EnviroLogix Inc v Ionian Technologies, Inc - [2020] APO 18

IP AUSTRALIA

AUSTRALIAN PATENT OFFICE

EnviroLogix Inc v Ionian Technologies, Inc [2020] APO 18

Patent Application: 2015202439

Title:Nicking and extension amplification reaction for the exponential amplification of nucleic acids

Patent Applicant: Ionian Technologies, Inc.

Opponent: EnviroLogix Inc.

Delegate: Felix White

Decision Date: 17 April 2020

Hearing Date: 5 July 2019, in Canberra
Further submissions from both parties were received on 24 July 2019 and 9 October 2019

Catchwords: PATENTS – section 59 opposition to grant of a patent – novelty – inventive step – no clear and unmistakeable direction or direct leading of the PSA with expectation of success – sufficiency – support – utility – claims broader than principle of general application – opposition successful on grounds of lack of sufficiency and support – opportunity to amend – re-examination may be necessary based on documents not pressed in hearing – costs awarded against applicant

Representation: Patent attorney for the applicant: Mr David Myers, Spruson & Ferguson
Counsel for the opponent: Mr Greg Arthur
Patent attorney for the opponent: Dr Caryn DeHoratius, InLegal Limited

IP AUSTRALIA

AUSTRALIAN PATENT OFFICE

Patent Application: 2015202439

Title:Nicking and extension amplification reaction for the exponential amplification of nucleic acids

Patent Applicant: Ionian Technologies Inc.

Date of Decision: 17 April 2020

DECISION

The opposition is successful on the grounds of lack of sufficiency and lack of support.

I allow Ionian Technologies, Inc. a period of two months from the date of this decision to
propose amendments to the specification.

In the event that D6 and D10 are relevant to the novelty and/or inventive step of the claims as proposed to be amended, the Commissioner may consider whether to initiate re-examination proceedings.

Costs according to Schedule 8 are awarded against Ionian Technologies Inc.

REASONS FOR DECISION

Background

Patent application 2015202439 (the application) was filed by Ionian Technologies, Inc (Ionian/the Applicant) on 6 May 2015 as a divisional application from patent application 2008276118 (the parent). The application claims the effective filing date of the parent (14 July 2007), and claims priority from US 11/778,018 with an earliest priority date of 14 July 2006.
The application was examined and advertised accepted on 21 September 2017. EnviroLogix Inc. (EnviroLogix/the Opponent) filed a notice of opposition under section 59 of the Patents Act 1990 (the Act) on 21 December 2017.
The statement of grounds and particulars (SG&P) was filed on 21 March 2018. The SG&P was accompanied by copies of sixteen citations (D1-D16).

Ionian filed amendments to the claims under section 104 on 24 September 2018. These amendments were allowed unopposed on 9 January 2019. This decision is in relation to the specification as amended.

Evidence

In addition to the citations filed with the SG&P, the parties relied upon evidence as set out in the table below.

Evidence	Declarant	Date of Declaration	Reference	Exhibits
In support	Prof Jeremy Edwards	21 June 2018	Edwards #1	JE-1 to JE-16
In answer	Dr Alison Todd	24 September 2018	Todd	AT-1 to AT-12
In reply	Prof Jeremy Edwards	25 November 2018	Edwards #2	JE-17

The opposition

Grounds of opposition

The grounds of opposition pressed by the opponent in the hearing were:

lack of novelty and lack of inventive step in view of each of:

Ehses, S. et al. (2005) “Optimization and design of oligonucleotide setup for strand displacement amplification”. J Biochem Biophys Methods. 30;63(3):170-86 (D1),

US 2005/0112631 A1 (D2), and

Ehses, S. “Isothermale in vitro Selektion und Amplifikation zur Untersuchung von Evolutionsvorgängen” (Dissertation, August 2005, Ruhr-Universität Bochum) (D3, with English translation as D3a);

lack of sufficiency, lack of support and lack of utility.

Standard of Proof

Examination of the application was requested after 15 April 2013. Consequently, the present opposition is governed by the Patents Act 1990 (the Act) and Patents Regulations 1991 (the Regulations) as amended by the Intellectual Property Laws Amendment (Raising the Bar) Act 2012 (Raising the Bar). Amendments to sections 7, 40 and 60(3A) of the Act apply to the present case as a consequence of Schedule 1, items 55(1)(d) and 55(4)(a), and Schedule 6, item 133(7)(d) of the Raising the Bar Act.
The Applicant submitted[1] that the propositions set out by Beach J in Meat & Livestock Australia Limited v Cargill, Inc[2], even though they relate to pre-Raising the Bar legislation[3] are still relevant to the current onus of proof due to the distinction between pre- and post-grant revocation proceedings. At the hearing the Applicant submitted that should a pre-grant revocation proceeding fail, a third party may still “have another shot” at challenging validity, but it an application is refused, the applicant’s options are exhausted.
[1] Applicant’s Submissions (AS) at [14]
[2] [2018] FCA 51 at [8-11]
[3] Op Cit at [5]

The Raising the Bar amendments gave the Commissioner the ability to refuse applications where the Commissioner is satisfied, on the balance of probabilities, that a granted patent would be invalid, rather than only permitting the Commissioner to refuse the patent application if she is practically certain the granted patent would be invalid[4].

[4] Explanatory Memorandum (EM) to the Raising the Bar Act, item 15
In view of this, any prior case law that is based on the pre-Raising the Bar “practically certain” standard must necessarily be set aside. The EM does not speak to any secondary considerations such as a distinction between pre- and post-grant revocation proceedings. I am also not convinced that there is any relevance to the suggestion that the Applicant’s options end with an opposition before the commissioner, given that not only does the Applicant have an opportunity to amend the application[5] but the Applicant also has the right to appeal a decision of the Commissioner to the Federal Court[6].

[5] s 60(3B) of the Act
[6] s 60(4) of the Act
Thus the standard of proof that applies in the present case is the balance of probabilities. Under subsection 60(3A) of the Act, if I am satisfied, on the balance of probabilities, that a ground of opposition to the grant of a patent exists, I may refuse the application.

Field of the application.

The application is titled “Nicking and extension amplification reaction for the exponential amplification of nucleic acids”.
The field of the invention is “… in general directed to the rapid exponential amplification of short DNA or RNA sequences at a constant temperature”[7]. The invention is in the field of in vitro diagnostics for detecting the presence of harmful species or determining the genetic sequence of a region of interest.
[7] Description, p. 1A l. 8-9

The person skilled in the art

It is well established that many of the issues in an Opposition are answered by reference to the person skilled in the art:

“He is the person to whom the patent is addressed and who must construe it. He is the person whose knowledge will determine whether a patent is novel. He is the person who will judge whether a patent is obvious.”[8]
[8]Root Quality Pty Ltd v Root Control Technologies Pty Ltd [2000] FCA 980 at [70].

The hypothetical skilled person works in the field with which the invention is connected and is a non-inventive person or team likely to have a practical interest in the subject matter of the invention.[9]
[9]Root Quality Pty Ltd v Root Control Technologies Pty Ltd [2000] FCA 980 at [70]-[72].

The Applicant presented evidence in the form of a declaration from Dr. Alison Todd. Dr. Todd has worked in the field of molecular diagnostics since the 1980s in both academia and industry and is a named inventor on patents relating to improvements in nucleic acid amplification techniques including inter alia PCR and LAMP[10]. Dr. Todd stated that she regularly kept herself apprised of the scientific, patent and commercial literature[11]. Dr. Todd would seem to be more inventive than the hypothetical skilled person, but her evidence can be used to inform me what said hypothetical construct would have known and/or done as of the priority date.

[10] Todd at [4-10]
[11] Todd at [11-12]
The Opponent presented evidence in the form of two declarations from Prof. Jeremy Edwards. Prof. Edwards has worked in the field of molecular genetics since the late 1990s and has been involved in developing inter alia non-PCR DNA amplification technologies for whole genome sequencing[12]. Prof. Edwards also is listed as an inventor on a number of biotechnology patents and patent applications. I likewise consider Prof. Edwards’ evidence can be used to inform me what the hypothetical skilled worker would have known and done as of the priority date.

[12] Edwards #1 at [1-6]
Both of the experts appear to be independent and well qualified to provide relevant evidence. Indeed, having reviewed all declarations I do not find there to be any significant points of disagreement with respect to the state of the prior art or common general knowledge. Any disagreements between the declarants seem to be restricted to the construction of the opposed specification and prior art citations. I will discuss these disagreements throughout the decision as required, bearing in mind that the job of construction is a matter of law and is one for the Delegate[13].

[13] Cf paragraph 34 infra
I note that the Opponent submitted at the hearing that Prof. Edwards’ evidence should be preferred since his first declaration is given with respect to the invention claimed. It is the case that Prof. Edwards’ declaration is structured as an analysis of the invention first, followed by comments as to why in his opinion the exhibits are anticipatory. However, it is the job of the Delegate to construe the claims, with respect to the available evidence. Indeed, the fact that Prof. Edwards’ evidence is given with the claimed invention in mind leads me to give less weight to his evidence relating to how the prior art citations would be read by the skilled worker, as it is somewhat tainted by hindsight knowledge of the invention[14].
[14] “… even the most honest and competent witnesses will tend to exaggerate what could have been anticipated once they have the advantage of knowing of the invention and the process involved in reaching that invention”. Eli Lilly and Company Limited v Apotex Pty Ltd [2013] FCA 214; 100 IPR 451 at [473].

Background of the invention

Before construing the specification, I note the comments of Middleton J in Eli Lilly and Company Limited v Apotex Pty Ltd[15]:

“It is well settled that the Court should, from the outset, approach the task of patent construction with a generous measure of common sense. The Court must place itself in the position of a person skilled in the relevant art, being the subject matter of the patent. From this perspective, the patent is to be read as a whole, in the context of the specification and in light of the prevailing common general knowledge and state of the relevant art at the priority date.”
[15][2013] FCA 214;100 IPR 451 at [139].
Nucleic acid tests are attractive due to the ability for fast and specific recognition of inter alia pathogens, genetically modified organisms and single nucleotide polymorphisms[16]. Although PCR is the gold standard for detection methods[17] the need for thermal cycling is seen as disadvantageous in some circumstances such as field applications[18].

[16] See p. 1A l 18-26 of the description
[17] Ibid. l 31
[18] Description at p. 2 l 1-2, Edwards #1 at [23].
To address this, various isothermal amplification techniques had been developed including Loop Mediated Amplification (LAMP), Helicase-dependent amplification (HDA), Recombinase Polymerase Amplification (RPA) and Strand Displacement Amplification (SDA). These are surveyed at the description at p. 2-4, Edwards #1 at [27]-[49], and Todd at [22]-[30].

