DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
This application 18/062,918 filed on 12/07/2022 claims the benefit of provisional U.S. Patent Application No. 63/286,717, filed on 12/07/2021.
The priority date of claim 1 and its dependent claims is determined to be 12/07/2021, the filing date of provisional U.S. Patent Application No. 63/286,717.
Status of Claims
Applicant’s amendments to claims filed 04/14/2026 in response to the Non-Final Rejection mailed 01/27/2026 are acknowledged.
Claims 1, 13, 15, 18, 20, and 21 are amended.
Claims 1-21, 30, and 43 are pending and claims 1-20 are under examination.
Response to Remarks filed 04/14/2026
The amendments and arguments presented in the papers filed 04/14/2026 ("Remarks”) have been thoroughly considered. The issues raised in the Office action dated 01/27/2026 listed below have been reconsidered as indicated.
a) The objections to the claims 1, 10, 13, 15, and 18 for informalities are withdrawn in view of the amendments to the specification.
b) The 35 USC 112(b) indefiniteness rejections of claim 1-20 have been withdrawn in view of the amendments to claims.
New and modified grounds of rejection necessitated by amendment are detailed below and this action is made FINAL.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 18-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 18 recites the limitations “iv) extending the hybridized second flap probe past the fourth cleavage site of the first cleavable probe using the first cleavable probe as a template, thereby forming a fourth cleavage site at the fourth cleavage region of the first cleavable probe” and “v) cleaving the fourth cleavage site of the first cleavable probe to form the first truncated probe” in step e). There is insufficient antecedent basis for the limitations the fourth cleavage site of the first cleavable probe, a fourth cleavage site, or the fourth cleavage region of the first cleavable probe. Claim 18 does not recite a first cleavable probe and claim 1, which claim 18 depends from, recites a first cleavable probe with a “second cleavage region --- capable of forming a second cleavage site”. For purposes of examination the limitations reciting a “fourth cleavage region” and “fourth cleavage site” of the first cleavable probe are interpreted as referring to the second cleavage region and second cleavage site of the first cleavable probe as recited in claim 1.
Claim 19 is similarly indefinite because it directly or indirectly depends from claim 18.
Claim 20 recites the limitations “iii) hybridizing the second flap probe to the fourth region of the second cleavable probe” in step d). There is insufficient antecedent basis for the limitation the second cleavable probe. Neither claim 20 nor claim 1, which claim 20 depends from, recites a second cleavable probe and claim 1.
Claim 20 further recites the limitation “iv) extending the hybridized second flap probe past the second cleavage region of the first cleavable probe using the first cleavable probe as a template, thereby forming a second cleavage site at the second cleavage region of the first cleavable probe”. It is unclear how the hybridized second flap probe would be extended using the first cleavable probe as a template after hybridizing to the second cleavable probe.
For purposes of examination the limitations reciting a second cleavable probe are interpreted as referring to the first cleavable probe as recited in claim 1.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-20 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Johnson et al. (US PGPub 2016/0040219) in view of Roth et al. (US PGPub 2014/0315747).
This maintained rejection has been modified to address claim amendments filed on 04/14/2026.
Regarding claim 1, Johnson teaches methods for the detection of nucleic acids. Regarding step (a) part (ii), Johnson teaches contacting a sample with a cleavable probe comprising a first sequence region comprising a label; a second sequence region; and a sequence that is the reverse complement of the second sequence region; and a sequence (a fourth region comprising a first cleavage region) that is complementary to a first region on a nucleic acid and cleavable after hybridization, i.e. when double-stranded (para 9). Johnson teaches the cleavable probe may further comprise a loop sequence of one or more nucleotides between the second sequence region and the sequence that is the reverse complement of the second sequence region, with a loop sequence that comprises one or more polymerase extension blocking moieties (para 12).
Regarding step (a) part (iii), Johnson teaches the use of endoribonucleases (Abstract and para 9).
