of which is incorporated herein in its entirety, discloses a method of synthesis of oligonucleotides onto the surface of a solidsubstrate under thermal control. Such a method can be used to form the primer(s) on the corresponding amplification site.Reagents

[0071] Any suitable reagents may be used. The reagents (a “PCR reaction mixture”) may comprise an enzyme for extension (e.g. a polymerase), one or more nucleotides (in some cases, a plurality) (deoxynucleotide triphosphates (dNTPs) and a buffering agent. A divalent cation source, such as magnesium ions, may also be present.

[0072] The concentration of each dNTP may range, for example, from about 20 pM to about 500 pM. The divalent salt, such as MgCL (or an equivalent salt), may be provided at a final concentration of about 1.0 mM to about 5.0 mM, as optimized for the particular reaction. The buffer may be formulated to provide a pH of approximately 7.5-9.0 at the reaction temperature, and can include additives (e.g., KC1 or (NH^SCh) that enhance polymerase activity. In certain embodiments, additional agents such as betaine, DMSO, formamide, or detergents may be incorporated to improve or facilitate amplification.Heating and individually controllable thermal devices

[0073] As set out above, the methods disclosed herein comprise setting a target temperature, namely a target hybridised temperature, and may further comprise setting other target temperatures, such as a target initial denature temperature, a target denature temperature and a target extension temperature. The methods may further comprise controlling the thermal devices such that each amplification site or the primer thereon is at these temperatures. In some cases, the target temperature (of any of the types listed above) for each amplification site is different. The target temperature may be a range, such that the range set for each of the plurality of sites is different. Where the target temperature is a range, the ranges may overlap in part provided that the ranges are different, for example, the central value of the range may be different and one range may not fully encompass the other. The extent of the ranges may be the same (i.e. each range may extend from a lower value to an upper value and have an extent of 1°C, 2°C) but the central value of the range may be different. Where this is a set temperature value - i.e. a single value - these are different values. For example, a different of at least 0. 1°C, such as at least 0.2°C, or at least 0.5°C. In some embodiments, the temperature (i.e. a set value) of each of a plurality of sites is different. It will be appreciated that, in some embodiments, there may be amplification sites set to the same temperature, but there will also be other amplification sites with a different target temperature.

[0074] The thermal devices can be or comprise a heater configured to heat the amplification site. This may be a resistive element, for example, to which a current or voltage may be applied to generate heat. The thermal device may additionally comprise a cooler so as to cool the amplification site. This may be a local heating and, where present, whereby the increase in temperature is confined to the reactor site (e.g. adjacent reactor sites have a temperature difference of less than 10% of that of the reactor site in question). Each thermal device is individually controllable (or actuatable) so that it can be used toheat or cool independently to the other devices. This may be under the control of a controller or processor. In some embodiments, the thermal devices can be operated together.

[0075] The thermal devices can be provided with or set at a target temperature for each amplification site. By this is it is meant that the thermal devices or the controller controlling each may aim to keep the amplification site at a particular temperature or within a particular range. A thermometer may be provided at each amplification site to provide a measurement signal indicative of the temperature and / or the thermal device may act as a thermometer. This may be used to guide operation of the thermal device (i.e. increase or decrease the heat or cooling provided).

[0076] Each of the individually controllable thermal devices of the plurality of amplification sites may be spaced apart from the other individually controllable thermal devices. This can assist with the application of a stimulus since the stimulus can be applied locally and is less likely to impact another site. This may be a spacing of at least 0.01 mm, such as at least 0.05 mm, at least 0.1 mm, at least 1 mm or at least 2mm. For example, from 0.01 mm to 10 cm, 0.01 mm to 5 cm or 0.05mm to 2cm. A spacing of 0.01 or 0.05 has been found to result in very little heat transfer for thermal devices provided within a substrate and having a diameter of 100 pm. The substrate, dielectric or insulator may be provided between a part of each of the reactor sites.Sensing and analysis

[0077] The method may further comprise monitoring the progression of a step of the PCR amplification process on each amplification site. The method may further comprise operating the corresponding thermal device of a specific amplification site based on the monitoring. This aids with termination or initiation of any of the denaturing, hybridizing or elongation steps during the PCR amplification process and further steps such as the release step. For example, this may be operating the corresponding thermal device of a specific amplification site in isolation to the other thermal devices of a specific amplification site (e.g. making a change to only one or a sub-set of the plurality of the specific amplification sites).

[0078] A sensing element can accordingly be used to monitor each amplification site. The sensing element may be provided on or beneath each amplification site (e.g. within the substrate). The sensing element may be an optical sensing element, thermal sensing element, pH sensing element, ion sensing element, conductivity sensing element, fluorescence sensing element or a sensing element configured to detect a chemical property. Electrochemical sensors such as amperometric, potentiometric or conductometric sensors may also be employed. This can then be used to provide information on a state of an amplification site. The method may further comprise performing additional steps based on this determination, such as the selective release of the amplification products from the amplification site in question.

[0079] The sensing element (or “sensing device”) may provide a measurement signal indicative of the property and therefore may be addressable in some methods to provide the measurement signal. The method may determine a property based on the measurement signal. For example, the property may bean intrinsic property of the product, such as the structure, melting point, etc. In particular, this may be analysis of the structure of the product, for example. In another embodiment, the property may be an extrinsic (or relational) property. In some embodiments, the property may be a concentration of the product on the capture site, or on the product surface. For example, this may be determination of a number of the individual product molecules or entities on the capture site.

[0080] For example, the sensing element may comprise an electrode, which electrode may be addressable to provide the measurement signal. In some embodiments, there may be plural electrodes. Where there are plural electrodes, each may be individually addressable. A sensing layer (e.g. a dielectric layer) may also be provided on or adjacent the electrode to enhance or enable the response of the electrode to a particular component or analyte. In other embodiments, the sensing element may comprise a layer or structure through which current is passed (for example, where the sensing element comprises is a resistive layer or structure located between first and second electrodes). In some embodiments, a portion of the substrate defining the surface and / or a portion of the solution or medium may define the sensing layer or structure between electrodes and the sensing element may comprise a pair of electrodes arranged to interrogate the portion of substrate and / or medium such that sample or a component in this particular region can be interrogated. In other embodiments, a ferrocene label can be used to electrochemically detect amplification products. Fc-dUTP has been used, for example, as a label which increases redox current with increased amplification product formation on a site.

[0081] The method may comprise sensing a property on each amplification site indicative of the progression of the PCR amplification. For example, this may be sensing an optical property or electrical property on each amplification site. This may be during the PCR amplification process.

[0082] This can be used for a number of different reasons, including operating the individual sites but also for providing an early indication as to a positive or negative indication as to the presence of a target nucleic acid.

[0083] Accordingly, where there are a plurality of reactor sites, each reactor site may comprise a sensing element configured to sense or analyse at least one of (i) each part of the sample on each reactor site during the application of the stimulus; and (ii) the product on the surface.

[0084] In some embodiments, it will be appreciated that a single element may be operable to perform the same function of the sensing element and the modification element. For example, an electrode may be operable to provide the measurement signal and provide the stimulus. Accordingly, in some embodiments the modification element may be a sensing element.

[0085] Sensing may comprise optical detection through fluorescently labelled probes (e.g., TaqMan®, Sybr Green I) or fluorescence-resonance energy transfer (FRET)-based techniques. Amplified products can be detected directly on the support using fluorescence scanning, chemiluminescence, or other labelbased or label-free methods (e.g., surface plasmon resonance). In other embodiments, enzymatic labels may be incorporated into the amplified DNA.

[0086] Accordingly, each amplification site further comprises at least one sensing element configured to provide a measurement signal indicative of the presence of nucleic acids on the corresponding amplification sites; and wherein the method further comprises monitoring the PCR amplification process on each amplification site using the sensing the sensing element.

[0087] In some embodiments, the method may further comprise determining a melt temperature for an amplification product produced on an amplification site by applying heat to the amplification product the using the individually controlling the thermal device. This can provide in situ analysis of the amplification products, and accordingly can be used as a confirmation of the presence of the amplification product on the site. The presence of the individually actuatable thermal devices allows for this to be carried out in situ on a site-by-site basis.Amplification sites

[0088] The method comprises providing an amplification device comprising a plurality of amplification sites. The amplification sites are each a distinct region of the surface and each comprise a portion of the surface. Each amplification site comprises a thermal device. The amplification sites may each be defined by the extent of the thermal device. Where the sensing element is also present on each site, this may be defined by the outer extent defined by the elements (including the thermal device) present. This is relative to the extent across the surface at the amplification site (i.e. perpendicular to the amplification site at the surface). This may be alternatively defined by the extent of the coverage of the primer(s) on the surface.