The specification states that “provided herein are methods of amplifying nucleic acid target sequences that rely on nicking and extension reactions to amplify shorter sequences in a quicker timeframe than traditional amplification reactions, such as, for example, strand displacement amplification reactions”[19].

[19] Description at p. 4 lines 17-20
Much of the description relates to the mechanism of the (known) technique of strand displacement amplification (SDA), which I will summarise briefly with reference to Fig 1A: A primer comprising a nickase target site at its 5’ end anneals to a target (1) and begins extension (2) to create a new strand with a nickase site at its 5’end (3). A second reverse primer also with a nickase target site binds to the new strand, displacing the original (4) and extends (5) to produce a double stranded amplicon (6). The nickase target sites allow self-priming of this amplicon without thermal cycling (Fig 1B, not shown here). Notably in this SDA technique, the initial target must be single stranded.

A “Detailed Mechanism of Amplification” is set out at p 37 of the description.

Amplification reactions of the present methods require the presence of a nucleic acid target, at least two template oligonucleotides, a nicking enzyme, for example, a thermophilic nicking enzyme, a thermophilic polymerase, and buffer components all held at the reaction temperature. The recognition region of the templates interacts with the complementary or substantially complementary target sequence. Since the melting temperature of the complementary or substantially complementary regions of the target and template is well below the reaction temperature, the interaction between the two nucleic acid strands is transient, but allows enough time for a thermophilic polymerase to extend from the 3' end of the template along the target strand[20].

…

For a double-stranded target, both templates can interact with the corresponding target strands simultaneously (forward template with the antisense strand and reverse template with the sense strand) during the normal breathing of double-stranded DNA. The target may also be generated by a [sic] single or double nick sites within the genome sequence[21].

[20] p 37 l. 10-19 of the description

[21] Ibid l. 22-24

These passages do contemplate alternative mechanisms, so the experts’ reading of them is instructive. Dr. Todd understands this to mean:

This mechanism takes advantage of the ‘breathing’ i.e. transient separation of short regions of complementary strands within the context of longer regions of double-stranded sequences which can occur at moderate temperatures. This distinguishes the methods of the patent application from the methods described in exhibits AT-5 to AT-10 (and other methods I was aware of at July 2007) by allowing primers to bind to the target without the need for a high-temperature thermal denaturation step, additional outer primer sets (e.g. bumper primers) or other alternative enzymatic pre-incubation steps[22].
[22] Todd at [78]
Prof. Edwards does not comment on this in his first declaration except to say that isothermal amplification reactions could be performed without an initial denaturation step[23]. In Evidence in Reply, Prof. Edwards disagrees with Dr. Todd’s understanding:

I am not aware of any evidence to suggest a shorter primer can interact more easily with double stranded DNA during normal breathing of DNA, and I would be surprised if that were indeed the case for typical primer lengths. In my view, the vast majority of primers interact by the creation of single stranded DNA from the nicking enzyme as in van Ness. This is consistent with the last sentence of the passage on page 37 extracted above which states that the target may also be generated by a single or double nick sites within the genome sequence[24].

[23] Edwards #1 at [62]
[24] Edwards #2 at [45]

Prof. Edwards goes on to say that:

I note that there are no examples of the Opposed Application showing amplification without the use of nicking enzymes and the nicking enzymes are a requirement of the claims[25].

This seems to me to involve a misunderstanding of the role of nicking enzymes in the SDA method. The nicking enzymes required in the claims (see also Fig 1A reproduced above) have target sites in that part of the template not complementary to the target, and so would not necessarily nick the target sequence[26]. However I understand the last sentence of the quoted passage at p. 37 to mean that the specification does not exclude the possibility of additional enzymes creating a single or double-stranded break, and this will become more important later in the decision.

[25] Ibid.

[26] Claims 4, 35, 36, 43 expressly exclude this.

The description goes on to state that amplification can take place without an initial thermal denaturation step, and that the reaction temperature can be chosen appropriately based on the length and GC concentration of the target as well as the presence of other agents that affect T_m.

The present methods do not require the use of temperature cycling, as often is
required in methods of amplification to dissociate the target sequence from the amplified nucleic acid. The temperature of the reaction may vary based on the length of the sequence, and the GC concentration, but, as understood by those of ordinary skill in the art, the temperature should be high enough to minimize non-specific binding. …. In certain embodiments, there is no denaturation step in
the process. The entire amplification process, including interacting templates with target nucleic acid, is conducted within substantially isothermal conditions, and without a denaturing step (e.g., no significant temperature increase (e.g., no increase in temperature to 90-110°C)), in some embodiments of the present methods[27].
[27] p. 41 l 11-24 of the description

In certain embodiments, one or more agents that destabilize nucleic acid interaction (e.g., inter-strand or intra-strand interactions) are included in an amplification process, and in alternative embodiments, one or more of such agents are not included in an amplification process[28].

Without being bound by theory, such agents lower the melting temperature (T_m) of nucleic acid interactions (e.g., lower duplex T_m). Those of ordinary skill in the art may determine the appropriate destabilizing agent and appropriate destabilizing agent concentration for the reaction, considering, for example, the amount of destabilization as well as the need to maintain enzymatic activity[29].

[28] p. 35 l 17-20
[29] p. 35 l 28-32

The detailed description of the invention also provides additional information regarding to template size, reaction temperature and time, and the provenance of the enzymes to be used in the assay. The specification also contains thirty[30] examples, which demonstrate the method of the application in a variety of applications, including different detection methods and target organisms. I will refer to relevant parts of the detailed description and examples where relevant later in this decision.

[30] An “Example 31” provides a statement that the invention is not limited to the targets and templates exemplified previously
Two examples which were relied upon by the Opponent in their submissions relating to s 40[31] are worthy of discussion at this point. Example 28 relates to experiments to determine the optimal number of spacer bases (the distance between the two primers[32]). In one experiment on a bacterial target, the optimal number of spacer bases was described as 1, 2, 3 or 4[33]. In a second experiment on a viral target, numbers of spacer bases of 2, 5, 6, 7, and 8 were tested, with 2 or 5 being optimal and no specific product detected with 6 or more spacer bases[34]. The effect of zero spacer bases was also tested: experiments on a miR target revealed that lack of spacer bases produced non-specific product[35], whereas in other experiments a spacer length of zero could result in a specific product[36]. The example concludes by suggesting that appropriate length of spacer bases for each should be determined by testing more than one set of templates[37].

[31] Opponent’s submissions (OS) at [310] and [319]
[32] Defined at p. 32 l 8 of the description
[33] p. 80, l. 13-20 of the description
[34] p. 81 l. 1-5 of the description
[35] Ibid. l. 7-8
[36] Ibid. l. 11
[37] Ibid. l. 11-14

Example 29 relates to experiments to determine the effect of stabilising regions using similar bacterial assays as performed in earlier examples. Templates with or without a 10-base stabilising region were prepared[38], and those without stabilising regions showed no amplification[39].
[38] Ibid. l. 17-18
[39] Ibid. l. 23

The claimed invention

The opposed application ends with 52 claims, of which claims 1, 35, 36 and 43 are independent.

Claim construction

The principles of construction have been well traversed by the courts, and a number of principles have been set down in various decisions. Principles most relevant to this particular case were summarised by the Full Court of the Federal Court in Kinabalu Investments Pty Ltd v Barron Rawson Pty Ltd[40].

“When determining the nature and extent of the monopoly claimed, the specification must be read as a whole. But as a whole it is made up of several parts which have different functions. The claims mark out the legal limits of the monopoly granted. The specification describes how to carry out the process claimed and the best method known to the patentee of doing that. Although the claims are construed in the context of the specification as a whole, it is not legitimate to narrow or expand the boundaries of monopoly as fixed by the words of a claim, by adding to those words glosses drawn from other parts of the specification. If a claim is clear and unambiguous, it is not to be varied, qualified or made obscure by statements found in other parts of the document. It is legitimate, however, to refer to the rest of the specification to explain the background of the claims, to ascertain the meaning of technical terms and resolve ambiguities in the construction of the claims. See Flexible Steel Lacing Co vBeltreco Ltd (2000) 49 IPR 331 at [73] – [75] (Hely J).
[40] [2008] FCAFC 178 at [44] – [45]:
Other more specific principles of construction collected in Flexible Steel at [81] are:
·a specification should be given a purposive construction rather than a purely literal one;
·the hypothetical addressee of the specification is the non-inventive person skilled in the art before the priority date;
·the words used in a specification are to be given the meaning the hypothetical addressee would attach to them, both in the light of the addressee’s own general knowledge and in the light of what is disclosed in the body of the specification;
·as a general rule, the terms of the specification should be according their ordinary English meaning;
·evidence can be given by experts on the meaning those skilled in the art would give to technical or scientific terms and phrases, and on unusual or special meanings given by such persons to words which might otherwise bear their ordinary meaning;
·however, the construction of the specification is for the court, not for the expert. In so far as a view expressed by an expert depends upon a reading of the patent, it cannot carry the day unless the court reads the patent in the same way.”
In order to give proper effect to a statement of claim, the presumption is that each claim is of differing scope and, where possible, claims should be construed accordingly[41]^.
[41] Parkinson v Simon (1894) 11 RPC 493

The independent claims

The four independent claims are each directed to nicking SDA and are characterised by the lack of a (thermal) denaturation step. The difference between the independent claims can be explained by choice of target (single or double stranded) and some functional limitations specifying the efficacy of the amplification.

The claims are attached in an appendix to this decision. Additionally, due to the significant overlap between the independent claims, I will deal first with claim 1 and then point out the respective differences between claim 1 and the other independent claims. I will not dwell particularly on the dependent claims except to the extent that they may influence the construction of the independent claims.
All claims are directed to methods for nucleic acid sequence amplification. Claim 1 is as follows:

1. A method for nucleotide sequence amplification, which comprises:
obtaining, from an animal, plant, or food, a sample comprising a target nucleic acid, the target nucleic acid comprising a target nucleotide sequence,
     without first subjecting the target nucleic acid molecule to a denaturation step associated with amplification of the target nucleotide sequence, combining, in a single step, the obtained sample directly with an amplification reagent mixture to form a reaction mixture or diluting the obtained sample and combining, in a single step, the diluted sample with an amplification reagent mixture to form a reaction mixture, in either case, the amplification reagent mixture comprising:
     (i) a polymerase,
     (ii) a first template nucleic acid that hybridizes to a first strand of the target nucleotide sequence, wherein the first template comprises a nucleic acid sequence comprising a first template recognition region at the 3' end that is complementary to the 3' end of the first strand of the target nucleotide sequence, a nicking enzyme binding site and a nicking site upstream of said recognition region; and
     (iii) a second template nucleic acid that hybridizes to the complement of the first strand of the target nucleotide sequence, wherein the second template comprises a nucleotide sequence comprising a second template recognition region at the 3' end that is complementary to the 3' end of the complement of the first strand of the target nucleotide sequence, a nicking enzyme binding site and a nicking site upstream of said recognition region, and
     (iv) one or more nicking enzymes;
     subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions to amplify the target nucleotide sequence; and
     detecting amplified target nucleotide sequence in real time within 10 minutes of subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions.