Regarding step (b), Johnson teaches forming a hairpin probe in the presence of a nucleic acid complementary to the cleavable probe (para 9), having a first Tm (Table 2 and para 163). Regarding step (b) parts (iii)-(vi), Johnson teaches hybridizing the cleavable probe with a target nucleic acid; cleaving the hybridized probe to form a truncated probe; extending the hairpin probe and allowing the truncated probe to hybridize to itself to form a hairpin probe (paras 9-11).
Regarding step (c), Johnson teaches detecting the signal (presence) of the hairpin probe to detect the target nucleic acid (paras 9-11).
Johnson does not teach the activation probe of claim 1 or the elements using the activation probe.
Regarding step (a) part (i), Johnson does not teach (I) the recited first activation probe. Regarding step (a) part (ii), Johnson teaches that the sequence of the cleavable probe is complementary to a first region of a target nucleic acid, but does not teach (II) that the nucleic acid is a first region of a first activation probe.
Roth teaches (I) a bifunctional mediator probe (activation probe) comprising, from 5’ to 3’, a mediator region (first region) and a probe region (second region). The probe region has affinity to a template molecule (target nucleic acid) and the mediator probe is cleaved at a cleavage site between the first and second regions during an amplification process (i.e. when double stranded), wherein interaction of the cleaved mediator region with a detection molecule triggers a detectable signal (para 36). The mediator region does not have any affinity for the template molecule (para 37) but has affinity to a detection molecule (para 36).
Roth further teaches (II) at the 3′ end, the detection molecule also contains a mediator hybridization site that is complementary to the mediator region (cleavable probe fourth region) (para 118).
Regarding step (b), Johnson teaches hybridizing the cleavable probe to a complementary nucleic acid, which reads in part on part (iii). However, Johnson does not teach parts (i) – (iii) elements directed towards the first activation probe.
Regarding step (b) part (i)-(iii), Roth teaches a reaction comprising binding the probe region of the mediator probe to a sequence of the template molecule (target nucleic acid), cleaving and splitting off the mediator probe at the cleavage site (releasing a first flap probe) and binding of the cleaved mediator region of the mediator probe to the detection molecule (cleavable probe fourth region) (para 97). Roth further teaches after adding the mediator region onto a sequence region of the unpaired 3' sequence segment, the mediator region is preferably elongated by a polymerase, which also reads on part (iv) of step (b).
Roth states that the use of a mediator probe separate from the detection molecule (hairpin) allows design of the detection molecule to be designed independently of the target molecule, making it possible to detect multiple target molecules in a sample and adapting the reaction inexpensively (para 62). Roth further states that the mediator probe is advantageous for multiplexing (para 146).
Neither Johnson nor Roth teach the released probe comprises at least one nucleotide of the second region of the first activation probe as in part b step (ii).
However, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention. The modification would have entailed (1) designing the sequence that is complementary to a first region on a target nucleic acid on the cleavable probe of Johnson to be complementary to the nucleic acids of the first region of the mediator probe of Roth and (2) using the mediator probe of Roth to hybridize to the target nucleic acid instead of the cleavable probe directly and release a mediator (flap) probe region that participates in the hairpin forming reaction. Additionally, the modification would have entailed using the cleavable probe of Johnson as the detection molecule of Roth. Roth teaches releasing the mediator region to interact with a complementary detection molecule, and Johnson teaches a fourth region complementary to a target nucleic acid. One would have been motivated to combine the two-part elements of Roth in order to decrease costs and increase multiplex detection potential, a stated goal of both, with the sensitivity of detection provided by the cleavable probe of Johnson. Both Johnson and Roth are silent regarding the released probe comprises at least one nucleotide of the second region of the first activation probe. This modification would have been a matter of routine modification and optimization as varying lengths of probes was well known in the art at the time as evidenced by the multiple examples of different probes and varying cleavage sites in both Roth and Johnson. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 2, Johnson teaches amplification of a target sequence. The amplification can be performed during the cleavage and extension of the cleavable probes (para 61).
Regarding claim 3, Johnson teaches the label can comprise one non-natural nucleotide labeled with a first member of a reporter-quencher pair and extension of the hairpin probe results in the incorporation of a complementary non-natural nucleotide labeled with a second member of the reporter-quencher pair (para 9).