[0089] Each of the amplification sites may be in fluid communication. In other words, these are such that a sample provided to the surface can interact with each of the amplification sites. This provides a single vessel or device solution which is easy to operate.

[0090] The structure of the amplification device lends itself to semiconductor manufacturing processes. As such, the number of amplification sites which can be provided on a substrate can be much larger than conventional amplification systems. In some embodiments, the number of amplification sites on the amplification device is at least 16, such as at least 96 or at least 300.Target copy number

[0091] The amplification device and method can be used to provide a quantitative PCR measurement and, therefore, a method to quantify target copy number. As set out above, the methods can be used to monitor progression during PCR, which can be used to determine target copy number. Further method steps may also enhance a determination.

[0092] For example, the method may further comprise providing a further amplification site and immobilising a first primer having the same structure as a first primer of one of the plurality of amplification sites, wherein at least one of: the number or density of first primers on the further amplification site and amplification site is different or the concentration of the corresponding targetnucleic acid provided to each amplification site is different. This can provide two amplification sites with first primers having the same structure (i.e. sequences) but to which different amounts of the primer or target is provided. This can give additional measurement data (e.g. using sensing elements on each site) which can be used as a data range to determine target copy number. This may also be useful where the quantity is unknown and having different amplification sites with different responses to an amount (for example, a different dynamic range) . These can be combined with varying the temperature on each of these sites to further provide data on the responses.Manipulation and separation

[0093] The methods may further comprise applying a force to at least one amplification product so as to migrate the amplification product relative to the surface.

[0094] The methods may further comprise applying a force to the sample to move at least a part of the sample relative to the surface. For example, during the step of providing the sample to the surface, a force may be applied so as to move species within the sample (i.e. nucleic acids) to and / or across the surface. This can be used to move the sample to each of the amplification sites, for example. Alternatively or additionally, a force may be applied during the step of recovering the released amplification product (where present) so as to move the isolated amplification product away from the amplification sites and recover the amplification product separately. For example, the systems and methods can use these electrodes to generate electric or magnetic fields that guide sample migration along predefined paths or affect a change in the environmental conditions which in turn create a force which causes movement of the along a pathway.

[0095] Use of such a force can advantageously quickly and efficiently transfer the nucleic acids (before, during and after PCR) without direct handling. Both large-scale and small-scale movements are possible within the system. In the methods disclosed herein, applying a force in the method may comprise applying an electric field and / or a magnetic field to the sample or amplification product(s). The use of an electric field for movement of charged species on the surface (and, more generally, in the device) is an efficient and effective means by which to manipulate the various species. It will be appreciated that various forces, such as those applies by electric and / or magnetic fields, can be used. With nucleic acids, these are charged and hence electric fields are a useful tool for manipulation and migration. This is also advantageous in that it can be combined with the other functionality disclosed herein.

[0096] The methods may further comprise applying a field (e.g. an electric field) to the target nucleic acids so as to cause movement of the target nucleic acids towards the surface, such as towards the amplification sites. Such a method can speed up measurement time by causing movement of the species in the sample to migrate to the sites at a movement speed which is greater than diffusion. This can reduce the time of or reduce the need for an incubation period.

[0097] Accordingly, the amplification device may comprise at least one manipulation assembly operable to cause an amplification product or sample to move on (e.g. along or towards) the surface. In other words, this may be operable to apply a force which causes an amplification product or a sample or a component thereof (e.g. a nucleic acid) to move towards a desired region of the device. Such a manipulation assembly may be operable to generate an electric field and / or a magnetic field to provide the respective force.

[0098] The first manipulation assembly may comprise a first electrode set arranged and operable to provide the respective force. Use of electrodes provides a precise control over the applied forces and allows for integration of the assemblies into a chip or integrated circuit for precise manufacture and miniaturisation. The methods disclosed herein may therefore further comprise operating a manipulation assembly to provide a force. The first electrode set can comprise a pair of electrodes provided as an electrically connectable or connected pair of field-generating electrodes for generating a field for influencing an amplification product, the sample or a part thereof (e .g . an electric field and / or a magnetic field). Each of the pair of electrodes can be provided at an opposing end of the pathway and to define the entire length of the pathway. In other words, these are spaced apart and medium is provided therebetween so that the region defined between them is the pathway. The pair of electrodes are electrically connectable or connected so that the field providing the force can be defined therebetween along the length of the pathway. Where a magnetic field is used, this can be applied using an electromagnetic manipulation assembly configured to apply a magnetic field across at least a portion of the respective pathway. The use of an electric field or magnetic field to provide the force is particularly advantageous with the structures and arrangements disclosed herein. For example, the application of an electric field or magnetic field can be achieved using electrodes, further allowing integration of this into a chip or integrated circuit structure and manufacture using traditional semiconductor manufacturing processes (e.g. CMOS manufacturing processes).

[0099] Variation of the force may be based on the strength or intensity (e.g. as measured by the input voltage (V / m or mV / m)) of the field applied, such as the electric field. The strength of the electric field may vary dependent on the species being moved. It will be appreciated that in any of the embodiments mentioned here, although the resultant force of the electric field acting on a particular component will depend on a number of factors, including the charge on the component, the magnitude of the force will be determined by the magnitude of the electric field such that a higher V / m value will lead to a greater force acting on the species in the sample.

[0100] The use of an electric field or magnetic field to provide the force is particularly advantageous with the structures and arrangements disclosed herein. For example, the application of an electric field or magnetic field can be achieved using electrodes, further allowing integration of this into a chip or integrated circuit structure and manufacture using traditional semiconductor manufacturing processes (e.g. CMOS manufacturing processes).

[0101] The method may further comprises applying a force to at least one amplification product (and optionally each amplification product) so as to migrate the at least one amplification product from the amplification site relative to (i.e. across or towards / away from) the surface, wherein migrating the amplification product relative to the surface is so as to (I) separate the amplification product from at least one additional species; and / or (II) collect the amplification product in a product region on the surface. Application of the force may be as set out above.

[0102] Separating at least one amplification product from at least one additional species may be from any other species. For example, it may be another amplification product. This may be an intended product, including by-products, unintended products (e.g. resulting from incomplete extension) or waste generated by the process. It may be any sample or components thereof which have not undergone a PCR - this may be that some nucleic acids have not been amplified or the components remaining were not nucleic acids - or any excess / or unreacted reagents. Or any other contaminant on the surface, for example. Accordingly, this provides a means of purifying or isolating the at least one amplification product so that it is separated from at least one additional species, for example the sample and any other amplification products formed on the surface, or indeed any other species, is meant that the at least one product is separated from any other components or species.

[0103] In some embodiments, applying a force or a plurality of forces is to a plurality of amplification products so as to migrate the plurality of amplification products relative to the surface. This feature enables simultaneous processing, and optionally separation, of multiple amplification products. This may be so as to separate the plurality of amplification products from one another. This can rely on electrophoresis or other existing methods, but without requiring transfer of the amplification products to another platform. This can reduce the risks associated with loss or contamination of the sample and product(s) present in existing reactors devices and, in turn, increases the effectiveness of the recovery of the product(s) and more accurate analysis of the resultant products. In such embodiments, the at least one additional species is another product of the PCR amplification process. Where plural forces are applied to different products or components, the plurality forces may differ in direction and / or intensity, for example.

[0104] Applying the force may additionally or alternatively be to collect the amplification product in a product region on the surface. The product region may be a distinct region of the surface separate to any of the reactor surfaces. It may comprise a portion of the surface of the device. These may be spaced apart from the amplification sites. This may be a spacing of at least 0.1 mm, at least 1 mm or at least 2mm or at least 1 cm. For example, from 0.1 mm to 10 cm. The substrate or a dielectric may be provided between a part of the product region and the nearest amplification site.

[0105] The methods and systems may additionally or alternatively comprise applying a force to the plurality of amplification products so as to migrate the plurality of amplification products to a product region. This can be a product region where the plurality of amplification products are mixed. This may be used prior to separation, for example, to enhance the resolution of subsequent separation since theamplification products can be collected at a single point and separated from the same point. This can reduce the spread of bands or overlap of separated components. Alternatively, this may be instead of a separation step, where the amplification products can be recovered and / or analysed from the product region.

[0106] Where there are a plurality of different amplification products, separating or purifying may comprise separating or purifying the plurality of amplification products together (i.e. relative to anything other than these products) so that they are provided as a single mixture of product or it may be separately to one other such that each of the plural amplification products is purified individually, or a combination (i.e. some may be purified together and at least one product purified separately). For the latter, the methods and system may further comprise migrating the amplification products relative to the surface so as to separate the amplification products from other species on the surface. This may be achievable using any suitable separation technique. For example, where the amplification products are sufficiently similar to one another in respect of at least one property and sufficiently different to other species on the surface, these may be separated on the basis of that property (e.g. size, charge, interaction with a particular medium or solid phase etc.). Alternatively, these may be distinguished in another way, such as by providing a tag. For the latter, the methods and system may further comprise migrating the amplification products relative to the surface so as to separate each amplification product from the other product(s). This may be achievable using any suitable separation technique, for example on the basis of a property (e.g. size, charge, interaction with a particular medium or solid phase etc.).