A summary of the key differences between the independent claims is set out here for convenience:

Claim 1	Claim 35	Claim 36	Claim 43
Double or single stranded target	Double stranded target	Single stranded target	Double stranded target, 22-35 nt long
Stabilising region
First and second nickases don’t nick inside target	First and second nickases don’t nick inside target	First and second nickases don’t nick inside target
Target not denatured	No thermal denaturation	No thermal denaturation	No thermal denaturation
Detection within 10 minutes	Detection within 10 minutes
Capability of 10⁷ fold amplification in 12 minutes

Sample

The first step of the method[42] comprises obtaining a sample from an animal, plant or food. The sample comprises a target nucleic acid which in turn comprises a target sequence.

[42] For the avoidance of doubt, any reference to “the method” or “the claim” refers to the method of claim 1 unless otherwise specified.
I note at this point that although a literal reading of the claim is that the sample must contain the target nucleic acid and target nucleotide sequence, this would be a nonsensical interpretation given that the field of the invention is diagnostics. It would seem absurd that a diagnostic method could only fall within the scope of the claim if a sample tested positive for the target. My understanding is that the introduction of “target nucleotide sequence” at this point of the claim is to provide an antecedent for the definition of the “templates” which must be designed with the intended target mind. It seems a more sensible construction is that the sample need only be suspected of comprising the target nucleic acid and target nucleotide sequence. There is precedent for this approach in construing biological assays: in CSL Limited v Pharmacia & Upjohn AB [2000] APO 58, a method for removing viruses from a sample was concluded to refer to carrying out the steps of the method even when no virus was present.

Nevertheless, the language “method for nucleic acid sequence amplification” and “to amplify the target nucleotide sequence” I take to be limiting insofar as if there is target nucleotide sequence present, the reagents and reaction conditions must be such as to be able to amplify it.

Prof. Edwards considered that the “sample” in the claim also includes synthetic samples, because of the reference in claims 9-11 that the target nucleic acid can be synthetic DNA or RNA[43]. I do not agree: the discussion of “samples” in the description[44] only appears to envision environmental samples. I also put this potential contradiction to the parties at the hearing and both agreed that there is no contradiction between the language of claim 1 as written and claims 9-11, as for example detection of genetically modified organisms could reasonably said to be detection of synthetic nucleic acid in a food sample.
[43] Edwards #1 at [82]
[44] p. 19 lines 13-35 of the description

Target nucleotide sequence

The target nucleic acid is not limited, and the target nucleotide sequence is not defined in terms of its length. The specification makes a number of references to suitable target lengths.

The “field of the invention” at p. 1A lines 8-9 couches the method as being directed to rapid exponential amplification of short DNA or RNA sequences. The summary of the invention refers to amplification of “shorter sequences in a quicker timeframe than traditional amplification reactions” such as SDA[45], and SDA had been characterised as usually amplifying products 60-100 bases in length[46]. Other references to the targets of the present invention are “can amplify 20-30mer products”[47], “for example, from 19-50 nucleotides in length” at reaction temperatures up to 60°C[48] and that the polymerase “does not, however, need to be very processive (30-40 nucleotides for a single synthesis are sufficient)”[49] which implies a target only 30-40 bp longer than one of the primers.
[45] p. 4 lines 18-19 of the description
[46] Ibid. lines 5-6
[47] Ibid. lines 29-30
[48] p. 19 lines 6-7 of the description
[49] p. 21 lines 24-25 of the description

The only working example that dealt with variation in target length was Example 28, in which 12-mer primers were able to amplify a targets with a maximum length of 28[50] or 29[51].
[50] C. trachomatis, 4 nt spacer, p. 80 l. 20 and Fig 29 of the description
[51] Viral RNA, 5 nt spacer, p. 81 line 4, data not shown

I also need to take into account that the specification would be read by the skilled worker. Dr. Todd’s evidence is she considers that the methods of the application rely on the region being short in order to take advantage of DNA “breathing”.[52] However she does not see any hard limit on target length either[53]:

“[I]f the reactions described in the patent application were carried out under conditions which either destabilise the DNA (such as higher temperatures, different pH), or include agents known to lower the melting temperature of DNA (such as betaine, DMSO or TMAC) then longer primer recognition sites and/or spaces between them, than those stipulated or implied in, for example, claims 2, 3, 16, and 43-44, could be employed”.

[52] Todd at [78-80] and also [115]

[53] Todd at [90]

In my view, none of these statements regarding target length can be taken to be absolutely limiting. To do so would run the risk of selectively importing a “gloss” from one part of the specification. I therefore conclude that the target nucleotide sequence is not limited to any particular length (or indeed other properties such as melting temperature or GC content).

Template recognition region

The term “template recognition region” refers to the sequence-specific parts of the two templates. The two templates are identical to the 5’ end of the target nucleotide sequence and the reverse complement of the 3’ end of the target sequence[54], and the lengths of the recognition regions are described as being for example 8-16 bases in length[55].
[54] p. 27 lines 9-17 of the description
[55] Ibid.

The lower limit of length appears to derive from the need for specificity (as set out on p. 28, a total recognition length of 16 will be found on average once in the human genome) and the upper limit appears to derive from reaction kinetics. At the bottom of p. 28, it is described that a length of 20 nucleotides per recognition region did not produce detectable products and 16 nucleotides had four-fold lower signal than 12-mer recognition regions.

This appears consistent with Dr. Todd’s reading that the access of the template to double-stranded nucleic acids is limited by the natural breathing of the DNA. The skilled worker would then understand that longer primer recognition sites may be possible by varying the reaction conditions[56].
[56] Todd at [90]

Furthermore, if the target is single-stranded or the target was to be exposed by another mechanism then there would not seem to be any need for an upper size limit on the target recognition sequence length.

Spacer bases

The term “spacer bases” is used in the application to refer to the bases in the target between the two recognition regions.

At p. 27 first paragraph the concept of spacer bases is mentioned by reference to less than total coverage of the templates on the target sequence. The discussion at p. 27 lines 7-8 that the templates “may be designed to bind to about 30% … or about 60% of the target sequence” implies a spacer lengths of up to 40% or more of the target sequence.

At p. 32 of the description, a number of embodiments with 1 to 5 “spacer bases” are described. However these have also been carefully drafted using language such as “in certain embodiments”, “in exemplary embodiments” to make it clear that the description does not intend to limit the number of spacer bases to any particular number.

The application contemplates that the skilled worker should optimise the spacer length according to the reaction conditions, as discussed above in relation to Example 28. The specification explicitly states that spacer bases are not required[57], and spacer bases are not listed in any of the independent claims.
[57] p. 33 l. 1-2 of the description

The declarants also did not consider there to be any absolute limit on the number of spacer bases. Although Dr. Todd does refer at various points in her declaration to the number of 1-5 spacer bases I did not get the impression that she considers this numerical range to be essential to the invention of all claims.

I therefore conclude that there is no limitation of the number of spacer bases present in the independent claims, as long as the template design is suitable for the reaction to proceed.

Nicking enzyme, binding site and nicking site

The specification defines a nicking enzyme as one that binds to double-stranded DNA and cleaves one strand of a double-stranded duplex[58]. This distinguishes a nickase from a double-stranded cleaving enzyme such as a restriction endonuclease. A number of exemplary nickases are disclosed in Table 3. The binding site and cleavage site of these enzymes have been characterised, and the declarants did not have any difficulty understanding these features[59].
[58] p. 24 l. 7-8 of the description
[59] Edwards at [41-42], [99-100]

Stabilising Region

A point of contention at the hearing was whether all “templates” in the independent claims possess a stabilising region. The Applicant pointed to the passage “Templates are defined as oligonucleotides that bind to a recognition region of a target sequence and also contain a nicking enzyme binding region upstream of the recognition region and a stabilizing region upstream to the nicking enzyme binding region” at p. 21 l 1-3 of the description, as well as the “Template Design” section at p. 26 l. 25 et seq, to argue that these features must be found in all templates. I do not agree for the following reasons:

All independent claims provide a structural definition of the template nucleic acids, which gives the impression that these features are not relying on a “dictionary” from the specification but are being defined ab initio. Independent claims 1, 35 and 36 do not recite a stabilising region but claim 43 does. This indicates an intent from the drafter to differentiate between these claims.

Additionally, dependent claims 33 and 34 explicitly add the feature of a stabilising region to claims 1, 35 and 36. This reinforces the presumption that no stabilising region was required in the independent claims where it is not mentioned.

Consequently, I do not consider that claims 1, 35 and 36 necessarily comprise a stabilising region.

The requisite structural features of a stabilising region are not explicitly defined in the description but the skilled worker would understand that it is a region of sufficient length and GC content to stabilise the double-stranded region after nickase cleavage[60].
[60] Cf Todd at [82]

Without denaturation step

There is no explicit definition of denaturation: there is an example given at p. 41 lines 23-24 that a denaturing step can be a significant temperature increase, such as to 90-110 °C. There are also consistent references in the description to denaturation at 94 °C being used to terminate reactions. In the discussion of prior art techniques, they are all described as requiring heat denaturation.

Prof. Edwards seems to treat “denaturation” purposively as a step of making the target region accessible to oligonucleotide primers[61].
[61] Edwards #1 at [83-84]

Claim 1 refers to “without … a denaturation step” whereas claims 35, 36, and 43 refer to “without a heat denaturation step”. Using the same principles of claim differentiation as applied above with respect to the stabilising region, I consider that claims 35, 36 and 43 do allow non-thermal denaturation. This is also consistent with the skilled worker’s understanding of other ways to destabilise double-stranded DNA[62] or indeed other ways of allowing primer access such as nicking[63].
[62] Cf Todd at [90]
[63] Cf. p. 37 l. 24 of the description

Essentially isothermal conditions – nicking SDA reaction

There was no controversy between the parties regarding the essential reagents and mechanism of the nicking SDA (or “NEAR”) reaction. I have discussed the mechanism of the SDA reaction earlier. The reaction proceeds substantially isothermally which is defined at p. 26 lines 5-16 to be a substantially constant temperature because the nicking and extension reactions both work in the same temperature range, however slight variations in temperature are not excluded. I am satisfied that the skilled worker understands an isothermal reaction is one where temperature fluctuations are not required for the reaction to proceed.