Regarding claim 4, Johnson teaches a label that comprises one non-natural nucleotide labeled with a first member of a reporter-quencher pair and a complementary non-natural nucleotide labeled with a second member of the reporter-quencher pair (para 9).
Regarding claim 5, Johnson teaches a non-natural nucleotide is an isobase, such as iso-guanine (isoG) or iso-cytosine (isoC) (para 20).
Regarding claim 6, Johnson teaches the cleavable probe comprises both a fluorophore (F) and a quencher (Q) with a confirmation such that when the probe is single-stranded the proximity of the fluorophore to the quencher results in detectable quenching of the signal from the fluorophore. And that extension of the cleaved probe onto the first sequence region creates a double-stranded molecule having a conformation that places the fluorophore at a greater distance from the quencher such that a detectable change in the signal can be observed (para 177 and Fig. 16).
Regarding claim 7, Johnson teaches performing a melt analysis on the hairpin probe (paras 9-11, 57).
Regarding claim 8, Johnson teaches detecting a signal by detecting a change in the signal from the reporter when increasing the temperature above or decreasing the temperature below the melt point of the hairpin (para 63).
Regarding claim 9, Johnson does not teach the first activation probe is cleaved by an invader assay cleavage event. Roth teaches the use of an invader assay but does not use the method for cleavage of the mediator probe (activation probe).
However, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention. The modification would have entailed substituting a different method of cleaving the mediator probe of Roth once bound to the target in order to release the mediator region (flap probe). Determining an appropriate means of cleavage is deemed a matter of judicious selection and routine optimization which is well within the purview of the skilled artisan. One of ordinary skill in the art would have been motivated to try different temperatures in order optimize conditions of cleavage and probe release. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 10, Johnson teaches the use of an endoribonuclease for cleavage of probes that comprise a ribonucleotide position (Abstract, para 76). However, Johnson teaches that the cleavable region that comprises at least one ribonucleotide and is cleaved by an endoribonuclease is within the cleavable probe and not within the activation probe.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention. The modification would have entailed performing the method of Johnson with the mediator probe of Roth which comprises a probe region complementary to the target nucleic acid. The inclusion of ribonucleotides in the cleavage region of the mediator probe and use of an endoribonuclease for cleavage would have been a simple substitution and constitutes a routine optimization. One of skill in the art would have been motivated to design the probe region of Roth, which is complementary to the target nucleic acid, to adapt the cleavage probe regions of Johnson that interact with the target nucleic acid. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 11, Johnson teaches an endoribonuclease can be RNase HII (para 76, claim 3).
Regarding claim 12, Johnson teaches using a polymerase possessing 5′ nuclease activity thereby cleaving the probe that is hybridized with target nucleic acid (paras 57, 58).
Regarding claim 13, Johnson teaches a cleavable probe with a fourth sequence comprising one or more ribonucleotide base that is complimentary to a first region on a first strand of the target nucleic acid, that is cleaved with an endoribonuclease (para 9), and the endoribonuclease can be RNase HII (para 76, claim 3). However, Johnson teaches that the cleavable probe complementary region is complementary to a target nucleic acid.
Roth teaches the detection molecule (cleavable probe) mediator hybridization site is complementary to the mediator region of the mediator probe (activation probe) (para 118).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention. The modification would have entailed performing the method of Johnson with the mediator probe of Roth which comprises a probe region complementary to the target nucleic acid. The inclusion of ribonucleotides in the cleavage region of the mediator probe and use of an endoribonuclease for cleavage would have been a simple substitution and constitutes a routine optimization. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 14, neither Johnson nor Roth Johnson teaches cleavage of the first cleavable probe is performed by a restriction enzyme or a nicking enzyme.