[0107] In some embodiments, migrating the amplification product relative to the surface so as to collect the amplification product in a product region on the surface may further comprise moving the amplification product off the surface. In other words, removing it from the surface. This may be to move through the substrate or off the surface onto another device or surface, such as a transfer membrane.

[0108] In some embodiments, collecting the amplification product in a product region on the surface is so as to concentrate the amplification product on the surface in the product region. Collection in a single region advantageously can be used to concentrate the amplification product in a single part of the device.

[0109] It will be appreciated that there are also numerous other ways in which forces (beyond those resulting from the direct application of electric or magnetic fields to species affected by these fields) can be applied and separation can be affected, including for example:

[0110] (i) Applying a force may comprise providing a medium in which a property of the medium varies along a pathway so as to provide a force which acts on at least a part of the sample so as to causes the sample or a part thereof to migrate through the medium. This can define the corresponding pathway. In other words, the application of force is through the creation of an environment which leads to osmotic pressure or which causes diffusion across the pathway. This may be a property of the medium or it may be imparted on the medium by a corresponding manipulation assembly (where present in the method).For example, the methods (and systems) may comprise applying a force (which acts on at least one component) by providing a concentration variation or gradient within the medium along the pathway of at least one species. This can be a property of the medium or can provided by creating these regions through manipulations of species in the medium. For example, charged species (e.g. dopants not forming a part of the sample) within a medium could be manipulated by an electric field to create a particular concentration or charge gradient).

[0111] (ii) Applying a force may comprise applying a force to cause sedimentation of a sample (i.e. the separation of at least one non-soluble component from a liquid and / or for separating a plurality of nonsoluble components from one another within a liquid). This may be application of a kinetic force to cause velocity sedimentation (measuring the speed at which a component settles) or isopycnic sedimentation (based on density of the components). The force acting on the particles may be gravity (i.e. the sample is provided at a first height in the medium such that gravity can move the samples to a plurality of different heights, thereby applying the force). The force may alternatively or additionally be a centrifugal or centripetal force device, such as applied by manipulation assembly (e.g. a centrifugal or centripetal force device (such as a zonal rotor)). One implementation of (ii) is separating cells.

[0112] (iii) Causing separation by a difference in at least one of adhesion, cohesion, and surface tension. This may be separating based on the difference in these features and interaction with the medium of the substrate surface or a feature provided on the substrate (such as a coating or functionalization). In some embodiments, these forces may also be used to apply the force so as to cause migration.

[0113] (iv) Applying a force may comprise applying a centrifugal or centripetal force to cause separation through the medium. This may be based on at least one of size of the components of the sample, shape of the components, density of the components, medium viscosity and rotor speed. For example, this can be used in combination with the separation of based on sedimentation (i.e. (ii)). At least one of (or a plurality or each) manipulation assembly may comprise a device configured to apply a centrifugal or centripetal force (such as a zonal rotor).

[0114] (v) applying a force may comprise applying a force using acoustic waves. For example, the respective manipulation assembly or assemblies may be configured to apply a force using sound-waves. These can also advantageously be used to apply a directional force on the sample or a component thereof. In one embodiment, the respective manipulation assembly or assemblies comprise an ultrasonic sound-wave generator, such as an ultrasonic transducer. By ultrasonic it is meant that the device generates high-frequency sound-waves which cause vibrations in a fluid, particularly in a liquid. For example, sound-waves with a frequency of greater than 18kHz, optionally 20-400 kHz and further optionally 40-80 kHz.

[0115] Combinations of any of the means for applying a force can be combined, including any of (i) to (v) with the use of an electrical field and / or magnetic field which acts directly on the sample. For example, the combination of (i) and an electric field can be used to move a sample or a part thereof through a gradient (which itself could be generated by a manipulation assembly) under the force of anelectric field. Similarly, changing pH across a gradient (e.g. (i)) can be combined with an electric field to separate based on isoelectric point.

[0116] At least one of the first, second or third manipulation assemblies (or a plurality of, or each) may be configured to modify a property of the medium so as to provide a force which causes the sample or a part thereof to migrate through the medium. This may be in addition to alternatively to a field acting on the sample or a part thereof. For example, the second or third manipulation assemblies may modify a property of the medium along the pathway to create a gradient of the property across the pathway. The property (also referred to herein as “environmental property” or “medium property”) may be of the medium across the pathway or just the environment adjacent (i.e. next to or on) one part of the manipulation assembly (e.g. an electrode). By modification of property, it is meant that it to modifies a physical (e.g. material) or chemical property of the medium, the environment or the sample, such as by applying electrical energy or an electric field directly to the sample to change the property or by providing another form of energy (e.g. thermal energy). In some embodiments, this may be a physical or chemical property of the medium. The property can include physical properties, such as thermal properties (e.g. temperature) and / or viscosity, and / or chemical properties selected from at least one of the pH, electrical conductivity, thermal conductivity and / or ionic strength of the sample or a part thereof. The property may be modified directly, e.g. by direct application of heat to change the temperature, or indirectly for example by causing a chemical change which in turn changes the property, such as hydrolysis or electrolysis due to application of electrical energy which can change the pH. For example, the manipulation elements can be configured to create an electrical field which can cause a localised change in a part of the medium.

[0117] Accordingly, at least one of (or a plurality of, or each) the first, second or third manipulation assemblies is configured to modify at least one of the concentration of at least one component of the medium.

[0118] For example, certain molecules are only charged within small pH ranges and may not bind to the corresponding moiety if not charged. Modification of the pH can change the propensity of the capture species and target analyte to bind, which can provide additional information on binding and kinetics of binding. In some cases, this can lead to dissociation. Changes in pH and / or ionic strength can also be used where the sample comprises other non-analyte species, by modifying the solution to a point where e.g. non-selective binding is reduced or eliminated. Moreover, pH is a critical factor in many aspects of biological processes and, indeed, is heavily susceptible to variation in biological samples, for example due to biological processes (e.g. CO2 or lactate production by cells). The ability to control this in situ and without requiring buffers or acids / bases provides a significant improvement.

[0119] In addition to the force, the system may be provided with a separation component provided on the sample substrate which interacts with at least a part of the sample as it is separated. This may be a component provided within the medium (e.g. particles) or provided on the sample surface (such as acoating or functionalization). Examples include antibodies provided on the sample surface, beads (which can be functionalized) or surface profiling on the sample surface.Electrodes

[0120] Numerous embodiments of the systems and method disclosed herein employ electrodes of various types, including for generating fields and as sensing elements. These electrodes may each be formed from or comprise gold, silver, copper, platinum, nickel, titanium, titanium nitride, ruthenium, a conducting polymer, a conductive carbon -based material (e.g. graphene) or combinations thereof. It will be appreciated that where an electrode is used, this may be a part of any suitable electrical circuit or system. For example, the electrode may be a working electrode and there may be a counter electrode provided in electrical connection with the working element, and there may further be a reference electrode (or pseudo reference electrode) provided as part of this electrical system. Where there are pairs of electrodes, these can act as working and counter electrodes.Sub str ate / su rf ace

[0121] The amplification device may comprise a substrate defining the surface. The substrate may be a planar surface or a non-planar surface. For example, the substrate may comprise multiple substrate portions, where the substrate portions are in different planes. For example, these may be angled with respect to each other or in opposing relationship (such that medium is provided therebetween). Each substrate portion may be planar. Where present, the substrate portions may abut one another or may be separated by joined by a liquid or gel medium.

[0122] The substrate may be formed from or by a layer or, where there are plural substrate portions, may be formed of or by a plurality of layers or sections forming the surface, for example.

[0123] Medium (e.g. solution) may be provided onto or over the surface but may further extend into the substrate, in some embodiments, and / or beyond the surface. The substrate may be formed of or comprise any suitable material. For example, it may be a polymer layer, a glass layer, a glass-ceramic layer, a ceramic layer, a metal oxide layer, a metal nitride layer, a silicon -containing layer (e.g. silicon, silicon dioxide, or silicon nitride), a gallium -containing layer (e.g. gallium nitride or gallium arsenide) or combinations thereof.Capture sites

[0124] The methods may further comprise providing at least one capture site provided on the sample surface, wherein the capture site is configured to selectively capture the product to retain an amplification product on the capture site. The method may further comprises selectively binding the amplification product to the capture site so as to retain the amplification product thereon. The amplification device may accordingly comprise such a capture site. The control unit of the systems may be configured to carry out this. As set out above, selective capture enables selective productcapture, allowing for separation from other components and for analysis. In certain embodiments, the amplification device further comprises at least one capture site provided and comprising a capture agent configured to retain the amplification product thereon.

