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 .
Election/Restrictions
Applicant’s election without traverse of Group I, 1) cell free DNA; (2) liquid biopsy; (3) a crowding reagent is added to the target nucleic acid molecule prior to or in
step (i); (4) in step (i) the polymerase is the Stoffel fragment of Taq polymerase and the dNTPs are provided at a concentration of no more than 1 M, preferably nor more than 0.5 M, and more preferably no more than 0.3, or 0.25 M; (5) the variant target nucleic acid sequence to be detected comprises a single variant base, and the variant base is not located at the position corresponding to the first or the last base of the gap between the hybridised ends of the first padlock probe; and (6) down to step (vi) the method is a homogenous method performed in solutionor suspension. in the reply filed on 12/10/2025 is acknowledged.
Claims 7, 12, 22, 28 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected inventions or species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 12/10/2025.
Claims 21, are being examined.
Priority
The instant application was filed 05/19/2023 and is a national stage entry of PCT/EP2021/084061 with an international filing date: 12/02/2021 and claims foreign priority to GB2019074.0, filed 12/03/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 5/19/2023 and 10/6/2023 is being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. The specification in 0211 and 0242teaches figure 10 is in color. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Claim Objections
Claims 1-6, 8-11, 13,16-17, 19-21, 23, 29 objected to because of the following informalities:
Claim 1 is objected to as it recites “RCA” but does not recite the full terminology for the acronym (or abbreviation). Claims are more concise when the first time an acronym (or abbreviation) is presented the full terminology is also presented. Finally an acronym (or abbreviation) may have alternative meanings to an artisan.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 1-6, 8-11, 13,16-17, 19-21, 23, 29 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 1 recites,” ii) optionally after cleavage of any unhybridised nucleotides at the 5' and/or 3' ends,extending the hybridised 3' end of the padlock probe using a polymerase to create a complementary copy of the target nucleic acid sequence, and ligating the extended 3 'end to the hybridised 5' end to circularise the padlock probe; (iii) performing a first RCA reaction using the circularised padlock probe as a first RCA template to generate a first RCA product (RCP) comprising multiple repeats of a copy of the target nucleic acid sequence;” Thus step ii) appears to be optional. However step iii) requires the padlock probe is circularized. Thus it is unclear how the padlock probe of i) is circularized if step ii) is optional. Alternatively it is unclear if all steps after the optional step are required or optional. If all steps after ii) are option it is unclear how the target nucleic acid is detected.
Claim 1 further recites, “regions specific for the target nucleic acid.” The recitation of specific for the target nucleic acid suggests there are regions non- specific for the target nucleic acid.” The specification and claims provide no standard to differentiate the two. Thus the metes and bounds are unclear.
Claim 2 depends from claim 1 and requires, “wherein in step (i), the 3' target binding region of the padlock probe is at least 6 bases shorter than the 5' target binding region and the padlock probe is contacted with the target nucleic acid molecule together with dNTPs, a polymerase and a ligase, and wherein the dNTPs are provided at a concentration of no more than 10 pM and the polymerase is provided at a concentration of no more than 0.025U/u.” This is confusing and unclear as optional step ii) appears to provide for the same outcome. Thus it is unclear how to practice claim 2 an step ii).
Claim 5 depends from claim 2 and recites, “wherein steps (i) and (ii) are cyclically repeated to generate the first RCA template.” The claim is confusing and unclear as it appears a single extension and ligation would provide for the RCA template. Further it is unclear what is require of cyclically as it is not an art accepted term and is not defined by the claims or the specification.
Regarding claim 13, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention or merely a preferred embodiment..
Regarding claim 17, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention or merely a preferred embodiment.
Claim 19 further recites, “a detection sequence which is specific to the padlock probe.” The recitation of specific to the padlock suggests there are regions non- specific for the padlock.” The specification and claims provide no standard to differentiate the two. Thus the metes and bounds are unclear.
Claim 19 recites, “the variant target nucleic acid sequence.” Neither claim 1 or claim 19 from which it depends previously recite, “ the variant target nucleic acid sequence.” Thus the metes and bounds are unclear what the limitation is refencing. This rejection can be overcome by amending the claim to provide an indefinite article prior to variant target nucleic.
Claim 19 recites, “the position corresponding.” Neither claim 1 or claim 19 from which it depends previously recite, “ the position corresponding.” Thus the metes and bounds are unclear what the limitation is refencing. This rejection can be overcome by amending the claim to provide an indefinite article prior to variant target nucleic.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-6, 8, 10-11, 13,16-17, 19-21, 23, 29 are rejected under 35 U.S.C. 102(a)(1)/102(a)(2) as being anticipated by Landegren (US20160289750) .