Detection within 10 minutes

Claims 1 and 36 specify a step of detecting amplified target nucleotide sequence in real time within 10 minutes of commencing the isothermal amplification reaction. In the section “readout” at p. 45 l. 26 et seq, a large number of non-limiting examples of target sequence detection methods are listed. A subset of these are “Real time” detection methods[64]. The skilled worker knows a variety of real-time detection methods; examples used in the specification are FRET, Molecular Beacons and intercalating dyes. The last method is non-specific to the target [65]and therefore detecting a target using a non-specific dye would be understood as carrying out the assay with reference to a negative control, whereas both FRET and molecular beacons could be expected to give a target-specific signal.
[64] p. 46 l. 25 et seq of the description
[65] Cf Example 6

When testing an unknown sample, it seems absurd to require that the target must be found within 10 minutes. Therefore I construe claims 1 and 36 to specify that real-time detection methods are employed during the first 10 minutes after the initiation of the isothermal reaction.

Mechanism of action/additional reaction components

The relationships between the integers in the claims are all defined as “comprising” or “comprises”. This is typically understood in the patent field to mean “includes” in a non-exhaustive or open sense. I note the passage at p. 83 l. 2-3 of the description which states that the invention may be practised in the absence of undisclosed elements, and that the applicants contemplate that the closed term “consisting of” could be used in place of the open term “comprising”. Since there has been a clear decision to use the open term “comprising” in the claims, it is clear that undisclosed elements may be included in the claimed method.

Dr. Todd understands that the invention, in using short recognition regions, takes advantage of the natural breathing of genomic DNA to initiate polymerisation without the need for thermal denaturation of the target nucleic acid[66].
[66] Todd [78]

The claims do not specify the mechanism of amplification; they only specify the structural features of the reagents and the running of the reaction under substantially isothermal conditions. As mentioned earlier, the description does have a section describing the mechanism of amplification at p. 37 et seq. This does refer to the templates interacting with the target during the natural breathing of double-stranded DNA[67], but also equivalently suggests that the target may be exposed by a single or double-stranded nick[68].
[67] p. 37 l. 22-24 of the description
[68] Ibid. l. 25-26

This latter embodiment is not excluded from the present claims, even though it would require the presence of an additional appropriately chosen restriction enzyme or nickase.

Another factor that suggests that other reaction components should be considered in the scope of claim 1 is the explicit exclusion of bumper primers[69] from claim 36.
[69] Cf. Edwards #1 at [31] and Todd at [30]

Other independent claims:

Claim 35 is specific for double stranded nucleic acids, and the two templates are defined correspondingly. The only other difference to claim 1 is the lack of the requirement for detection within ten minutes. Therefore there is no explicit requirement to detect the nucleic acid at all. However given the field of diagnostics there is clearly an implicit requirement that the sample be assayed for the presence of amplified nucleic acid at some point, and likewise there is an implicit requirement that the method be capable of amplifying its target.

Claim 36 is specific for single stranded nucleic acids, and has the further exclusion that bumper primers are not to be used in the method. Otherwise the scope is the same as claim 1.

Claim 43 is specific for double-stranded nucleic acids, explicitly points out the presence of a stabilising region in the template molecules, and instead of the requirement for detection in 10 minutes has a feature relating to the 10⁷ fold amplification of a 22-35 mer target sequence within 12 minutes.

10⁷-fold amplification within 12 minutes (Claim 43)

Claim 43 specifies that 10⁷ fold amplification of a 22-35 nucleotide target is obtained within 12 minutes. It is only possible to determine if this limitation is satisfied if quantitative measurement of nucleic acids is carried out before and after the amplification process. However, since the purpose of the amplification in the real world would be to determine the presence of (unknown) nucleic acid in an environmental sample, this limitation could never be borne out, and so I feel I must reject the literal construction of this term for the claim to have any real world meaning.

A more compelling construction of this term is that the assay system must be capable of producing this level of amplification for a positive control. The only demonstration of measurement of fold increase is found in Example 27 and table 5 of the description. After a 5 minute amplification using a 25mer B subtilis target sequence and 12mer recognition sequences, approx. 9 logs of amplification were observed for 50 copy starting samples and approx. 8 logs amplification for 500 copies. This implies that the amount of amplification is dependent on the number of starting copies (and presumably the amount of reagents available). This leads further credence to the construction that the method must be capable of attaining those levels of amplification for an appropriate control rather than in an uncharacterised sample.

I also note that the fold amplification limitation is to “a” 22-35 nucleotide target. This has no antecedent so could be construed to not be the same as the double-stranded target sequence. However, given that the templates must be designed with a specific target in mind, the method is only capable of amplifying “a” 22-35 nt target if the target does in fact have that length.

Novelty

It is well established that the general test for anticipation is the reverse infringement test. The classic formulation of this test is that given by Aicken J[70]:

“The basic test for anticipation or want of novelty is the same as that for infringement and generally one can properly ask oneself whether the alleged anticipation would, if the patent were valid, constitute an infringement.”

[70] Meyers Taylor Pty Ltd v Vicarr Industries Ltd [1977] HCA 19; 137 CLR 228 at 235

This test is satisfied if the alleged anticipation discloses all the essential features of the invention claimed[71].
[71] Nicaro Holdings Pty Ltd v Martin Engineering Company [1990] FCA 40; 16 IPR 545 at 549

Both parties identified the oft-cited decision of the UK Court of Appeal in The General Tire & Rubber Company v The Firestone Tyre and Rubber Company Limited[72] (the General Tire case):

"If the prior inventor's publication contains a clear description of, or clear instructions to do or make, something that would infringe the patentee’s claim if carried out after the grant of the patentee's patent, the patentee's claim will have been shown to lack the necessary novelty, that is to say, it will have been anticipated. The prior inventor, however, and the patentee may have approached the same device from different starting points and may for this reason, or it may be for other reasons, have so described their devices that it cannot be immediately discerned from a reading of the language which they have respectively used that they have discovered in truth the same device; but if carrying out the directions contained in the prior inventor's publication will inevitably result in something being made or done which, if the patentee's patent were valid, would constitute an infringement of the patentee's claim, this circumstance demonstrates that the patentee's claim has in fact been anticipated.
If, on the other hand, the prior publication contains a direction which is capable of being carried out in a manner which would infringe the patentee's claim, but would be at least as likely to be carried out in a way which would not do so, the patentee's claim will not have been anticipated, although it may fail on the ground of obviousness. To anticipate the patentee's claim the prior publication must contain clear and unmistakeable directions to do what the patentee claims to have invented. A signpost, however clear, upon the road to the patentee's invention will not suffice. The prior inventor must be clearly shown to have planted his flag at the precise destination before the patentee." (citations omitted)
[72] [1972] RPC 457

At the hearing, the Applicant pressed the relevance of Bristol-Myers Squibb Co v F H Faulding & Co Ltd[73], such that the citation needs to “teach” the claimed invention. This decision dealt with a “paper anticipation” and (to paraphrase) whether a publication of an unsuccessful trial constituted an enabling disclosure[74]. However, as will become evident, it does not seem necessary to consider whether the facts of this case mirror the situation in Bristol-Myers Squibb.
[73] (2000) FCA 316
[74] Op. cit at 62-63

The opponents pressed grounds of lack of novelty in view of D1 alone, D3 [D3a is an English translation of D3] alone, D1 and D3 read as a single document, and D2 alone.

I will deal initially with the assertion that D1 and D3 should be read as a single document. Subsection 7(1)(b) provides that for novelty, prior art information made publicly available in two or more related documents, or through doing two or more related acts, can be used, if the relationship between the documents or acts is such that a person skilled in the relevant art would treat them as a single source of that information. The factors for determining this relationship are very much on a case by case basis[75].
[75] Nicaro Holdings, supra at 570

D3 is a dissertation by one of the authors of D1. At p.3 of D3a it is acknowledged that parts of the thesis were published in D1. This bald statement does not lend me to consider that the non-overlapping parts of D1 and D3 would be treated by the skilled reader as a single document. Certainly, there are parts of D3 that are not found in D1 and are likely to represent different work, and any parts of D1 not found in D3 could well be contributions from different authors. I do not see any reason why the skilled worker would consider D1 and D3 so related as to be able to mosaic different parts of the document.

D1: (Ehses, S. et al. (2005) “Optimization and design of oligonucleotide setup for strand displacement amplification”. J Biochem Biophys Methods. 30;63(3):170-86)

The Opponent submitted during the hearing that applying the reverse infringement test to D1: “if the authors of D1 were to repeat their experiments today, they would infringe the claims”. For the reasons below, I disagree.

D1 is a publication directed to “Optimisation and design of oligonucleotide setup for strand displacement amplification”. In particular, D1 seeks to address “[t]he major problem of isothermal amplification mechanism [which] is the accumulation of non-predictable byproduct especially for longer incubation time and low concentrations of initial template DNA”[76].

[76] D1, abstract
D1 seeks to address this by “Besides improving the reaction conditions, the stringency of primer hybridization can be distinctly improved by computer based sequence prediction algorithms based on the thermodynamic stability of DNA hybrid a [sic] described by the partition function of the hybridization reaction. An alternative SDA mechanism, with sequences developed by this means is also investigated”[77].
[77] Ibid.

D1 discloses three types of SDA reactions: a “Standard SDA”, “Nicking SDA” and “Minimal SDA”. It was not disputed by the parties that the reaction scheme set out in Fig.1 of D1 is the same as the amplification mechanism used in the present invention.

The primers for “Nicking SDA” contain a nickase recognition site GAGTC [Table 1] but primers for standard and minimal SDA do not.

For the purposes of novelty, it is necessary to consider what D1 discloses with respect to Nicking SDA. The “Oligonucleotides (Table 1) were synthesized and purified by IBA and MWG”[78] (which are biotech companies). The experimental conditions for Nicking SDA are disclosed at Section 2.1.3

Nicking SDA reactions were performed in a 30 µl volume with final concentrations of 100 mM KCl, 35 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.4 mM each dATP, dGTP, dTTP and dCTP, 0.1 mg/ml BSA, 0.5 µM of each primer, either 1 µM TO-PRO-1 or 1 :5 SYBR Gold, 0.24 U/µl exo-Bst 1.7 U/ul N.BstNBI). After addition of template DNA into final volume of 24 µl and before addition of any enzymes, the reaction sample was incubated for 3 min at 95°C, followed by 1 min at 55°C. Upon addition of the enzymes, the amplification mixture was incubated 15–60 min in an ICycler (BioRAD) and the increase in fluorescence intensity was monitored. [emphasis added]

[78] D1 at 2.1.1

As can be seen from the above, the Nicking SDA reactions were performed with a preliminary heat denaturation step, and were performed on purified synthetic template samples.