[0125] The capture sites are each a distinct region of the surface and each comprise a portion of the surface of the device. Each capture site may accordingly be a region of the surface with an array or arrangement of one type of capture species. Each capture site is adapted so that it captures only one of the amplification products (i.e. one specific nucleic acid). Where there are plural components to capture, such as a plurality of amplification products, there may be plural capture sites, each being configured to capture a different amplification product, so that the amplification device overall is capable of capturing or configured to capture multiple different species (i.e. a plurality of capture sites, each configured to selective capture a different component, such as one of a plurality of amplification products). It will be appreciated that each capture site may capture a plurality of this one type of species, for example plural species molecules. That is, each site is not limited to a single molecule or compound, thus there may be a plurality of capture species (of one type) to capture the component. The device may further comprise plural capture sites configured to capture the same type of component, e.g. amplification product. It may be advantageous from an incubation time perspective to have plural capture sites across the surface configured to capture the same component.

[0126] Each capture site and, in some embodiments, the capture site comprises a capture agent configured to bind to a component, such as an amplification product. Examples include capture species (i.e. a molecular species or functional group) (or “capture probe”), which are selective so as to selectively bind to the respective component or may include non-selective (or non-specific) capture agents such as gels, matrices, or porous bodies. For example, the capture agent may encapsulate the component.

[0127] In some embodiments, the capture agent may comprise a capture species (i.e. a molecular species or functional group) (or “capture probe”) adhered to the respective capture site, each capture species configured to selectively bind to the respective component (e.g. amplification product). This can form a bound pair and retain the corresponding species on the capture site. The capture species may be configured to bind to the amplification product to immobilise it on the capture site through covalent- like interactions (e.g., chemisorption of anchor species onto the surface through chemical bond formation) and / or non-covalent-like interactions (e.g., physisorption of capture species onto the surface through weaker, often van der Waals, interactions) depending on the identity of the capture species. The capture species may be selected from or comprise a protein, a peptide (e.g. an enzyme), a carbohydrate, a nucleic acid, an aptamer or a combination thereof. Some examples include a single or double stranded DNA chain. An aptamer may be defined as an oligonucleotide, polynucleotide or peptide configured to selectively bind to the species. In some cases, there may be a plurality of capture species (i.e. plural of the same capture species). Capture species may be plural, such that in some cases,there may be a plurality of capture species (i.e. plural of the same capture species) on each reactor / capture site.

[0128] The capture species may be covalently or non-covalently bonded to the capture site. The capture species (for example, a plurality of individual capture species) can be located adjacent to (i.e. next to or abutting) or on the surface, a modification element, a sensing element or any other component provided on the substrate (where these may also define at least a part of the surface) and interact with species provided on the sample surface. In some examples, the capture species is provided on (e.g. adhered to or bound to) the surface, a modification element, a sensing element or any other component provided on the substrate. In some examples, the surface is functionalized with the capture species. Such functionalization can be achieved in any suitable manner, such as by covalently or non-covalently immobilizing the capture species to the surface.

[0129] The capture species can be provided to the sites either by forming the capture species in situ (i.e. on a reactor surface) or these can be produced separately and applied to the capture sites.

[0130] As set out below, selective release from the sample surface may also comprise release of a part of the corresponding capture site. For example, a capture species configured to bind to a product may be detachable from the reminder of capture site (and the sample surface) so as to selectively release the product. In such cases, the capture species may be bound to the remainder of the capture site (e.g. the surface) by a cleavable group, such as a linker which can be cleaved. This may be cleavable by the application of a stimulus, such as heat or a change in environment (e.g. pH), which can be applied by the modification element, for example. In some embodiments, the or each capture site may comprise an individually actuatable modification element configured to cause the selectively releasing of the species bound to the capture site. This may be to provide the stimulus.Uses

[0131] The provision of plural amplification sites, each with their own thermal control (i.e. such that the sequence design of the primer is not temperature restricted in respect to annealing or melting (Taand Tm) because the temperature is controlled for each electrode, allows for a much broader PCR platform. For example, this can be used to detect a broader array of nucleic acids, in turn providing broader diagnostics screening methods. This combined with the ability to monitor progression in situ can also improve the speed of diagnostics. These can be used, for example, in gene validation or pathogen detection / diagnostics. For example, mutation detection in breast cancer, where cDNA can be provided to the surface and amplification sites can be provided with primers for gene targets, for example one site for BRCA1, one for BRCA2, one for ERBB2 and one for TP53. Similarly, a respiratory virus panel could comprise primers for different viruses on each amplification site.

[0132] Further, the ability to build the primers on the amplification sites, due to the ability to individually control these sites with the heaters, allows for custom primers in situ enabling custom screening, such as targeting genes for pathogens or cancer genes etc.Specific Implementations

[0133] Fig. 1 depicts a first method 100 of amplifying target nucleic acids in a sample. Figs. 2A to 2E provides a schematic depiction of an exemplary amplification device 150 for use in the method 100. Fig. 2A provides a schematic plan view, Fig. 2B provides a schematic cross-sectional view through the line A-A of Fig. 2A and Figs. 2C to 2F provide schematic cross-sections through line A-A of Fig. 2A but for the first amplification site 165 A only.

[0134] The method 100 comprises providing 105 a solid-phase amplification device 150. The amplification device 150 is for performing a solid-phase PCR amplification process. The amplification device 150 depicted in Figs. 2A-2F comprises a substrate 155 and a plurality of amplification sites 165A-C arranged in a grid - in this embodiment a 3 x 3 grid (where only the left hand column is labelled for clarity). Each amplification site 165A-C comprises a circular sensing element 160A-C in the form of an electrode embedded in the substrate 355. Each of the sensing elements 160A-C is individually addressable so that a measurement signal can be obtained for each amplification site 165A-C.

[0135] Each amplification site 165A-C further comprises a corresponding heater 162A-C formed in the substrate 155 and located directly beneath the corresponding sensing element 160A-C (visible in dashed lines in Fig. 2A and through the cross-section of Fig. 2B). Each of the heaters 162A-C is individually actuatable. The heaters 162A-C can be used to locally heat the region on and above each corresponding amplification site 165A-C (e.g. the medium adjacent and on the corresponding amplification site 165A- C). This can be used for a number of reasons, including first increasing the temperature of the solution around the amplification sites 165A-C to promote hybridization, as set out in more detail below.

[0136] The method 100 further comprises immobilising 110 aprimer 161A-C corresponding to atarget nucleic acid 101A-C to each amplification site 165A-C, wherein the primer 161A-C on each amplification site 165 A-C corresponds to a part of a different target nucleic acid 101 A-C such that each amplification site 165 A-C corresponds to a different one of a plurality of target nucleic acids 101A-C.

[0137] In particular and as schematically depicted in Fig. 6B, a first amplification site 165 A comprises a primer 161 A functionalised on the surface of a first sensing element 160A, the primer 161 A having a structure which is complimentary to (i.e. will specifically bind to) to a first target nucleic acid 302A. Similarly, a second amplification site 165B comprises another, different primer 161B functionalised on a second sensing element 360B having a structure which is complimentary to (i.e. will specifically bind to) to the second, different target nucleic acid 101 B . A third amplification site 165 C comprises another, different primer 161C functionalised on a third sensing element 160C having sequence which is complimentary to (i.e. will specifically bind to) to a third target nucleic acid 101C.

[0138] Although not depicted, each of the sensing elements 160A-C and the plurality of heaters 162A- C are electrically connected to a control unit (not shown) so that they can be operated under the control of the control unit.

[0139] The method 100 further comprises providing reagents 115 (not depicted for the sake of clarity) for thermal amplification of each of the plurality of different target nucleic acids 101 A-C to the surface 156. Any suitable reagents may be used. The reagents (a “PCR reaction mixture”) may comprise an enzyme for extension (e.g. a polymerase), one or more nucleotides (in some cases, a plurality) (deoxynucleotide triphosphates (dNTPs) and a buffering agent. A divalent cation source, such as magnesium ions, may also be present.

[0140] The method 100 further comprises providing 120 the sample to the surface 156, which sample may or may not contain the target nucleic acids 101A-C. For example, the DNA or RNA from which they are derived may not be present in the sample. It will be appreciated that the methods of the present disclosure can be used for diagnostic tools including screening for the presence of a particular nucleic acid (e.g. for virus screening, or gene diagnostics) in which case the target nucleic acids 101A-C may not be present in the sample, if the screen is negative. In other cases, some or all of the target nucleic acids 101A-C may be present. This can be a screening process. However, in other embodiments, this can be used as a nucleic acid synthesis process (e.g. a DNA synthesis process) where a small amount of a nucleic acid can be amplified to generate more.