Roth teaches using a restriction enzyme to cleave the mediator probe (para 89).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention. The modification would have entailed substituting the restriction enzyme of Roth for the endoribonuclease of Johnson. Such a modification would have been a simple substitution and choosing a particular nuclease for cleaving constitutes a design choice that constitutes routine optimization. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 15, Johnson teaches use of a second cleavable probe comprising, from 5′ to 3′, (i) a first sequence region comprising at least a one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a second sequence region; (iii) a sequence that is the reverse complement of the second sequence region; and (iv) a sequence comprising one or more ribonucleotide base that is complimentary to a first region on a first strand of a second target nucleic acid (para 55, claim 51), which reads on step (d) part (ii).
Regarding step (d) part (iii), Johnson teaches the use of endoribonucleases (Abstract and para 9).
Regarding step (e) parts (iv)- (vi), Johnson teaches forming a hairpin probe (para 9), having a first Tm (Table 2 and para 173). Johnson teaches hybridizing the cleavable probe with a target nucleic acid; cleaving the hybridized probe to form a second truncated probe; extending the second hairpin probe and allowing the second truncated probe to hybridize to itself to form a hairpin probe (para 21). Johnson further teaches first and second cleavable probes with distinguishable melting temperatures (paras 21, 49, claim 149).
Regarding step (f), Johnson teaches detecting the signal (presence) of a second hairpin probe to detect a second target nucleic acid (para 21).
Johnson does not teach the second activation probe of claim 15 or the elements using the second activation probe.
Regarding step (d) part (i), Johnson does not teach (I) the recited second activation probe. Regarding step (d) part (ii), Johnson teaches that the sequence of the cleavable probe is complementary to a first region of a target nucleic acid, but does not teach (II) that the nucleic acid is a first region of a first activation probe.
Roth teaches (I) a mediator probe (activation probe) comprising, from 5’ to 3’, a mediator region (first region) and a probe region (second region). The probe region has affinity to a template molecule (target nucleic acid) and the mediator probe is cleaved at a cleavage site between the regions during an amplification process (i.e. when double stranded), wherein interaction of the cleaved mediator region with a detection molecule triggers a detectable signal (para 36). The mediator region does not have any affinity for the template molecule (para 37) and has affinity to a detection molecule (para 36).
Roth further teaches (II) at the 3′ end, the detection molecule also contains a mediator hybridization site that is complementary to the mediator region (cleavable probe fourth region) (para 118).
Roth teaches the use of "n" different mediator probes for "n" different target molecules (i.e.. a second mediator probe and a second target nucleic acid), wherein the mediator region represents a specific interaction to a defined detection molecule, i.e. a second cleavable probe paired with a second mediator probe (para 146). Roth further teaches the use two different mediator probes (activation probes) and two different detection molecules for detecting different nucleic acids in parallel (para 127), detecting multiple targets.
Regarding step (e), Johnson teaches hybridizing the cleavable probe to a complementary nucleic acid , which reads in part on part (iii). However, Johnson does not teach parts (i) – (iii) elements directed towards a second activation probe.
As described above Roth teaches "n" different mediator probes, which is encompassed by a second activation probe. Regarding step (e) parts (i)-(iii), Roth teaches a reaction comprising binding the probe region of the mediator probe to a sequence of the template molecule (target nucleic acid), cleaving and splitting off the mediator probe at the cleavage site (releasing a second flap probe) and binding of the cleaved mediator region of the mediator probe to the detection molecule (para 97). Roth further teaches after adding the mediator region onto a sequence region of the unpaired 3' sequence segment, the mediator region is preferably elongated by a polymerase, which also reads on part (iv) of step (e).
Roth states that the use of a mediator probe separate from the detection molecule (hairpin) allows design of the detection molecule to be designed independently of the target molecule, making it possible to detect multiple target molecules in a sample and adapting the reaction inexpensively (para 62). Roth further states that the mediator probe is advantageous for multiplexing and allows multiparameter analysis (para 146).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention. The modification would have entailed using two mediator probes of Roth to hybridize to two target nucleic acids instead of the cleavable probe directly and release mediator (flap) probe regions specific to two detection molecules (cleavable probes) and designing the sequence that is complementary to a first region on a target nucleic acid on two cleavable probes of Johnson to be complementary to the nucleic acids of the first region of the two mediator probes of Roth, with one mediator probe paired to one cleavable probe. Alternatively the modification would have entailed using the two cleavable probes of Johnson as the detection molecules of Roth. The modification would further have entailed following the method of Roth to bind the cleavable probe of Johnson to form the hairpin probe for detecting the target nucleic acid and performing the same reactions for each pair of mediator and cleavable probes. One would have been motivated to combine the elements of Roth and Johnson and add a second pair of probes in order to increase multiplex target nucleic acid detection, a stated goal of both, with the sensitivity of detection provided by the cleavable probe of Johnson. Further, a two part system as in Roth provides added flexibility in terms of design and cost. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 16, Johnson teaches amplification of a target sequence. The amplification can be performed during the cleavage and extension of the cleavable probes (para 61 and claim 42).