The Opponent drew attention to the observation at 3.2 of D1 that “[w]hen starting with less template, omitting the initial denaturation step or increasing the reaction time, the standard SDA system as well as the nicking system shows the tendency to side-reactions”. The Opponent conceded during the hearing that D1 does not directly disclose the results of nicking SDA without an initial denaturation step[79] but contended that the discussion of the consequences of omitting the initial denaturation step demonstrates that nicking SDA had in fact been performed without said denaturation step. This was also Prof. Edwards’ reading of the citation, and indeed he considers that disclosure demonstrates to him that a denaturation step is not required for nicking SDA to successfully amplify a target[80].
[79] Figure 3c, which relates to the above quotation, discloses standard and nicking SDA with and without template after 90 mins amplification
[80] Edwards #1 at 173-174

It seems to me that this reading is at odds with the disclosure of D1 in the figure legend to Fig 3c “Because of the increased side-reactions, the desired product cannot be detected by subsequent staining with SYBR Gold”. Nevertheless, Prof. Edwards subsequently performed a nicking SDA reaction as per section 2.1.3 of D1 but without the initial denaturation step and demonstrated detection of product by both SYBR fluorescence and a sequence specific molecular beacon[81].
[81] Exhibit JE-17.

Taking all the above into account, I construe D1 to disclose that standard or nicking SDA could have been carried without an initial denaturation step. The dual choices here seem to enliven the alternative proposition in General Tire:

If, on the other hand, the prior publication contains a direction which is capable of being carried out in a manner which would infringe the patentee's claim, but would be at least as likely to be carried out in a way which would not do so, the patentee's claim will not have been anticipated.

However, regardless of the above, D1 uses an entirely synthetic template and does not perform amplification on a sample derived from an animal, plant, or food, as required by each of the claims. Prof. Edwards considers this feature is provided by the mention of diagnostics and detection of mycobacteria in D1[82] however these are general statements as to the applicability of SDA in general. There is no clear and unmistakeable direction to carry out a nicking SDA reaction on an environmental sample, and there is certainly no clear and unmistakeable direction to carry out the specific combination claimed, which is a nicking SDA reaction on an environmental sample in the absence of a thermal denaturation step.
[82] Edwards #1 at 172 and 190

Couched in the terms of the Opponent’s original contention, if the authors of D1 were to reproduce their experiments today, they would not infringe the present claims for at least the reason that the experimental samples are not derived from an animal, plant or food.

D3 (Ehses, S. “Isothermale in vitro Selektion und Amplifikation zur Untersuchung von Evolutionsvorgängen” (Dissertation, August 2005, Ruhr-Universität Bochum), published online 1 August 2005) and D3a (English translation thereof)

D3 also discloses a nicking SDA reaction apparently identical to D1 (see methods at 2.3.1 and 2.3.2). Prof. Edwards considers that D3 successfully performs the method without an initial thermal denaturation step[83] apparently based on a single line at [134] of D3a that “A denaturation step prior to the addition of the enzyme delays the formation of this site [sic] product”.
[83] Edwards #1 at [220]

However similar considerations apply as to D1: it is not clear whether this line refers to standard or nicking SDA and therefore does not constitute a clear and unmistakeable direction to carry out nicking SDA without denaturation; and D3 does not use environmental samples, so that should the author of the dissertation repeat her experiments today, there would be no danger of infringing the present claims for at least that reason.

D2 (US2005/0112631 A1 to Piepenburg et al)

D2 is generally directed to a method called “Recombinase Polymerase Amplification” in which a complex of a recombinase (such as RecA) and a primer is used to invade double stranded DNA and initiate additional strand synthesis without need for initial thermal denaturation[84].
[84] See Fig 2 of D2, OS [175-176]

The Opponent’s contention that D2 deprives the claims of novelty centres on the backfire synthesis embodiment disclosed in Figure 11B and paragraphs [0411-0412], which relate to the presence of a nickase target site. This Figure is reproduced below.

Although this figure only discloses a single template nucleic acid comprising a nicking site, Prof. Edwards considers that a second primer having the same structure and function is implicit in this disclosure because it would be required for exponential amplification[85]. The Opponent also pointed to the Todd declaration at [133] and to paragraphs [0185-0186] of D2[86]. These passages disclose that lsRPA (leading strand recombinase polymerase amplification) involves contacting a recombinase with two primers to carry out continuous amplification with reference to Fig 6-7.
[85] Edwards #1 at [264-266]
[86] OS at [189]

I queried during the hearing whether the embodiment relating to backfire synthesis could necessarily be read as the same embodiment as leading strand RPA amplification. The parties sought additional time to answer this question.

Therefore in correspondence of 10 July 2019, I asked the parties:

Prof. Edwards’ declaration at [264] discusses Figure 11B of US2005/0112631 to Piepenburg (D2), and concludes that “while Figure 11B shows only one primer, it would be apparent that a second primer having the same structure and function would be required for amplification”. However the discussion of this Figure at [0412] of D2 does not appear to be related to amplification, and neither is the general discussion of “backfire synthesis” at [0408]-[0410] and Fig 10. Is there any basis elsewhere in D2 for Figure 11B to be understood to be involved in amplification and/or require a second primer

The Opponent responded to this query on 24 July 2019 and highlighted that all of the amplification reactions in D2 involved a pair of primers, with reference to Figs 16-18, and pointed to the discussion of lsRPA as being similar to PCR[87]. The Opponent also pointed to the advantage of backfire synthesis as providing a branch migration resistant structure, where branch migration is discussed as being undesirable at e.g. [0171] and [0269].
[87] D2 at [0167]

The Applicant responded to this query on 24 July 2019 by pointing to [0408] of D2 which discloses that backfire synthesis can only occur at the end of a duplex which the Applicant considers differentiates backfire synthesis from the invention as claimed.

I am not satisfied that Fig 11B of D2 contains a direction which is only capable of being carried out in a manner which would infringe the patentee's claim. In particular the discussion of Fig 11B at [0412] does not appear to relate to lsRPA. This passage describes that “…a suitable polymerase, for example the Klenow fragment, can extend from the nick and displace a DNA strand.” This differs from lsRPA in that no recombinase is involved. This passage concludes with the statement that “Multiple strands may be run-off by successive nicking and elongation from a single template.” This does not appear to describe exponential amplification of a double stranded target.

Therefore even if Fig 11B could be read as an improvement of lsRPA, at best it is a “signpost” within the meaning of General Tire. The disclosure of D2 does not contain a “planted flag” within the scope of the claim and therefore cannot deprive the claims of novelty.

Inventive step

The Opponent framed the inventive step analysis in terms of the reformulated Cripps question at [256] in their submissions

Applying the present situation to the reformulated Cripps question: Would the person skilled in the relevant art in all the circumstances which would include knowledge of the publication directly be led as a matter of course to try the claimed method for nucleotide sequence amplification (in particular, without a denaturation step) in the expectation that it might well produce amplification of DNA or RNA sequences at a constant temperature?

Although the reformulated Cripps question is the appropriate framework, it is important that in framing the question, only common general knowledge or the disclosure of the prior art base should be taken into account, not the inventors own starting point[88]. However in this case it is evident that nicking SDA reactions were known to be capable of isothermal amplification of DNA or RNA once in train. Therefore the question can be simplified as to whether the skilled worker would directly be led as a matter of course to omit the initial denaturation step from such reactions with the expectation that the reaction would still be functional.
[88] AstraZeneca AB v Apotex Pty Ltd [2014] FCAFC 99 at [202]–[203]

Common general knowledge

Common general knowledge is the background knowledge and experience available to all those working in the relevant art:

"The notion of common general knowledge itself involves the use of that which is known or used by those in the relevant trade. It forms the background knowledge and experience which is available to all in the trade in considering the making of new products, or the making of improvements in old, and it must be treated as being used by an individual as a general body of knowledge.”[89]

[89] .Minnesota Mining & Manufacturing Co v Beiersdorf (Australia) Ltd [1980] HCA 9; 144 CLR 253 at 292

Guidance on this consideration was provided in British Acoustic Films Ld v Nettlefold Productions (1936) RPC 221 at 250:

“In my judgement it is not sufficient to prove common general knowledge that a particular disclosure is made in an article, or a series of articles, in a scientific journal, no matter how wide the circulation of that journal may be, in the absence of any evidence that the disclosure is accepted generally by those who are engaged in the art to which the disclosure relates. A piece of particular knowledge as disclosed in a scientific paper does not become common general knowledge merely because it is widely read, and still less merely because it is widely circulated. Such a piece of knowledge only becomes common general knowledge when it is generally known and accepted without question by the bulk of those who are engaged in the particular art; in other words, when it becomes part of their common stock of knowledge relating to the art.”

To summarise, if it can be demonstrated by evidence that something is generally accepted by the hypothetical skilled worker, then this can be treated as common general knowledge. Scientific papers can be used to support such evidence but they do not themselves constitute common general knowledge.

The principal point of contention between the experts related to whether omitting a denaturation step prior to an amplification reaction was common general knowledge. However, the ground of lack of inventive step over common general knowledge alone was not pressed in the hearing, and as will be seen later, this factor does not come into play in my consideration of inventive step in view of D1-D3, which were the only grounds pressed by the Opponent.

D1/D3

Bearing in mind the differences identified with respect to D1 (and D3), the question becomes whether the skilled worker would have been directly led to try as a matter of course the nicking SDA method disclosed in those citations, to test an environmental sample, without a denaturation step.

Although the Opponent submits that Prof Edwards is a person skilled in the relevant art, and Prof Edwards’ opinion is that D1 (and D3) demonstrate that amplification can be successfully performed without a denaturation step, it appears that his reading may have been tainted by hindsight of the claimed invention and as such I must reduce the weight of this evidence and I am not satisfied that it tips the balance of probabilities.

Reading D1 as a whole, the problem identified in the abstract is the presence of unwanted side products. The gist of D1 as a whole in my view is design of appropriate primer sequences to reduce said side products[90]. In the legend to Fig 3 (in which an assay without a denaturation step did not appear to be tested, although other conditions such as low target and long duration were tested) it is disclosed that the presence of side products made detection of target impossible.
[90] This is also consistent with Dr. Todd’s reading of D1, see [45-46] of her declaration. Notably, Dr Todd read D1 before being shown a copy of the opposed application.

In view of this identified problem and given that D1 discloses that omitting a denaturation step increases the tendency to form side products, the skilled worker would be led away from performing the assay of D1 in the field without a denaturation step.

Similar considerations apply to D3.