[0141] The method 100 further comprises amplifying 125, using a polymerase chain reaction (PCR) amplification, any of the plurality of target nucleic acids 101 A-C present in the sample at the corresponding amplification site 165 A-C using the corresponding first primer 161, the corresponding reagents and a process to form amplification products 103 A-C corresponding to each of the target nucleic acids 101 A-C present in the sample.

[0142] The PCR amplification process is schematically depicted, in part, in Figs. 2C to 2F. In general, PCR amplification process comprises a plurality of PCR cycles comprising the steps of (i) denaturing / release to form target nucleic acids 101 A-C; (ii) hybridizing or “annealing” the target or template nucleic acids 101A-C to corresponding primers 161 A-C to form respective bound pairs; and (iii) extension of the primers 161A-C while bound to the target or template nucleic acid(s) 101A-C.

[0143] For the first cycle of the PCR amplification process, denaturing may only be carried out if the sample provided includes or may include double -stranded nucleic acids, where denaturing a doublestranded nucleic acid would lead to two target nucleic acids 101A-C.

[0144] In this embodiment, the sample comprises double stranded nucleic acids and so the method 100 comprises a first PCR cycles comprising the steps of (i) denaturing / release of any double stranded nucleic acids in the sample to form a plurality of target nucleic acids 101A-C. This is achieved by heating the heaters 162A-C on each of the plurality of amplification sites 162A-C across the whole surface 156 so as to raise the temperature to a denature temperature. This results in a plurality of single stranded target nucleic acids 101A-C above the surface 156, as depicted in Fig. 2B.These target nucleic acids 101 A-C can then be hybridised or bound with the corresponding primer 161A-C on the corresponding amplification site 165A-C. The PCR amplification process in the methods disclosed herein are carried out so that this hybridization comprises hybridizing the targetnucleic acids 101A-C in the sample to the corresponding primer 161A-C of the corresponding amplification site 165A-C using the heater 162A-C provided as part of each amplification site 165A-C. Specifically, each of the heaters 162A-C is controlled so that a target temperature for each of the amplification sites 165A-C is different and corresponds to a temperature at which the primer 161A-C on the amplification site 165 A-C can selectively bind to a corresponding part of the corresponding target nucleic acid 101 A-C so that the primer 161 A-C and the part of the corresponding target nucleic acid 101A-C can hybridize to form a bound pair. In other words, the heaters 162A-C are each set to a specific temperature or temperature range optimised and selected for the specific primer 161 A-C on that site (and hence for the primer 161 A-C and target nucleic acid 101 A-C duplex or pair). The temperature can be determined using the methods set out above. The same method may be used for each primer 161A-C on the different amplification sites 165A-C. Where this is based on the Tm, the same adjustment to a target hybridisation temperature (or an assumed Ta) may be used.

[0145] As an example, the primer 161A of the first amplification site 165A may have a Tmdetermined to be 50 °C. The method may comprise using a target hybridisation temperature by the equation Tm- 5 °C, giving a target hybridisation temperature for the first amplification site 165 A of 45 °C. Accordingly, the heater 162A of this first amplification site 165A may be set to a temperature of 45 °C. The control unit may control the heater 162A to maintain this temperature. In this method, the denaturing temperature is higher than the target hybridisation temperature and so initially the temperature on the first amplification site 165 A is higher than the target hybridization temperature; however, as the temperature is reduced (active cooling may be used), the heater 162A may then maintain the target hybridisation temperature. Turning to the other amplification sites 165B-C, primer 161B of the second amplification site 165B may have a Tmdetermined to be 53 °C. Using the same process, this gives a target hybridisation temperature for the second amplification site 165B of 48 °C. Accordingly, the heater 162B of this second amplification site 165B may be set to a temperature of 45 °C. Similarly, the primer 161C of the third amplification site 165C may have a Tmdetermined to be 58 °C. Using the same process, this gives a target hybridisation temperature for the third amplification site 165 C of 53 °C. Accordingly, the heater 162C of this third amplification site 165C may be set to a temperature of 53 °C. Similarly, the further amplification sites may be set to other temperatures. For example, one may comprise a primer having a Tmof 60 °C and so has a target hybridisation temperature of 55 °C It will be appreciated that these are simply exemplary and other temperatures and primers may be used. The presence of individually controllable heaters 162A-C enables this range of temperatures to be applied locally to each amplification site 165 A-C, whereas the range of Tmacross these primers 161 A-C would otherwise have prevented their use in a single, system.

[0146] This process is depicted in Figs. 2C for the first amplification site 165A only, where Fig. 2C shows the first target nucleic acid 101 A and primer 161 A bound as a bound pair on the first amplification site 165 A. It will be appreciated that this will also have occurred for all of the other amplification sites 165A-C on the amplification device 150 (see Fig. 2A), including those in the othertwo columns, where each of the other amplification sites have primer which correspond to the other target nucleic acids. In this first cycle, the target nucleic acids 101A-C bound to the surface 156 are directly obtained from the sample.

[0147] The PCR cycle then comprises an extension step using a polymerase present in the reagents and a plurality of dNTPs, which are inserted onto the end of the primer 161A. This comprises heating each amplification site 165A-C to a target extension temperature at which an extension on the primer 161A- C of the bound pair can occur to form an amplification product. In this embodiment, heating each amplification site 165A-C comprises individually controlling each of the heaters 161A-C to heat the amplification sites 165A-C.

[0148] The result is depicted in Fig. 2D. Here a corresponding immobilised extended nucleic acid strand 102 A has been created on the template provided by the first target nucleic acid 101 A. In the configuration shown in Fig. 2D, these are provided as a bound pair 103A, which itself can also be considered to be an amplification product and is a copy of the double stranded nucleic acid of the sample.

[0149] The first PCR cycle is complete. The method 100 may then comprise further PCR cycles. These may each comprise (i) denaturing / release; (ii) hybridizing or “annealing”; and (iii) extension, as set out above. However, it will be appreciated that denature in respect of the second PCR cycle onwards may also refer to denaturing the amplification product of the previous cycle: that is, the target nucleic acid 101 A bound to the extended nucleic acid strand 102A as a bound pair 103 A.

[0150] For example, Fig. 2D shows the completed extended amplification product in the form of the bound pair 103A. A further PCR cycle may denature this bound pair 103A (and for all of the amplification sites 165A-C), releasing the target nucleic acid 101 A (which can be used as a template for a further cycle) and leaving the extended nucleic acid strand 102A immobilized on the amplification site 165A-C (which itself can be considered to be an amplification product). This is shown in Fig. 2E. It will, of course, be appreciated that the representation of primers 161 A is schematic, such that there may be significantly more unused primer 161A molecules on the surface 156 for further cycles.

[0151] The further PCR cycles may then be carried out.

[0152] Alternatively or additionally, for example at the end of the PCR amplification process, the method 100 may comprise selectively releasing the amplification products in the form of the bound pair 103A or the extended nucleic acid strand 102A from the corresponding amplification site 165A. Fig. 2F shows the release of a bound pair 103 A (i.e. the release from the arrangement of Fig. 2D). As depicted in Fig. 2F, this comprises releasing the amplification products 103A from the respective amplification site 165A-C - in Fig. 2F, the first amplification site 165 A - while still retaining the other amplification products on their respective amplification sites 165B-C. This provides a solution above the surface 156 in which only the first amplification product 103 A is present and which can then be removed from the surface 156 in isolation to the other amplification products. In this embodiment, the selective release can be achieved using the electrode forming the sensing element 160A forming a partof the corresponding amplification site 165 A. In particular, the electrode can be actuated to provide a release stimulus which causes the covalent bond holding the primer 161 A (now incorporated into the amplification product 103 A to break. The location of the electrode on each amplification site 165 A and the spaced apart nature of the amplification sites 165A-C means that the stimulus is local only such that this will only release the amplification product 103 A on the first amplification site 165 A and leave the remaining amplification products bound to the surface 156. This process can then be repeated for the other amplification products.

[0153] Throughout the above PCR amplification process, monitoring the progression of each PCR process on each amplification site 165A-C may be carried out using the corresponding sensing element 160A-C. In particular, the charged nature of the nucleic acids will interact with the sensing element such that there will be signals corresponding to the build-up of nucleic acids on the amplification site, which can give an indication as to the progression of the process. As the process progresses, there will be more nucleic acids on the site, leading to a higher signal. This process may be enhanced by tagging dNTPs with a reporter element, such as ferrocene, which can be used to increase the interaction with the electrode (for example through a redox process). The control unit may be configured to use this to control the process. For example, this may be operating the corresponding thermal device of a specific amplification site in isolation to the other thermal devices of a specific amplification site (e.g. making a change to only one or a sub-set of the plurality of the specific amplification sites).