Regarding claim 17, Johnson teaches first and second cleavable probes with the same reporter and distinguishable melting temperatures that differ by 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30° C from one another (paras 21,49) and detecting a signal by detecting a change in the signal from the reporter when increasing the temperature above or decreasing the temperature below the melt point of the hairpin of one more of the primers in the sample (para 63), which reads on steps (g) and (i) and the limitation in step (h) “detecting signal from the reporter at a third temperature that is below the second TM and a fourth temperature that is above the second TM”.
Johnson does not explicitly teach in step (h) wherein the third temperature is equal to or greater than the second temperature.
However, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Johnson and Roth to arrive at the instantly claimed invention. The method of Johnson comprising two cleavable probes is directed to cleavable probes with distinguishable melting temperatures. It would have been obvious to use temperatures that do not overlap (i.e. the third temperature is equal to or greater than the second temperature). Determining an appropriate temperature range is deemed merely a matter of judicious selection and routine optimization which is well within the purview of the skilled artisan. One of ordinary skill in the art would have been motivated to try different temperatures in order to optimize conditions for distinguishing melting temperatures. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 18, for purposes of examination the limitations reciting a “fourth cleavage region” and “fourth cleavage site” of the first cleavable probe are interpreted as referring to the second cleavage region and second cleavage site of the first cleavable probe as recited in claim 1.
Regarding step (d) part (ii) Johnson teaches the use of endoribonucleases (Abstract and para 9).
Regarding step (e) parts (iv)- (vi), Johnson teaches forming a hairpin probe (para), having a first Tm (Table 2 and para 173). Johnson teaches hybridizing the cleavable probe with a target nucleic acid; cleaving the hybridized probe to form a second truncated probe; extending the second hairpin probe and allowing the second truncated probe to hybridize to itself to form a hairpin probe (para 21).
Regarding step (e), Johnson teaches detecting the signal (presence) of the first hairpin probe (paras 9-11).
Regarding step (d) part (i), Johnson does not teach the recited second activation probe.
Roth teaches a mediator probe (activation probe) comprising, from 5’ to 3’, a mediator region (first region) and a probe region (second region). The probe region has affinity to a template molecule (target nucleic acid) and the mediator probe is cleaved at a cleavage site between the regions during an amplification process (i.e. when double stranded), wherein interaction of the cleaved mediator region with a detection molecule triggers a detectable signal (para 36). The mediator region does not have any affinity for the template molecule (para 37) and has affinity to a detection molecule (para 36).
Roth teaches the use of "n" different mediator probes for "n" different target molecules (i.e.. a second mediator probe and a second target nucleic acid) (para 146)., which reads on a second mediator probe with a probe region complementary to a second target nucleic acid.
Roth further teaches that is possible to use a standardized detection molecule (i.e. a cleavable probe with a region complementary to the identical first mediator probe region and second mediator probe mediator region), which can be produced in large quantities to minimize production costs (para 123). Roth also teaches that coupling between the presence of a target molecule and the detection reaction depends only on the properties of the mediator region and/or the mediator probe and thus allows free coupling between any target molecule and any detection reaction and/or detection molecule (para 37).
Roth states that the detection molecule can therefore be used universally and is not bound to a specific target which greatly reduces cost because the detection molecule need not be tailored to each reaction and each target molecule (para 117).
Regarding step (e), Johnson does not teach parts (i) – (iii) directed towards the second activation probe.