I have found above that the embodiment of Fig 11B of D2 cannot be read as being something that would necessarily infringe the claims i.e. a method with two primers each with nickase sites. There still remains the question about whether the uninventive person skilled in the art would be directly led as a matter of course to modify this embodiment to include a second primer and carry out an amplification reaction. If so, it would seem to me that all the claim elements of claim 1 would be taken, because the recombinase-mediated invasion method disclosed in D2 does not require a denaturation step and can be carried out isothermally[91].
[91] See e.g. [0371] of D2

It is Prof. Edwards’ view that a second structure having the same structure and function would be required for amplification[92]. However this does not address the question about whether the uninventive skilled worker would have considered modifying this embodiment for amplification in the first place. Indeed, it seems to me that Prof. Edwards’ evidence cannot be informative as to what a skilled person would have done when reading D2 in isolation, because in preparing his evidence, he had already been made aware of the nature of the claimed invention[93], and in my view had read D2 with nickase mediated amplification reactions already in mind. This can be seen from Prof Edwards’ immediate focus on Fig 11B and his explanation as to why this discloses each and every feature of claim 1[94].
[92] Edwards #1 at [264], my emphasis
[93] As discussed in para 20 earlier.
[94] Edwards #1 at [252]

The following factors lead me to conclude that Fig 11B of D2 would not necessarily have led the skilled worker as a matter of course to try this embodiment in an amplification reaction:
Even though the context of D2 as a whole is amplification using paired primers, the embodiment of Fig 11 (and the discussion of backfire synthesis as a whole) is entirely silent as to amplification or paired primers.
The discussion of running off multiple displaced strands from a single template at [0412] in D2 seems on the balance of probabilities to refer to linear generation of single strands, rather than exponential generation of multiple strands as in lsRPA (or PCR).
As pointed out by the Applicant, backfire synthesis requires invasion at the end of a duplex, whereas all of the figures in D2 relating to lsRPA (Fig 1, 2, 7-8, 15-17) show paired primers invading inside double stranded DNA.
It therefore seems more likely on the balance of probabilities that the skilled reader would take this section as alternative uses of recombinase mediated invasion rather than improvements to lsRPA.
Furthermore, in order to carry out the embodiment of Fig 11B with two primers, it would be necessary to modify the disclosure of D2 to create double-strand breaks at both ends, since backfire synthesis is described to require the end of a duplex. I do not have any evidence as to how this modification would have been arrived at by an uninventive skilled worker.
Even though the Opponent has argued that backfire synthesis has properties that would lend it to be of use in RPA (in particular, reduced branch migration), the embodiment of Fig 11B is only one possible application of backfire synthesis and as discussed above the strand run-off embodiment is not related to RPA. Therefore even if the skilled worker were to apply a backfire synthesis method from Fig 11 to the RPA methods discussed elsewhere in D2, there is no evidence as to why they would choose the particular backfire embodiment of Fig 11B.

Therefore on the balance of probabilities in view of the available evidence, no claims lack inventive step in view of D1, D3/D3a or D2.

Documents presented in evidence but not pressed – potential relevance to novelty and inventive step

During my consideration of the evidence, it came to my attention that several documents raised in evidence, but not pressed at the hearing, are potentially relevant to novelty and inventive step of independent claims 1, 35 and 36.

This was due to my construction of claims 1 and 36 relating to amplification of single stranded targets, and claims 35, 36 and 41 not excluding other forms of denaturation, such as pH mediated denaturation.

I wrote to the parties on 11 September 2019 to advise them of my concerns as follows, and to allow them the opportunity to comment:

D6 (WO2000/028084) – amplification of single stranded target in two minutes without denaturation.
D6 is directed to nicking strand displacement amplification (SDA) (See Figs1A/1B). During prosecution, the examiner apparently withdrew this document after the applicant argued that this document is not relevant to novelty or inventive step because it requires a thermal denaturation step. However D6 discloses that the method only requires denaturation if the target nucleic acid is in a double stranded form (p. 17 lines 27-29 of D6).
Example 4 of D6 discloses isothermal detection of a single-stranded 18S RNA of C. parvum, which does not involve a separate denaturation step, and which produces a large amount (five micrograms) within two minutes. Although this is not an animal, plant or food sample, the skilled reader would take this as a clear and unmistakeable direction to detect at least C. parvum RNA in an environmental sample (see e.g. p. 3 line 20 and p. 6 line 25 of D6).
D6 would therefore seem to be prima facie novelty destroying for independent claims 1 and 36 insofar as they detect single-stranded nucleic acid targets.
D10 (US5712124) – inventive step of “lacking a thermal denaturation step”
D10 is directed to isothermal nicking SDA (See Figs 1-2). D10 does require denaturation of target double-stranded nucleic acids (see e.g. col. 6 lines 57-59) but teaches that alternative methods of denaturation involve raising and lowering pH (col. 6 lines 62-64).
Therefore it would seem that independent claim 35, to the extent that it does not exclude pH mediated denaturation and does not specify the reaction duration, includes uninventive alternatives of the technique disclosed in D10.

The Opponent provided detailed comments in a response filed on 9 October 2019, which extended beyond my concerns expressed above. These included an element by element analysis of D6 and D10 and contended that D6 was novelty destroying for claims 1, 35, and 36, and D10 was novelty destroying for claim 35. This submission also included an analysis of which dependent claims the Opponent contended lack novelty and/or inventiveness in view of D6 or D10.

The Applicant provided comments on 9 October 2019 and also 15 October 2019. With respect to D6, the Applicant’s contention in essence was that the demonstration of amplification of C. parvum 18S RNA in vitro would not be considered a clear and unmistakeable direction to detect it in an environmental sample. With respect to D10, the Applicant’s contention in essence was that D10 requires fragmentation of a sample before amplification (not “direct amplification”) and that the skilled worker would not choose pH denaturation as an option in D10 due to technical inconvenience.

With respect to process, the Applicant at that stage requested the opportunity to make further comments and file evidence in response to the Opponent’s comments, in the interests of procedural fairness.
At that stage, after weighing up the need for both parties to address statements from the other, the need for decisions to be made with respect to evidence, and the purpose of the Opposition proceeding as a relatively expeditious decision making process, I communicated to the parties on 22 October 2019 as follows.
In consideration of the circumstances, I will limit the findings in my decision to the grounds pressed during the oral hearing. Nevertheless, I will include preliminary observations on D6 and D10 in my decision. As suggested by the Applicant, a more detailed consideration of D6 and D10 will occur in a re-examination proceeding, should that prove necessary.

The Commissioner has the power under s 60(3) to take into account additional grounds and it is prima facie apparent to me that D6 and D10 would be prejudicial to the novelty and inventive step of subject matter relating to detection of single stranded targets or non-thermal denaturation for the reasons set forth above. I will nevertheless refrain from making a decision on these grounds, as the parties have not been afforded sufficient opportunity to adduce evidence addressing these grounds.

The Applicant is being provided with the opportunity to amend the claims as a result of other findings in this decision. At the conclusion of the Opposition process, should subject matter relating to amplification of single stranded targets, or non-thermal denaturation remain in the scope of the claims, the relevance of D6 and D10 could be addressed in a re-examination proceeding. The Opponent’s submissions of 9 October 2019 could provide a convenient starting point if this were necessary.

Support, sufficiency and utility

The Opponent advanced effectively identical arguments for why the application lacks support, sufficiency and utility. The two features attacked by the opponent were the lack of limitation to i) the number of spacer bases (given that Example 28 demonstrates that using more than 6 spacer bases does not result in amplification) and ii) the requirement for a stabilising region.

Since more consideration has been given to the ground of sufficiency post-Raising the Bar I will commence with the analysis of that ground.

Sufficiency (clear enough and complete enough disclosure)

Sufficiency post-Raising the Bar was surveyed in Evolva[95]. The approach adopted by the Deputy Commissioner in that case was:

What is the scope of the invention as claimed?
What does the specification disclose to the skilled person?
Does the specification provide an enabling disclosure of all the things that fall within the scope of the claims, and in particular:
(a) Is it plausible that the invention can be worked across the full scope of the claim?
[95] Evolva SA [2017] APO 57 (14 November 2017)
[96] Op. Cit at [45]
(b) Can the invention be performed across the full scope of the claim without undue burden?[96]

Factors indicative of undue burden include the need for the skilled addressee to undertake a “research programme” to carry out the invention across the full scope.[97]
[97] Op. Cit at [33], [36], [44]

Factors indicative of sufficient disclosure include the presence of a “principle of general application” that can be applied across the full scope[98].
[98] Op. Cit at [37], [38], [43]

Spacer bases

The Opponent’s argument relating to spacer bases was essentially that the number of such spacers is not limited whereas Example 28 indicates that there is a cap of 6[99]. The Applicant’s position as stated in the hearing was essentially that they are entitled to some “wriggle room”[100] and not to be limited by examples.
[99] OS [310-316]
[100] [sic] argument presented in the oral hearing. Also see AS [122] “Dr Todd is of the understanding … that the broader claims should not be limited to specific recognition region lengths or spacer lengths, as the ranges required may vary depending on the conditions. See, for example, TODD @ 90, and see also 78 and 115.”

As discussed in my construction of the number of spacer bases above, the number of spacer bases appears to be simply one variable in an empirical optimisation[101] that involves target sequence length, recognition region length, spacer length (which is a function of the first two), inherent properties such as GC content and melting temperature of the target sequence, reaction temperature, and potentially other factors such as the properties of the polymerase chosen in view of the chosen reaction temperature, and the presence of other interfering factors in the reaction (such as betaine as suggested by Dr. Todd[102]).
[101] Cf p. 81 l 11-14 of the description
[102] Todd at [70]

The specification does provide guidance with regard to the optimisation that would be required for amplification in just the presence of one nickase and polymerase:

choice of enzyme stability, reaction temperature, target length and GC concentration[103]

[103] p. 19 l 1-12:
choice of nickase and polymerase with similar temperature stability[104]

[104] p. 24 l.15
optimisation of recognition sequence length[105]

[105] p. 27-28
optimisation of nickase recognition site[106]

[106] p. 35 l.2
optimisation of reagent concentrations[107]

[107] p. 43-44
optimisation of reaction temperature[108]

[108] p. 45. l.18
optimisation of molecular beacon recognition sequences[109]

[109] p. 47. l.20
optimisation of recognition sequence and reaction conditions to distinguish target side products[110]
optimisation of spacer length[111]
optimisation of [Mg²⁺][112]
[110] p. 67 (example 5)
[111] p. 81 (example 28)
[112] p. 81 (example 30)

It seems to me that the skilled worker could be able to systematically change reaction conditions in order to work the invention with larger spacer lengths than in Example 28. Although the application does not particularly bind itself to any theory, it identifies factors such as DNA breathing (if this is a limiting factor then increasing the reaction temperature would increase the length of DNA unwound per breath to enable longer target sequence and hence longer spacer regions) and polymerase processivity (if this is a limiting factor then choosing a faster polymerase could enable lengthening of the spacer regions). However Dr. Todd is also aware of other factors that may destabilise or lower the melting temperature of the DNA and enable some variation in target length[113].
[113] Todd at [90]

The Applicant submitted at the hearing that the application does provide a “principle of general application” insofar as it demonstrated that the principle that templates are able to access a target without denaturation. The description prima facie appears to provide a principle of general application that short templates can initiate nicking SDA on a non-denatured target, with an appropriate choice of template length, target size and reaction conditions providing some “wriggle room” around those exemplified in the thirty examples, i.e. with a single nickase and single polymerase.
However the claims in my construction allow significantly more variability than this.