[0154] Although in the above method 100, the thermal devices were heaters 162A-C, it will be appreciated that the thermal devices may additionally include cooling devices such that reaching the target temperatures may include actively cooling each amplification site 165A-C using the corresponding cooling device on the amplification site 165A-C.

[0155] Figs. 3A to 3C provides a schematic depiction of an exemplary amplification device 250 for use in a method. Fig. 3A provides a schematic plan view and Figs. 3B and 3C provide schematic crosssections through line A-A of Fig. 3A but for the first amplification site 265A only.

[0156] The solid-phase amplification device 250 of Figs. 3 A to 3 C has the same device structure as the amplification device 150 of Figs. 2A-2F. It comprises a substrate 255 and a plurality of amplification sites 265A-C arranged in a grid. Each amplification site 265A-C comprises a circular sensing element 260A-C in the form of an electrode embedded in the substrate 255. Each of the sensing elements 260A- C is individually addressable so that a measurement signal can be obtained for each amplification site 265A-C.

[0157] Each amplification site 265A-C further comprises a corresponding heater 262A-C formed in the substrate 255 and located directly beneath the corresponding sensing element 260A-C (visible in dashed lines in Fig. 3A and through the cross-section of Fig. 3B). Each of the heaters 262A-C is individually actuatable. The heaters 262A-C can be used to locally heat the region on and above each corresponding amplification site 265A-C (e.g. the medium adjacent and on the corresponding amplification site 265A- C).

[0158] However, in this embodiment, each amplification site 265 A-C is provided with a primer set comprises two different primers. In particular, the method of using the amplification device comprises immobilising a first primer to each amplification site 265A-C, in Fig. 3B that is a first primer 261 A to the first amplification site 265 A, and further a second primer to each amplification site, which in Fig. 3B is second primer 264A to the first amplification site 265A. The same process is carried out for all amplification sites 265A-C.

[0159] The first primer on each amplification site 265A-C corresponds to a first target nucleic acid of one double stranded nucleic acid and the second primer on each amplification site 265A-C corresponds to a second target nucleic acid of the same double stranded nucleic acid. In other words, the first primer and the second primer may correspond to forward and reverse primers for the double stranded nucleic acid. For the first amplification site 265 A, the first primer 261 A is therefore a primer for a first target nucleic acid strand 201 A of a double stranded nucleic acid 201 and the second primer 26 IB is a primer for the other, second target nucleic acid strand 204A of the same double stranded nucleic acid 201 . Accordingly, amplification of the double stranded nucleic acid 201 may occur on one amplification site 265A.

[0160] The PCR process may then proceed as above, with heating leading to denaturing of the double stranded nucleic acid 201 to the first and second target nucleic acid strands 201A, 204A (Fig. 3B). These are then hybridised to the respective primer 261A-B through individual control of the temperature using heater 262A (carried out across all amplification sites 265A-C), as shown in Fig. 3C. Since there are two different primers on each amplification site 265A-C, the hybridizing step of the PCR amplification process further comprises hybridizing a part of the second target nucleic acid 204A to the corresponding second primer 264A of the corresponding amplification site 265A. This too comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the second primer 654A on the amplification site 265A can bind to a corresponding part of the second target nucleic acid 204A so that the second primer and the part of the second target nucleic acid can hybridize to form a bound pair. It will be appreciated that the Tmand Tamay vary, and hence the target hybridisation temperature for each may differ. In such a case, either a composite target temperature at which both may hybridize may be used or, in other embodiments, the method may comprise individually controlling each of the heaters so that a first target temperature is used, followed by a second target temperature.

[0161] Extension can then occur, and the PCR cycle repeated until sufficient amplification has occurred.

[0162] Figs. 4 to 6 schematically depict a further amplification device 350 which can be used in the methods disclosed herein. Fig. 4 provides a schematic plan view of the amplification device 350, Fig. 5 shows an expanded view of a part of the amplification device 350 and Fig. 6 provides a cross-section along line A-A in Fig. 4. Although not shown, the amplification device 350 comprises a control unit (not shown) which can be used in embodiments to perform the device-related steps of the methods.

[0163] The amplification device 350 comprises a substrate 355 in part defining an upper surface 356. The amplification device 350 also comprises a plurality of capture sites 369 (only some of the electrode arrays 390 are labelled in FIG. 6 for the sake of clarity) arranged in a 4 x 4 grid in part also defining the upper surface 356 of the substrate 355 (although it will be appreciated that any number of the capture sites 369 may be provided in any configuration). Each capture site 369 comprises a modification element 360 in the form of a heater embedded in the substrate 355. Each of the modification elements 360 is individually actuatable and is functionalised with a capture species 368 specific to a particular amplification product generated in a PCR process. Accordingly, each of the modification elements 360 and capture species 368 defines a capture site 369 for a corresponding amplification product.

[0164] Although not depicted, each of the plurality of modification elements 360 are electrically connected to a control unit so that the plurality of modification elements 360 can be actuated under the control of the control unit. The modification elements 360 can act as individual modification elements and can be used to locally heat the region on and above each corresponding capture site 369 (e.g. the solution adjacent and on the corresponding capture site 369). As set out above, this can be used to aid incubation (i.e. to promote the binding of the amplification product generated in a PCR process to the corresponding capture species 368). Due to the individual actuation of the modification elements 360 and the local nature of the modification element 360 (i.e. that each provides and is limited to a single capture site 369), the temperature on each capture site 369 can be different, which can be particularly useful for providing appropriate environments for each pair of amplification product and capture species. It can also be further be used for the selective release of the bound amplification product, where this is present.

[0165] The amplification device 350 further comprises a plurality of electrode arrays 390 arranged around each of the capture sites 369, where each electrode array 390 comprises four individual electrodes 391A-D (see Fig. 10) arranged so that each of the four electrodes 391A-D defines the edge of a square surrounding the capture site 369. Fig. 10 shows an expanded view of a part of the amplification device 350 in which the upper left electrode array 390 (as it is shown in Fig. 6 - i.e. the first electrode array 390 in the 4 x 4 grid) is more clearly visible. Here it can be seen that this (and each) electrode array 390 comprises four elongated electrodes 391A-D defining the edges of a square shape. This provides two sets of opposing pairs of electrodes. Specifically, there is a first pair comprising a first electrode 391A arranged spaced apart and opposing from a third electrode 391C (i.e. defining the opposite side of the square). The first electrode 391A and third electrode 391C of the first electrode array 390 are electrically connected or connectable to one another so that an electric field can be formed therebetween. There is also a second pair comprising a second electrode 39 IB (in this arrangement, this extends between the top ends of the first electrode 391 A and third electrode 391C). This opposes and is spaced apart from a fourth electrode 39 ID (i.e. defining the opposite side of the square), which is also arranged to extend across the bottom edge of the square between the bottom ends of the first electrode 391A and third electrode 391C. The second electrode 391B and fourth electrode 391D of thiselectrode array 390 are electrically connected or connectable to one another so that an electric field can be formed therebetween. Each of the electrode arrays 390 of the amplification device 350 has a corresponding structure. Although we have noted that the opposing pairs of electrodes can be electrically connected, it should be noted that, in some configurations, all of the electrodes of the electrode arrays 390 may be connectable to or connected to all of the other electrodes of the electrode arrays 390 in a particular row or column of the grid to provide the ability to generate an electric field therebetween. This allows movement therebetween and sensing. In some configurations, all of the electrodes of the electrode arrays 390 may be connectable to or connected to all of the other electrodes of the electrode arrays 390.

[0166] These electrode arrays 390 of the amplification device 350 can serve multiple purposes and, accordingly, the control unit may be configured to operate these for a number of different reasons and operations during implementation of the methods disclosed herein.

[0167] First, these may be used to manipulate the amplification products (or, indeed, any other species) on the surface 356 so as to migrate these across the surface 356. That is, the electrodes 391A-D of each electrode array 390 may act as manipulation assemblies which can be used to apply a force to charged species. In particular, any combination of two electrodes 391A-D on the amplification device 350 can act as a manipulation assembly by providing an electric field therebetween. For example, a first manipulation assembly 693 could be considered to be formed across the top row defined by the first and third electrodes 391 A, 391C of each electrode array 390 extending across the width of the substrate 355. An electric field formed between the first and third electrodes 391A, 391C of each electrode array 390 will cause charged species to move along the surface 356 between the corresponding first and third electrodes 391A, 391C (i.e. from right to left or left to right, as depicted in FIG. 6). Because of the provision of electrodes 391A-D between each of the capture sites 369, this provides granular control of the movement of the amplification product such that fine manipulation within individually controllable regions is possible across small regions of the surface 356.