Roth teaches a reaction comprising binding the probe region of the mediator probe to a sequence of the template molecule (target nucleic acid), cleaving and splitting off the mediator probe at the cleavage site (releasing a first flap probe) and binding of the cleaved mediator region of the mediator probe to the detection molecule (para 97). Roth further teaches after adding the mediator region onto a sequence region of the unpaired 3' sequence segment, the mediator region is preferably elongated by a polymerase, which also reads on part (iv) of step (e).
Roth states that the use of a mediator probe separate from the detection molecule (hairpin) allows design of the detection molecule to be designed independently of the target molecule, making it possible to detect multiple target molecules in a sample and adapting the reaction inexpensively (para 62). Roth further states that the mediator probe is advantageous for multiplexing.
Roth does not explicitly teach a second mediator probe with a mediator region identical to the first region of the first activation probe.
However, It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention using two mediator probes capable of binding to the same cleavable probe. The modification would have entailed using two mediator probes of Roth to hybridize to two target nucleic acids. The mediator probes of Roth would be designed with identical mediator regions, resulting in the release of mediator (flap) probe regions complementary to the same detection molecules (first cleavable probe) and designing the sequence that is complementary to a first region on a target nucleic acid on the cleavable probe of Johnson to be complementary to the nucleic acids of the probe region of the two mediator probes of Roth. The modification would further have entailed following the method of Roth to bind mediator regions from a first and second mediator probe to the cleavable probe of Johnson to form the hairpin probe for detecting the target nucleic acid and performing the same reactions for each pair of mediator and cleavable probes. Roth teaches a strength of the bifunctional system is ease of multiplexing design. One would have been motivated to design identical probe regions in order to benefit from the inexpensive adaptive design made possible with the modular probes of Roth. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 19, Johnson teaches amplification of a target sequence. The amplification can be performed during the cleavage and extension of the cleavable probes (para 61).
Regarding claim 20, for purposes of examination the limitations reciting a second cleavable probe are interpreted as referring to the first cleavable probe as recited in claim 1.
Roth teaches a mediator probe (activation probe) comprising, from 5’ to 3’, a mediator region (first region) and a probe region (second region). The probe region has affinity to a template molecule (target nucleic acid) and the mediator probe is cleaved at a cleavage site between the regions during an amplification process (i.e. when double stranded), wherein interaction of the cleaved mediator region with a detection molecule triggers a detectable signal (para 36). The mediator region does not have any affinity for the template molecule (para 37) and has affinity to a detection molecule (para 36).
Roth teaches the use of "n" different mediator probes for "n" different target molecule (para 146), which reads on a second activation probe with a probe region complementary to a second target nucleic acid.
Roth teaches that coupling between the presence of a target molecule and the detection reaction depends only on the properties of the mediator region and/or the mediator probe and thus allows free coupling between any target molecule and any detection reaction and/or detection molecule (para 37). Roth further teaches that is possible to use a standardized detection molecule (e.g. a cleavable probe with a region complementary to the identical first mediator probe region and second mediator probe mediator region), which can be produced in large quantities to minimize production costs (para 123).
Roth states that the detection molecule can therefore be used universally and is not bound to a specific target which greatly reduces cost because the detection molecule need not be tailored to each reaction and each target molecule (para 117).
Johnson teaches forming a hairpin probe (para), having a first Tm (Table 2 and para 173).
Regarding steps (iv)-(vii), Johnson teaches hybridizing a cleavable probe with a target nucleic acid; cleaving the hybridized cleavable probe to form a truncated probe; allowing the second truncated probe to hybridize to itself to form a hairpin probe and extending the hairpin probe (para 21). Johnson further teaches detecting the signal (presence) of the first hairpin probe (paras 9-11).
Regarding steps (i)-(iii), Roth teaches a reaction comprising binding the probe region of the mediator probe to a sequence of the template molecule (target nucleic acid), cleaving and splitting off the mediator probe at the cleavage site (releasing a first flap probe) and binding of the cleaved mediator region of the mediator probe to the detection molecule (para 97). Roth further teaches after adding the mediator region onto a sequence region of the unpaired 3' sequence segment, the mediator region is preferably elongated by a polymerase, which also reads on part (iv).