In particular, the independent claims and specification deliberately seek to not limit the size of the target and/or templates. Furthermore, the claims do not exclude additional reagents that could potentially make the invention work with much longer target and/or template sizes than those exemplified.

Additional reagents

The claims are clearly written in open “comprising” language. The description clearly envisages that factors not explicitly mentioned could be excluded from the claimed methods by using conventional language such as “consisting of” or “consisting essentially of”[114] but this language is not used in the present claims. Therefore the claimed methods encompass the use of additional reagents such as:
[114] p. 83 lines 2-5 explicitly contemplates that the invention could be practiced in the absence of non-disclosed elements, and that this would be signalled by the conventional language “consisting of” or “consisting essentially of”.

additional primers – all claims (except for claims 36 and 50-52 which explicitly disclaim them), include the use of additional primers such as bumper primers. Helper oligonucleotides are also explicitly discussed in the description[115].

[115] p. 36 l.27 – p. 37 l. 9
additional nickases – even though the references to nickases in claims 35, 36, 43 specify that the first and second nickase do not nick within the target sequence, there is no exclusion from any claim of an additional nickase or restriction enzyme that nicks upstream of the target sequence so as to expose the target sequence without the need for natural breathing. These are explicitly contemplated in the description at p. 37 l. 25.
additional additives are mentioned at p. 44 lines 21-24. Although this list is relatively short, other additives that could significantly affect the reaction include T_m modifying agents such as such as betaine, DMSO or TMAC[116] or even strand-opening enzymes such as recombinases[117] or helicases[118].

[116] Todd at [90]
[117] Cf D2
[118] Cf helicase-directed amplification that does not require thermal denaturation discussed at p. 3 l.9-13

The specification does not provide any guidance with regards to optimisation that would be required to incorporate the above additional reagents.

It seems implicit that the effect of the additional factors may be to significantly increase the range of target lengths that could be contemplated, or to enable the reaction to take place at lower temperatures. If this is indeed the case, then by omitting any limits on the length of the target, recognition regions and spacers (and indeed the reaction temperature) the Applicant seems to be drafting the claims to encompass a much broader range of reaction conditions that could perhaps be achieved by using additional reagents, but without providing any instruction on how much breadth could even be achieved, let alone how to carry this out.

The need for a “research project” is set forth in Evolva[119] as an indicator that undue burden is required to carry out the invention over the full scope of the claims. Guidance from the authorities regarding the nature of a “research project” include:
“EP and UK decisions have provided some general guidance on factors that come into consideration, including: uncertainty and a lack of predictability, incomplete experimental details and a lack of guidance in the specification including instructions on how to proceed in case of failure.”[120]
“There is a difference between on the one hand a specification which requires the skilled person to use his skill and application to perform the invention and, on the other, a specification which requires the skilled person to go to the expense and labour of trying to ascertain whether some product has the required properties. When carrying out the former the skilled person is trying to perform the invention, whereas the latter requires him to go further and to carry out research to ascertain how the invention is to be performed. If the latter is required the specification would appear to be insufficient.”[121]
[119] Evolva at [36]
[120] Evolva at [34], citations omitted
[121] American Home Products Corporation v Novartis Pharmaceuticals UK Limited [2001] RPC 8 at [40], cited in Evolva at [33]
The CSR case[122], also discussed in Evolva, seems analogous to the present situation in that the specification provides basis for optimising some but not all parameters.
“In the case of the “high expansion particulate” component, only high expansion vermiculite was exemplified, but the preparation of panels using other non-vermiculite particulates was not considered an undue burden. However other parameters (core density, core hardness, Thermal Insulation Index and fire resistance) were determined by a number of process and starting material variables, and the specification provided no real guidance on how these could be adjusted to achieve the full combination of properties defined by the claims. The lack of guidance, the uncertainty in the research and the degree of trial and error required led to the determination that there was an undue burden required to perform the invention across the full scope of the claims.”[123]
[122] CSR Building Products Limited v United States Gypsum Company [2015] APO 72.
[123] Evolva at [35], discussing CSR.
I have construed the independent method claims to be:

open with respect to presence of additional reagents;
not particularly limited by template lengths or reaction temperature; and
“capable of” performing an amplification, i.e. must be capable of working.

I can accept there is a principle disclosed that can be applied to the situation where the templates access the target through natural breathing, but given that the claims are broader than this, I do not accept that it does in fact have “general application” over the entire scope of the claims, which permit the presence of additional reagents affecting primer access or DNA duplex stability.

In my view the skilled worker would be left with a research project to find out which methods within the broad scope of the claims will in fact be capable of effective amplification.

The dependent claims include limitations to individual factors such as recognition region length or spacer length (claims 2, 3, 41-42, 44), target length (claim 32 as well as independent claim 43), and reaction temperature (claims 19-20) but it seems to me that simply limiting one of these factors at a time does not reduce the scope of the research project sufficiently.

Therefore all claims lack sufficiency.

Stabilising region

The Opponent asserted that claims 1, 35 and 36 and all claims dependent therefrom (apart from claims 33 and 34) were not supported, insufficiently disclosed and not useful because they do not require a stabilising region.

It was not apparently in dispute that templates without some kind of stabilising region are not functional, as demonstrated in Example 29. The Applicant’s position was that the presence of a stabilising region is implicit in all claims. However, this is not my construction of the claims[124]. Seeing as the description does not provide any disclosure as to how the method can be performed without a stabilising region all claims that do not require stabilising regions in both templates include subject matter are not sufficiently disclosed.
[124] Paragraph [63] supra.

Support

To determine whether the requirements of support are satisfied the following steps were set out in CSR:
i. construe the claims to determine the scope of the invention as claimed,
ii. construe the description to determine the technical contribution to the art, and
iii. decide whether the claims are supported by the technical contribution to the art[125].
[125] CSR at [95]
The claims have been construed in detail above.

In terms of construction of the description to determine the technical contribution to the art, it is evident to me that the description has taken some care not to limit or restrict the nature of the invention disclosed. Not only are the statements describing the invention generally non-limiting in terms of ranges and conditions, there are also repeated references for the need of the skilled worker to optimise reaction conditions for whichever target is chosen. Given this explicit reference to routine experimental trial and error, it seems to me that the specification’s technical contribution to the art is the general concept that nicking SDA can be performed without the need for a thermal denaturation step, within conditions ascertainable by routine trial and error. However, such routine trial and error cannot include a “research project” which I have found is necessary to carry out the full scope of the claims if additional reagents are present.

That is, the technical contribution is in my mind identical to the scope that is enabled, and I therefore come to the same conclusion as for sufficiency.

Utility

The ground of utility is somewhat different from support and sufficiency above. Since I have construed all claims as to be capable of performing amplification, the invention in all claims must necessarily have utility. This does not affect my conclusions as to why these claims fail the support and sufficiency requirements however[126].
[126] Cf Gary B Cox v MacroGenics, Inc. [2019] APO 13 at [120] “While drafting claims to exclude non-functional embodiments does not avoid a lack of enablement, this conclusion does not follow with respect to the ground of lack of utility”

Conclusion

The opposition is successful on the grounds of lack of sufficiency and lack of support, and fails on grounds of lack of utility, lack of novelty and lack of inventive step in view of D1‑D3.

Objections may be addressed by amendment

The grounds identified above appear amenable to being addressed by amendment. I therefore allow the applicant a period of two (2) months to file amendments under s 104. Please note that following resolution of any such amendment the Commissioner may consider whether to initiate re-examination proceedings in view of D6 and D10.

Costs:

It is conventional that costs follow the event. I therefore award costs according to Schedule 8 against the applicant.

Felix White
Delegate of the Commissioner of Patents

ANNEX: The claims

1. A method for nucleotide sequence amplification, which comprises:
obtaining, from an animal, plant, or food, a sample comprising a target nucleic acid, the target nucleic acid comprising a target nucleotide sequence,
without first subjecting the target nucleic acid molecule to a denaturation step associated with amplification of the target nucleotide sequence, combining, in a single step, the obtained sample directly with an amplification reagent mixture to form a reaction mixture or diluting the obtained sample and combining, in a single step, the diluted sample with an amplification reagent mixture to form a reaction mixture, in either case, the amplification reagent mixture comprising:
(i) a polymerase,
(ii) a first template nucleic acid that hybridizes to a first strand of the target nucleotide sequence, wherein the first template comprises a nucleic acid sequence comprising a first template recognition region at the 3' end that is complementary to the 3' end of the first strand of the target nucleotide sequence, a nicking enzyme binding site and a nicking site upstream of said recognition region; and
(iii) a second template nucleic acid that hybridizes to the complement of the first strand of the target nucleotide sequence, wherein the second template comprises a nucleotide sequence comprising a second template recognition region at the 3' end that is complementary to the 3' end of the complement of the first strand of the target nucleotide sequence, a nicking enzyme binding site and a nicking site upstream of said
recognition region, and
(iv) one or more nicking enzymes;
subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions to amplify the target nucleotide sequence; and
detecting amplified target nucleotide sequence in real time within 10 minutes of subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions.

2. The method of claim 1, wherein:
the recognition region of the first template is 8-15 nucleotides in length and the recognition region of the second template is 8-15 nucleotides in length.

3. The method of claim 1, 2, 35 or 36, wherein the target nucleotide sequence comprises from 1 to 5 nucleotides more than the sum of the nucleotides of the first or forward template recognition region and the second or reverse template recognition region.

4. The method of any one of claims 1 to 3, wherein the one or more nicking enzymes nick at the nicking site of said first and said second templates, wherein either one nicking enzyme nicks both of said templates, or two or more nicking enzymes nick each template, and wherein said one or more nicking enzymes do not nick within said target sequence.