[0168] These electrodes 391A-D (and the manipulation assemblies they form) can be used to aid the specific steps of the methods disclosed herein. For example, this can be used to move an amplification product to a product region or to cause separation, as set out above, by defining a pathway through the medium through which the product can be caused to migrate and separate out from at least one other species. More generally, the electrodes 391A-D can be used to move species around the surface 356. For example, the sample may be provided to one part of the surface 356 and the electrodes 391A-D can be actuated to cause the sample to migrate across the surface 356 to the relevant amplification site (set out below). Similarly for amplification products, it can be used to ensure that this is provided to the relevant product region and ensure that the capture sites 369 are exposed to the amplification products. For example, a first electrode 391 A (e.g. the upper left most electrode) and a third electrode 391C (e.g. the upper right most electrode) along the same row of the grid could be used to generate an electric field therebetween causing amplification product to migrate from one side of the amplification device 350 tothe other side. It will be further be appreciated that the grid structure of the electrode arrays 390 is such that a species could be manipulated in more than one dimension - i.e. in two or more dimensions. For example, it is possible for a species to be migrated along a first dimension, followed by migration along a second dimension. At this point, the control unit may be further configured to cause another electrode pair to cause the species to be moved along another (third) dimension. Accordingly, the amplification device 350 provides a multi-dimensional manipulation system with a vast degree of flexibility to perform complex separations and manipulations.

[0169] Further, these can be used to remove any unbound species or non-specifically bound species prior to selective release and / or analysis. This can also be used to strip off non-specifically bound species from the amplification sites 365 or capture species 368 prior to selective release and / or analysis. The provision of the electrode arrays 390 surrounding each capture site 369 - and, accordingly, providing each capture site 369 with its own electrode array 390 - allows for different electric field strengths to be provided to different capture sites 369. This allows for greater control and for the electric field strength to be tailored to the capture species / bound species relationship (i.e. the strength of the bound pair). This allows for more accurate removal of non-specifically bound species, for example. Alternatively or additionally, this can be used as part of the selective release, where present. For example, by applying an electric field of an appropriate strength, this can be used to pull the bound product off the capture site 369. As noted above, the provision of the electrode arrays 390 surrounding each capture site 369 - and, accordingly, providing each capture site 369 with its own electrode array 390 - allows for different electric field strengths to be provided to different capture sites 369. This allows for greater control of the selective release stage. With respect to removal of a released product, the electrodes 391A-C can be used to apply a force and remove the product from the surface where it can be recovered. This can also be achieved on the amplification site 365. Because of the provision of electrodes 391A-D between each of the capture sites 369, this provides granular control of the movement of the released product such that fine manipulation is possible across small regions of the surface 356.

[0170] Each electrode array 390 could also be used to influence a part of the amplification product with respect to the capture site 369. For example, at least two opposing electrodes 391A-D of an electrode array 390 could be used to drive or concentrate the product to the appropriate capture site 369. It is possible that movement of the amplification product will lead to diffusion of the amplification product within the medium / solution, the electrodes 391A-C can be used (e.g. during the recovery step) to concentrate the amplification product on a particular part of the surface 356, from which it can be recovered.

[0171] Second, the electrodes 391A-D can be used for sensing - i.e. as sensing elements - or modifications - i.e. as modification elements. For example, the configuration and location of the electrodes arrays 390 is such that these can be operated to (e.g. by the control unit) to serve as sensing elements or modification elements. For instance, the electrodes 391A-D can be used to interrogatespecies located between any of the two electrodes 391A-D. For example, it may be that the amplification product bound to the capture site 369 will change the permittivity of the medium located therebetween and the electrodes 391A-D are calibrated or arranged so that the change in permittivity can be determined. Individual electrodes 391 A-D could also be used to determine the location of species. Each electrode 391 A is individually addressable so that it can provide its own measurement signal. These could further be operated to act as a modification element, for example for applying a stimulus.

[0172] The amplification device 350 further comprises amplification sites 365 for use in a PCR process. The amplification sites 365 each comprise a heater 360. As with the amplification devices 150, 250 of the previous embodiments, each of the amplification sites is provided with a primer 361 specific to a target nucleic acid. Each heater 360 is individually controllable.

[0173] The apparatus comprises four additional manipulation assemblies arranged as four parallel rows from the top (as depicted in Fig. 10) to the bottom of the substrate 355. Each additional manipulation assembly has a similar structure comprises a pair of first and second field electrodes 693A, 693B extending across the width of the substrate 355. For each additional manipulation assembly, the first field electrode 693A is provided on the outermost left hand side of the substrate 355, on one side of the amplification site 365 on this row, as depicted in Fig. 10 delimiting a pathway and a second field electrode 693B provided on the outermost right hand side of the substrate 355 delimiting the opposite end of the corresponding pathway. The pathway formed therebetween accordingly encompasses both the amplification site 365 of each row and the capture sites 369. An electric field formed between the first and second field electrodes 693A, B of each first manipulation assembly will cause charged species within the amplification product to move along the corresponding pathway between the corresponding first and second field electrodes 693A, 693B (i.e. from right to left or left to right, as depicted in FIG. 10). Further, between the first and second field electrode 694A, B of each additional manipulation assembly are the electrodes of the electrode arrays 390, each of which can further interact with the first and second field electrodes 693 A, B, defining additional individually controllable regions (beyond those formed by the electrode arrays 390 themselves).

[0174] The use of these electrodes and the arrays leads to a grid of multiple dimensions in which components can be separated and detected. For example, amplification products can be produced on the amplification sites 365 and separated from other components or products in the horizontal direction. Where the same amplification product(s) are produced on different amplification sites 365, these could be caused to align (e.g. by the separation or by further manipulation) with each other on a column, at which point the electrode arrays 390 could be actuated so as to manipulate the amplification product in question along a column (as it is depicted in Fig. 10). This could be used to concentrate the product on a particular part of the surface 565.

[0175] Although not depicted, it will be appreciated that the amplification device 350 of Figs. 4 to 6 may further include other components. For example, each of the capture sites 369 may comprise a corresponding electrode provided in the substrate 355. Each of the electrodes can be individuallyactuatable and can be functionalised with the capture species 368 specific to an amplification product. Each of the plurality of electrodes can be electrically connected to a control unit (optionally via a signal processing unit) so that the plurality of electrodes can be individually actuated and addressed by the control unit. This can be used to provide stimuli, such as a debinding stimulus, and also to address the electrodes to obtain measurement signals which can indicate whether the amplification product(s) are bound to the surface 356. Accordingly, the electrodes can act as both a modification element and a sensing element. These can be used in conjunction with the modification elements 360 such that both are present on the device. For example, the electrodes can be provided at the surface and be functionalized, with the heaters located beneath the electrodes within or beneath the substrate. Each capture site 369 in this arrangement will accordingly have a corresponding modification element 360 and electrode.

[0176] Figs 7 to 9 schematically depict a further amplification device 450, which can be used in the methods disclosed herein. Fig. 7 provides a schematic plan view of the amplification device 450, Fig. 8 shows an expanded view of a part of the amplification device 450 and Fig. 9 provides a cross-section along line A-A in Fig. 6. Although not shown, the amplification device 450 comprises a control unit (not shown) which can be used in embodiments to perform the device-related steps of the methods.

[0177] The amplification device 450 has a structure very similar to the amplification device 350 of Figs. 4 to 6. However, instead of having an array of capture sites 365, the amplification device 450 comprises a grid of amplification sites 465.

[0178] The amplification device 450 comprises a substrate 455 in part defining an upper surface 456. The amplification device 450 also comprises a plurality of amplification sites 465 (only the outer electrode arrays 490 are labelled in FIG. 7 for the sake of clarity) arranged in a 4 x 4 grid in part also defining the upper surface 456 of the substrate 455 (although it will be appreciated that any number of the amplification sites 465 may be provided in any configuration). Each amplification site 465 comprises an individually controllable heater 460 embedded in the substrate 455. Each of the heaters 460 is individually actuatable.

[0179] A primer set comprising primers 461 specific to a target nucleic acid is provided on each amplification site 465. In some embodiments, the primers 461 may be different on each amplification site 465 of the amplification device 450. In other embodiments, there are different primers 461 on different amplification sites 465, but there may be plural amplification sites 465 for each type of primer 461. This can be used for screening of multiple samples, for example simultaneously. For example, the top row of four sites may each have a different primer 461 provided thereon (e.g. Primer A on the first amplification site (from left to right), Primer B on the second amplification site, Primer C on the third amplification site, Primer D on the fourth amplification site). Then each row may have this same pattern, such that the first column of amplification sites comprises Primer A, the second column Primer B, and so on. The same sample can be provided to each site on each row or at the start of each row and manipulated using the electrode arrays 490, discussed below.