With regards to claim 1, Landegren discloses methods and compositions
for performing rolling circle amplification (RCA) reaction for at least two rounds of RCA using padlock probes. A target nucleic acid is hybridized with the complementary 5' and 3' binding sites of a padlock probe. 3' end of the padlock probe is extended with a polymerase using the target nucleic acid sequence as a template to fill a gap between the hybridized ends of the 5' and 3' binding sites. The padlock probe is ligated after a successful hybridisation to the target in order to generate a circular RCA template from which to produce a first DNA product. One or more subsequent padlock probes and their circularization for targeting the multiple repeats of the first RCA product to produce multiple second RCA products from the second circularized padlock probes is disclosed. Multiple rounds of RCA may be performed using the RCA product of previous round hybridized to a padlock probe. The second RCA products are detected with fluorescently labeled oligonucleotides. Multiple different nucleic acid sequences may be detected in a multiplex assay using different padlock probes with unique recognition sequences to produce different RCA products simultaneously. The target nucleic acid can be gDNA or RNA. The sample containing the target nucleic acid can be blood or plasma or other biopsy sample such as spinal fluid, tissue or clinical sample. In situ detection in cell samples or tissue is also disclosed. The target nucleic acid is hybridized with the padlock probe at 60 °C. The gap between the binding regions of the padlock probe can be 4 nucleotides. Microscopy imaging and flow cytometry are disclosed as a detection method for the RCA products. The method can be performed as a homogenous process in solution or on a solid surface with washing steps. “(Figures 1 and 3, Description, paragraphs [0028], [0029], [0030], [0031],
[0032], [0039], [0041], [0042], [0043], [0046], [004 7], [0052], [0067], [0078],
[0086], [0089], [0095], [0102], [0103], [0108], [011 O], [0112], [0129], [0133],
[0162], [0163], [0223], [0214] Example 2)
With regards to claim 3, Landegren teaches The padlock probe is ligated after a successful hybridisation to the target in order to generate a circular RCA template from which to produce a first DNA product. “(Figures 1 and 3, Description, paragraphs [0028], [0029], [0030], [0031], [0032], [0039], [0041], [0042], [0043], [0046], [004 7], [0052], [0067], [0078], [0086], [0089], [0095], [0102], [0103], [0108], [011 O], [0112], [0129], [0133], [0162], [0163], [0223], [0214] Example 2)
With regards to claim 4, 8 Landegren teaches, “[0172] An example of a method to detect rare mutations utilising the sRCA reaction of the present invention may involve isolating DNA from target cells, fragmenting the DNA, isolating a fragment which may contain a target sequence (e.g. mutation) of interest using a “selector” probe which is designed to have two target-specific ends which bind specifically to a target region of interest and allow the target fragment to be circularised, and performing a first RCA reaction using the circularised target fragment as first RCA template. The first RCA product thus generated may be subjected to the sRCA method of the present invention. The probe for the second RCA can be designed to recognise (e.g. bind to, and/or be ligated by) a mutant sequence it is desired to detect. Thus, by isolating target sequences to generate a first RCA template, the opportunity is afforded to generate a first RCA product comprising multiple (complementary) copies of the target sequence. Thus, an increased number of “targets” can be created in the concatemeric first RCA product. Each of these may be bound by a probe, which may be designed to be specific for the mutation it is desired to detect, to initiate a second RCA reaction, and the second RCA product can be detected to detect the mutation.”
With regards to claim 6, Landegren teaches, “Analytes of particular interest may thus include nucleic acid molecules, such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA etc)” (0110)
With regards to claims 10-11, 23Landegren teaches plasma samples (0112)
With regards to claim 13, Landegren teaches hybridization and ligation at 60oC (example 10)
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.