5. The method of any one of claims 1 to 4, wherein the target nucleic acid is double stranded or single stranded.

6. The method of claim 5, wherein the target nucleic acid is double-stranded DNA.

7. The method of claim 5, wherein the target nucleic acid is single-stranded DNA.

8. The method of claim 5, wherein the target nucleic acid is RNA.

9. The method of claim 6, wherein the target nucleic acid is selected from thegroup consisting of genomic DNA, plasmid DNA, viral DNA, mitochondrial DNA, and synthetic double-stranded DNA.

10. The method of claim 7, wherein the target nucleic acid is selected from the group consisting of viral DNA, cDNA, and synthetic single-stranded DNA.

11. The method of claim 8, wherein the target nucleic acid is selected from the group consisting of messenger RNA, viral RNA, ribosomal RNA, transfer RNA, micro RNA, micro RNA precursor, and synthetic RNA.

12. The method of claim 1, 35, or 36, wherein said polymerase is a DNA polymerase, and said DNA polymerase is a thermophilic polymerase.

13. The method of any one of claims 1 to 12, 35, or 36, wherein said polymerase is selected from the group consisting of Bst (large fragment), 9°N, VentR® (exo-) DNA Polymerase, Therminator, and Therminator II.

14. The method of claim 4, wherein the first and second templates comprise nicking enzyme binding sites recognized by the same nicking enzyme and said nicking enzyme nicks both templates.

15. The method of claim 14, 35 or 36, wherein said nicking enzyme is selected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NtAIwI, NtBbvCI, NtBstNBI, Nt.CviPII, Nb.BpulOI, and Nt.BpulOI.

16. The method of any one of claims 1 to 15, wherein the portion of the nucleic acid sequence of the first template that is complementary to the first strand of the target nucleotide sequence is 8-15 nucleotides in length and wherein the portion of the second template that is complementary to the complement of the first strand of target nucleotide sequence is 8-15 nucleotides in length.

17. The method of any of claims 1 to 16, 35 or 36, further comprising a second polymerase.

18. The method of claim 17, wherein at least one of the first or second polymerases comprises reverse transcriptase activity.

19. The method of any of claims 1 to 18, 35 or 36, wherein the temperature of the essentially isothermal conditions is between 54°C and 60°C.

20. The method of any of claims 1 to 18, 35, or 36, wherein the temperature of the essentially isothermal conditions is between 56°C and 58°C.

21. The method of any one of claims 1-20, 35 or 36, wherein the amplification reaction is held at a constant temperature for 1 to 10 minutes.

22. The method of any of claims 1 to 20, 35 or 36, wherein the amplification reaction is held at a constant temperature for 1 to 20 minutes.

23. The method of claim 1 or 40, wherein detecting amplified target nucleotide sequence is performed by a method selected from the group consisting of fluorescence, intercalating dye detection, fluorescence resonance energy transfer (FRET), molecular beacon detection, and incorporation of labeled nucleotides.

24. The method of any one of claims 1 to 23, 35 or 36, wherein at least two target sequences are being amplified.

25. The method of any one of claims 1 to 24, 35 or 36, wherein at least one of said templates comprises a spacer, blocking group, or a modified nucleotide.

26. The method of any one of claims 1 to 25, wherein the target nucleotide sequence is amplified 1 X 10⁶ fold or more in about ten minutes.

27. The method of any one of claims 1 to 25, wherein the target nucleotide sequenceis amplified 1 X 10⁶ fold or more in about five minutes.

28. The method of any one of claims 1 to 25, wherein the target nucleotide sequence is amplified 1 X 10⁶ fold or more in about 2.5 minutes.

29. The method of any one of claims 1 to 25, wherein the target nucleotide sequence is amplified 1 X 10⁷ fold or more in about five minutes.

30. The method of any one of claims 1 to 25, wherein the target nucleotide sequence is amplified I X 10⁸ fold or more in about five minutes.

31. The method of any one of claims 1 to 25, wherein the target nucleotide sequence
is amplified 1 X 10⁹ fold or more in about five minutes.

32. The method of any one of claims 1 to 31, wherein the target nucleotide sequence is between 20 and 40 nucleotides in length.

33. The method of any one of claims 1 to 32, 35, and 36, wherein the first or forward template nucleic acid comprises a stabilizing region upstream of said nicking site.

34. The method of any one of claims 1 to 32, 35, and 36, wherein the second or reverse template nucleic acid comprises a stabilizing region upstream of said nicking site.

35. A method for amplifying a double-stranded nucleic acid target sequence without a heat denaturation step, comprising
a) directly adding an optionally diluted sample from an animal, plant, or food, said sample comprising a target DNA molecule comprising a double-stranded target sequence, having a sense strand and an antisense strand, with an amplification reagent mixture comprising a forward template and a reverse template, a first nicking enzyme, a second nicking enzyme and a polymerase to form a reaction mixture, wherein:
i) said forward template comprises a nucleic acid sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence antisense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region;
ii) said reverse template comprises a nucleotide sequence comprising recognition region at the 3 ' end that is complementary to the 3 ' end of the target sequence sense strand, a nicking enzyme binding site and a nicking site upstream of said recognition region; and
wherein said first nicking enzyme nicks upstream, downstream, or at the nicking site of said forward template, and does not nick within said target sequence;
and said second nicking enzyme nicks upstream, downstream, or at the nicking site of said reverse template and does not nick within said target sequence; and
b) subjecting the reaction mixture to essentially isothermal conditions to amplify the double-stranded nucleic acid target sequence;
wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse templates along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, or amplified copies of said sites, producing an amplification product.

36. A method for amplifying a single-stranded nucleic acid target sequence without a heat denaturation step and without the assistance of bumper primers, comprising
a) adding an optionally diluted sample from an animal, plant, or food, said sample comprising a target nucleic acid comprising a single-stranded target sequence, to an amplification reagent mixture comprising a reverse template, a first nicking enzyme, and a polymerase to form a reaction mixture, wherein:
(i) said reverse template comprises a nucleic acid sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence, a nicking enzyme binding site and a nicking site upstream of said recognition region;
and wherein said first nicking enzyme nicks at the nicking site of said reverse template, and does not nick within said target sequence; and
b) amplifying the single-stranded nucleic acid target sequence by subjecting the reaction mixture to essentially isothermal conditions, from [sic] steps comprising:
(i) extending, with the polymerase, under essentially isothermal conditions, the reverse template along the single-stranded nucleic acid molecule to create an extended reverse template molecule;
(ii) contacting said extended reverse template with a forward template, wherein said forward template comprises a recognition region at the 3' end that is complementary to the 3' end of the extended reverse template, a nicking enzyme binding site and a nicking site upstream of said recognition region; and

(iii) providing a second nicking enzyme that nicks at the nicking site of said forward template and does not nick within said target sequence or within the complement of said target sequence;
wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse templates along said target sequence, producing double-stranded nicking sites, and said nicking enzymes nicking at said nicking sites, producing an amplification product; and
detecting amplified target nucleotide sequence in real time within 10 minutes of subjecting the reaction mixture to essentially isothermal conditions.

37. The method of claim 35 or 36, wherein said forward and reverse templates comprise nicking enzyme binding sites recognized by the same nicking enzyme and said first and said second nicking enzymes are the same.

38. The method of claim 35, wherein the target DNA molecule is selected from the group consisting of genomic DNA, plasmid, mitochondrial, and viral DNA.

39. The method of claim 36, wherein the target nucleic acid is selected from the group consisting of viral DNA, messenger RNA, microRNA, and microRNA precursors.

40. The method of claim 35 or 36, further comprising detecting amplified target nucleotide sequence in real time within 10 minutes of subjecting the reaction mixture to essentially isothermal conditions.

41. The method of claim 35, wherein either:
(i) the portion of the forward template nucleic acid sequence that is complementary to the 3' end of the target antisense strand is 8-15 nucleotides in length and the portion of the reverse template nucleic acid sequence that is complementary to the 3' end of the target sense strand is 8-15 nucleotides in length; or
(ii) the target sequence comprises from 1 to 5 nucleotides more than the sum of the nucleotides of said forward template recognition region and said reverse template recognition region.

42. The method of claim 36, wherein either
(i) the portion of the nucleic acid sequence that is complementary to the 3' end of the target sequence is 8-15 nucleotides in length and the portion of the nucleic acid sequence that is complementary to the 3' end of the extended reverse template is 8-15 nucleotides in length; or
(ii) the target sequence comprises from 1 to 5 nucleotides more than the sum of the nucleotides of said forward template recognition region and said reverse template recognition region.

43. A method for amplifying a double-stranded nucleic acid target sequence without a heat denaturation step, comprising
a) directly contacting an optionally diluted sample from an animal, plant, or food, said sample comprising a target DNA molecule comprising a double-stranded target sequence, having a sense strand and an antisense strand, with an amplification reagent mixture comprising a forward template and a reverse template, a first nicking enzyme, a second nicking enzyme and a polymerase to form a reaction mixture,
wherein:
i) said forward template comprises a nucleic acid sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence antisense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site;
ii) said reverse template comprises a nucleotide sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence sense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site; and
wherein said first nicking enzyme nicks at the nicking site of said forward template, and does not nick within said target sequence; and said second nicking enzyme nicks at the nicking site of said reverse template and does not nick within said target sequence; and
b) subjecting the reaction mixture to essentially isothermal conditions to amplify the double-stranded nucleic acid target sequence, wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse templates along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, or amplified copies of said sites, producing an amplification product,
wherein at least a 1 X 10⁷ fold amplification of a 22-35 nucleotide long target sequence is obtained when the amplification reaction is run for twelve minutes.

44. The method of claim 43, wherein the portion of the forward template that is complementary to the 3' end of the target antisense strand is 8-15 nucleotides in length; and
wherein the portion of the reverse template that is complementary to the 3' end of the target antisense strand is 8-15 nucleotides in length.

45. The method of any one of claims 1, 35, or 36, wherein the sample is obtained from an animal and is blood, mucus, sputum, saliva, tears, feces, or urine.

46. The method of any one of claims 1, 35, or 36, wherein the sample is obtained from an animal and the animal is a human.

47. The method of any one of claims 1, 35, or 36, wherein the sample is obtained from an animal and the target nucleic acid is a nucleic acid of an animal pathogen.

48. The method of claim 47, wherein the animal pathogen is a single-stranded DNA virus, double-stranded DNA virus, or single-stranded RNA virus.

49. The method of claim 47, wherein the animal pathogen is a bacterium.

50. The method of any one of claims 1 or 35, wherein the target nucleotide sequence is amplified without the assistance of bumper primers.

51. The method of any one of claims 1 or 35, wherein the amplification reagent mixture is free of bumper primers.

52. The method of any one of claims 1, or 35, wherein the reaction mixture is free of bumper primers.