Neither Johnson nor Roth teach the released probe comprises at least one nucleotide of the second region of the first activation probe as in step (ii).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Johnson and Roth to arrive at the instantly claimed invention using two mediator probes capable of binding to the same cleavable probe. The modification would have entailed using two mediator probes of Roth to hybridize to two target nucleic acids and identical mediator regions, releasing mediator (flap) probe regions specific to two detection molecules (cleavable probes) and designing the sequence that is complementary to a first region on a target nucleic acid on the cleavable probe of Johnson to be complementary to the nucleic acids of the first region of the two mediator probes of Roth. One would have been motivated to combine the elements of Roth and Johnson and add a second pair of mediator probes in order to increase multiplex target nucleic acid detection, a stated goal of both. Further, a two part system as in Roth provides added flexibility in terms of design and cost and a shared detection molecule (cleavable probe) would simplify design by only requiring change to the mediator probe complementary to a second target nucleic acid. Both Johnson and Roth are silent regarding the released probe comprises at least one nucleotide of the second region of the first activation probe. This modification would have been a matter of routine modification and optimization as varying lengths of probes was well known in the art at the time as evidenced by the multiple examples of different probes and varying cleavage sites in both Roth and Johnson. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Response to Arguments against Claim Rejection - 35 U.S. C § 103
The response asserts that the cleavable probe of Johnson is different from the cleavable probe of the instant methods, because the fourth sequence in Johnson is complementary to a target nucleic acid for detection, whereas the cleavable probe in the instant methods comprises a sequence complementary to a region of the activation probe. Therefore, the cleavable probes in Johnson and the instant methods have entirely different structures (p. 15).
Applicant's arguments have been fully considered but are not persuasive.
While Johnson does not teach a fourth region complementary to an activation probe, Johnson does teach a fourth region complementary to a nucleic acid sequence, that is cleavable after hybridization (double-stranded), and further that is cleaved to form a truncated probe and form a hairpin probe for melt analysis. The probe of Johnson is functionally equivalent to the instant probe – forming a hairpin probe as a reporter of the presence of a nucleic acid sequence. The only necessary design choice is to select as the complementary nucleic acid sequence of the fourth region, the sequence of an intermediary such as the mediator probe of Roth. As taught by Roth, and presented in the 103 rejection above, the addition of an intermediary probe permits multiplexing and increases adaptability. One of skill in the art would have seen the benefits to making the cleavable probe of Johnson an indirect reporter, hybridizing to an intermediary rather than directly to the target nucleic acid. Further, Roth teaches the benefits of a two-part system using a probe that detects a target nucleic acid and releases a probe region to subsequently trigger a detection molecule such as the cleavable probe of Johnson.
The response asserts that Roth does not describe the activation probes in the present methods but differs fundamentally from the activation probes used in the instant methods. Instead, Roth's probes consist of a probe region and a mediator region, separated by a cleavage site located between the probe and mediator regions, which necessitates an amplification reaction to generate the cleavage site before cleavage can occur. In contrast, the activation probe structure (compris[ing] a second region that comprises a cleavage region and is complementary to the first target nucleic acid), allows the activation probe to be directly cleaved upon hybridization to the target nucleic acid, eliminating the requirement for a separate amplification step. The response asserts that using the mediator probe of Roth in the methods of Johnson would not lead to the claimed methods, because the activation probes and cleavable probes in both cited references are entirely different from those in the instantly claimed methods (p. 15-16).
Applicant's arguments have been fully considered but are not persuasive.
The method of Roth uses probes with a region complementary to a target nucleic acid as required by the instant claims that is capable of being cleaved when double-stranded and an additional region that is not complementary to the target nucleic acid but is released to interact with a second reporter molecule. The methods of the instant claims do not exclude amplification steps. In particular, claim 1 recites “performing a reaction -- wherein the reaction includes the steps of” . The phrase “includes the steps of” constitutes open claim language that does not exclude the presence of additional steps.