[0180] Although not depicted, each of the plurality of heaters 460 are electrically connected to a control unit so that the plurality of heaters 460 can be actuated under the control of the control unit. The heaters 460 can be used to locally heat the region around each corresponding amplification site 465 (e.g. the solution adjacent and on the corresponding amplification site 465). As set out above, this is used for the PCR process, including hybridization.

[0181] The functionality and use of the amplification sites 465 is accordingly the same as for the other amplification devices 150, 150 disclosed herein. It will, therefore, be appreciated how such a device can be used with the methods disclosed herein.

[0182] However, the amplification device 450 of Figs. 7 to 9 includes some of the additional functionality of the amplification device 350 of the Figs. 4 to 6 to aid this process. In particular, the amplification device 450 is provided with a plurality of electrode arrays 490 arranged around each of the amplification sites 465, where each electrode array 490 comprises four individual electrodes 491A- D (see Fig. 7) arranged so that each of the four electrodes 491A-D defines the edge of a square surrounding the amplification site 465. Fig. 8 shows an expanded view of a part of the amplification device 450 in which the upper left electrode array 490 (as it is shown in Fig. 7 - i.e. the first electrode array 490 in the 4 x 4 grid) is more clearly visible. Here it can be seen that this (and each) electrode array 490 comprises four elongated electrodes 491A-D defining the edges of a square shape. This provides two sets of opposing pairs of electrodes. Specifically, there is a first pair comprising a first electrode 491A arranged spaced apart and opposing from a third electrode 491C (i.e. defining the opposite side of the square). The first electrode 491 A and third electrode 322A3 of the first electrode array 490 are electrically connected or connectable to one another so that an electric field can be formed therebetween. There is also a second pair comprising a second electrode 49 IB (in this arrangement, this extends between the top ends of the first electrode 491 A and third electrode 491C). This opposes and is spaced apart from a fourth electrode 49 ID (i.e. defining the opposite side of the square), which is also arranged to extend across the bottom edge of the square between the bottom ends of the first electrode 491A and third electrode 491C. The second electrode 491B and fourth electrode 491D of this electrode array 490 are electrically connected or connectable to one another so that an electric field can be formed therebetween. Each of the electrode arrays 490 of the amplification device 450 has a corresponding structure. Although we have noted that the opposing pairs of electrodes can be electrically connected, it should be noted that, in some configurations, all of the electrodes of the electrode arrays 490 may be connectable to or connected to all of the other electrodes of the electrode arrays 490 in a particular row or column of the grid to provide the ability to generate an electric field therebetween. This allows movement therebetween and sensing. In some configurations, all of the electrodes of the electrode arrays 490 may be connectable to or connected to all of the other electrodes of the electrode arrays 490.

[0183] These electrode arrays 490 of the amplification device 450 can serve multiple purposes as set out for the amplification device 350 for Figs. 4 to 6. In this case, the electrode arrays may also be usedto apply a force to the nucleic acids in the sample. Each electrode array 490 could be used to influence location of the sample with respect to the amplification site 465. For example, at least two opposing electrodes 491 A-D of an electrode array 490 could be used to direct the sample to the amplification site 465. This can be useful to overcome diffusion. Second, the electrodes 491 A-D can be used for sensing - i.e. as sensing elements - or modifications - i.e. as modification elements, as detailed above.

[0184] It will be appreciated that this provides a particularly useful platform for PCR amplification processes and manipulations of the products produced thereon.

[0185] Variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the disclosed systems and methods, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfil the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. If a computer program is discussed above, it may be stored / distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems. If the term "adapted to" is used in the claims or description, it is noted the term "adapted to" is intended to be equivalent to the term "configured to". Any reference signs in the claims should not be construed as limiting the scope.

[0186] The flow diagrams and block diagrams in the Figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the disclosed systems and methods. In this regard, each block in the flow diagrams orblock diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and / or flow diagrams and combinations of blocks in the block diagrams and / or flow diagrams, can be implemented by special purpose hardware-based systems that perform the specified functions or acts or carry out combinations of special purpose hardware and computer instructions.

Claims

CLAIMS1. A method of amplifying target nucleic acids in a sample, comprising: providing a solid-phase amplification device comprising a plurality of amplification sites provided on a surface, each of the plurality of amplification sites comprising an individually controllable thermal device configured to control the temperature at a corresponding amplification site; immobilising a first primer corresponding to a target nucleic acid to each amplification site, wherein the first primer on each amplification site corresponds to a part of a different target nucleic acid such that each amplification site corresponds to a different one of a plurality of target nucleic acids; providing reagents for thermal amplification of each of the plurality of different target nucleic acids to the surface; providing the sample to the surface; and amplifying any of the plurality of target nucleic acids present in the sample at the corresponding amplification site using the corresponding first primer, the corresponding reagents and a polymerase chain reaction (PCR) amplification process to form amplification products corresponding to each of the plurality of target nucleic acids present in the sample, wherein the PCR amplification process comprises hybridizing a part of any target nucleic acid present in the sample to the corresponding first primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the first primer on the amplification site selective binds to a corresponding part of the target nucleic acid so that the first primer and the part of the target nucleic acid can hybridize to form a bound pair.

2. The method of claim 1, wherein the sample comprises a plurality of double stranded nucleic acids and wherein providing the sample to the surface comprises heating the sample to a denature temperature at which the plurality of double stranded nucleic acids will denature to form the plurality of target nucleic acids.

3. The method of claim 2, wherein heating the sample to a denature temperature comprises controlling the individually controllable thermal devices to heat the sample to the temperature.

4. The method of claim 2 or claim 3, further comprising immobilising a second primer to each amplification site, wherein the first primer on each amplification site corresponds to a first target nucleic acid of one double stranded nucleic acid and the second primer on each amplification site corresponds to a second target nucleic acid of the same double stranded nucleic acid.

5. The method of claim 4, wherein hybridizing further comprises hybridizing a part of the second target nucleic acid to the corresponding second primer of the corresponding amplification site; and wherein hybridizing comprises individually controlling each of the thermal devices so that a target temperature for each amplification site is different and corresponds to a temperature at which the second primer on the amplification site can selectively bind to a corresponding part of the second target nucleic acid so that the second primer and the part of the second target nucleic acid can hybridize to form a bound pair.

6. The method of any preceding claim, wherein the PCR amplification process comprises an extension step comprising heating each amplification site to a target extension temperature at which an extension on the first primer of the bound pair can occur to form a corresponding immobilised extended nucleic acid strand.

7. The method of claim 6, wherein heating each amplification site comprises individually controlling each of the thermal devices so that a target extension temperature for each amplification site is different and corresponds to a temperature at which the first primer of the bound pair on each amplification site can be extended to form the corresponding extended nucleic acid strand.

8. The method of claim 6 or claim 7, wherein the PCR amplification process further comprises a postextension denature step comprising heating each amplification site to a target denature temperature at which the target nucleic acid is released from the immobilised extended nucleic acid strand.

9. The method of claim 6 or claim 7, wherein the PCR amplification process further comprises a release step comprising applying a stimulus to at least one amplification site such that a bound target nucleic acid and extended nucleic acid strand on the amplification site are released together from the amplification site.

10. The method of any preceding claim, wherein each of the individually controllable thermal devices of the plurality of amplification sites is spaced apart from the other individually controllable thermal devices.

11. The method of any preceding claim, wherein each amplification site further comprises at least one sensing element configured to provide a measurement signal indicative of the presence of nucleic acids on the corresponding amplification sites; and wherein the method further comprises monitoring the PCR amplification process on each amplification site using the sensing the sensing element.

12. The method of any preceding claim, wherein immobilising a first primer corresponding to a target nucleic acid to each amplification site comprises synthesising the first primer on each amplification site.

13. The method of any preceding claim, further comprising applying a force to the sample to move at least a part of the sample relative to the surface.

14. The method of any preceding claim, further comprising applying a force to at least one amplification product so as to migrate the amplification product relative to the surface.

15. The method of any preceding claim, further comprising determining a melt temperature for an amplification product produced on an amplification site by applying heat to the amplification product the using the individually controlling the thermal device.

16. The method of any preceding claim, further comprising providing a further amplification site and immobilising a first primer having the same structure as a first primer of one of the plurality of amplification sites, wherein at least one of: the number or density of first primers on the further amplification site and amplification site is different or the concentration of the corresponding target nucleic acid provided to each amplification site is different.

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