Further, in response to applicant's arguments against the references individually (regarding the structures of the Johnson probes and the Roth probes), one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-20 remain/are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 9,982,291 in view of Johnson et al. (US PGPub 2016/0040219) and Roth et al. (US PGPub 2014/0315747).
Although the claims at issue are not identical, they are not patentably distinct from each other because both methods are directed to methods using a cleavable probe that forms a hairpin reporter to detecting a target nucleic acids by detecting a signal and performing melt analysis. The additional limitations of the ‘291 claims are encompassed by the open claim language "comprising" found in the instant claims.
Claim 1 of the ‘291 patent requires (a) contacting a sample with a first cleavable probe, said probe comprising, from 5′ to 3′, (i) a first sequence region comprising at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a second sequence region; (iii) a sequence that is the reverse complement of the second sequence region; and (iv) a sequence comprising one or more ribonucleotide base(s) that is complementary to a first region on a first strand of the target nucleic acid; (b) contacting the cleavable probe with an endoribonuclease, thereby cleaving probe that is hybridized with target nucleic acid to form a truncated cleavable probe; (c) allowing the truncated cleavable probe to hybridize to itself to form a hairpin probe; (d) extending the hairpin probe in the presence of a non-natural nucleotide labeled with a second member of a reporter-quencher pair that is capable of base-pairing with the at least one non-natural nucleotide of the first sequence region; and (e) detecting the target nucleic acid by detecting a change in signal from the label on the cleavable probe and the hairpin probe, which satisfies the requirements of instant claims 1, 3, 4,5, and 10.
Regarding instant claim 1, claim 5 of the ‘291 patent further requires the cleavable probe further comprises (v) a loop sequence of one or more nucleotide(s) between the second sequence region and the sequence that is the reverse complement of the second sequence region and claim 9 of the ‘291 patent requires the cleavable probe comprises an extension-blocking modification in the loop sequence.
Claims of the ‘291 patent do not require an activation probe comprising a region complementary to a target nucleic acid and a region complementary to the cleavable probe or a cleavable probe comprising a region complementary to the activation probe.
The teachings of Johnson and Roth as they relate to these claims are given previously in this office action and are fully incorporated here.
Regarding instant claim 2, claim 12 of the ‘291 patent requires amplifying the target nucleic acid.
Regarding instant claim 6, claims of the ‘291 patent do not require the label comprises a reporter-quencher pair arranged such that the quencher quenches the reporter signal when the first region of the first cleavable probe is single stranded and extension of the hybridized third region separates the reporter and quencher to release the reporter from quenching.
The teachings of Johnson as they relate to these claims are given previously in this office action and are fully incorporated here.
Regarding instant claims 7 and 8, claim 10 of the ‘291 patent requires detecting a change in signal from the label comprises detecting a change in signal from a reporter as the temperature of the sample is changed and claim 11 of the ‘291 patent requires detecting a change in signal from the reporter comprises detecting a change in signal from the reporter as the temperature of the sample is increased above the melt point of the hairpin probe.
Regarding instant claim 11, claim 3 of the ‘291 patent requires the endoribonuclease is RNase HII.
Regarding instant claim 12, claims of the ‘291 patent do not require
The teachings of Johnson as they relate to these claims are given previously in this office action and are fully incorporated here.
Regarding instant claims 9 and 13-20, the claims of the ‘291 patent do not require the recited limitations.
The teachings of Johnson and Roth as they relate to these claims are given previously in this office action and are fully incorporated here.
Response to Arguments against Double Patenting
The response asserts that the claims of the '291 Patent do not teach an activation probe, as is required by and Johnson and Roth do not cure these deficiencies (p. 16-17).
Applicant's arguments have been fully considered but are not persuasive.
Response to arguments against Johnson and Roth are presented above in the 103 rejection. Thus, for the reasons stated above, and those already of the record, the rejection is maintained.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA GRAY whose telephone number is (571)272-0116. The examiner can normally be reached Monday-Friday 8-5 with second Fridays off.
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/JESSICA GRAY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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