Claim(s) 1-6, 8, 10-11, 13,16-17, 19-21, 23, 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Landegren (US20160289750), CHEN (NUCLEIC ACIDS RESEARCH, vol. 46, no. 4, 28 November 2017 (2017-11-28), pages e22-e22) and Li (GENOME RESEARCH, vol. 19, no. 9, 1 September 2009 (2009-09-01), pages 1606-1615)
With regards to claim 1, Landegren discloses methods and compositions
for performing rolling circle amplification (RCA) reaction for at least two rounds of RCA using padlock probes. A target nucleic acid is hybridized with the complementary 5' and 3' binding sites of a padlock probe. 3' end of the padlock probe is extended with a polymerase using the target nucleic acid sequence as a template to fill a gap between the hybridized ends of the 5' and 3' binding sites. The padlock probe is ligated after a successful hybridisation to the target in order to generate a circular RCA template from which to produce a first DNA product. One or more subsequent padlock probes and their circularization for targeting the multiple repeats of the first RCA product to produce multiple second RCA products from the second circularized padlock probes is disclosed. Multiple rounds of RCA may be performed using the RCA product of previous round hybridized to a padlock probe. The second RCA products are detected with fluorescently labeled oligonucleotides. Multiple different nucleic acid sequences may be detected in a multiplex assay using different padlock probes with unique recognition sequences to produce different RCA products simultaneously. The target nucleic acid can be gDNA or RNA. The sample containing the target nucleic acid can be blood or plasma or other biopsy sample such as spinal fluid, tissue or clinical sample. In situ detection in cell samples or tissue is also disclosed. The target nucleic acid is hybridized with the padlock probe at 60 °C. The gap between the binding regions of the padlock probe can be 4 nucleotides. Microscopy imaging and flow cytometry are disclosed as a detection method for the RCA products. The method can be performed as a homogenous process in solution or on a solid surface with washing steps. “(Figures 1 and 3, Description, paragraphs [0028], [0029], [0030], [0031],
[0032], [0039], [0041], [0042], [0043], [0046], [004 7], [0052], [0067], [0078],
[0086], [0089], [0095], [0102], [0103], [0108], [011 O], [0112], [0129], [0133],
[0162], [0163], [0223], [0214] Example 2)
Landegren does not specifically teach, “the 3' target binding region of the padlock probe is at least 6 bases shorter than the 5' target binding region and the padlock probe is contacted with the target nucleic acid molecule together with dNTPs, a polymerase and a ligase, and wherein the dNTPs are provided at a concentration of no more than 10 pM and the polymerase is provided at a concentration of no more than 0.025U/u.”
However, Chen teaches optimization of padlock-based technique for in situ barcode sequencing. The optimization results in an five-fold increase of the amplification efficiency. Chen identifies causes for the lower efficiency of the gap-filling padlock-based method. One of the causes is identified as the overextension of the padlock probe's 3' end, preventing ligation. Various ways to optimize the gap-fill ligation are identified. Among them are dNTP concentration and the polymerase choice. Stoffel fragment, Phusion polymerase and Taq polymerase are disclosed as options for the gap-fill ligation. The polymerase concentration is optimized and used at 0.012 U/μI and the dNTP concentration is optimized and used at 20 μM. (Introduction, Materials and methods, Results, Figure 1)
Li teaches the utilizationand optimization of the use of padlock probes in high-throughput sequencing forenrichment of genomic loci and for detection of SNP's. The document identifies factors that contribute to substantial improvements. These factors include probe design and synthesis and the careful adjustment of dNTP concentration. Among the opimization parameters, a concentration of 2 μM is disclosed for dNTP mix in the gap-fill reaction and a concentration of 0.12 U/μI is disclosed for the polymerase. dNTP concentrations are tested in a range of 0.02 μM, 0.2 μM, 2 μM, 20 μM. A design aspect of padlock probe is identified where a more tightly bound 5' end of a padlock probe promotes ligase activity over a polymerase strand displacement and overextension.
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to recognize the problem with overextension of the padlock probe and optimize the concentration of the polymerase and the dNTP mix and based on the teachings of Chen. The artisan would be motivated to avoid overextension and optimize RCA ligation. The artisan would have a reasonable expectation of success as the teachings of Chen are within the same magnitude, the experiments would easily lead to the range of the claims. Further one of ordinary skill in the art prior to the effective tasked with designing a padlock probe that hybridizes more tightly from its 5' end would make a longer target binding region to that end. Thus one of skill in the art would have a reasonable expectation of success.(claims 2, 5, 16-17, 19 and 29)
With regards to claim 3, Landegren teaches The padlock probe is ligated after a successful hybridisation to the target in order to generate a circular RCA template from which to produce a first DNA product. “(Figures 1 and 3, Description, paragraphs [0028], [0029], [0030], [0031], [0032], [0039], [0041], [0042], [0043], [0046], [004 7], [0052], [0067], [0078], [0086], [0089], [0095], [0102], [0103], [0108], [011 O], [0112], [0129], [0133], [0162], [0163], [0223], [0214] Example 2)
With regards to claim 4, 8 Landegren teaches, “[0172] An example of a method to detect rare mutations utilising the sRCA reaction of the present invention may involve isolating DNA from target cells, fragmenting the DNA, isolating a fragment which may contain a target sequence (e.g. mutation) of interest using a “selector” probe which is designed to have two target-specific ends which bind specifically to a target region of interest and allow the target fragment to be circularised, and performing a first RCA reaction using the circularised target fragment as first RCA template. The first RCA product thus generated may be subjected to the sRCA method of the present invention. The probe for the second RCA can be designed to recognise (e.g. bind to, and/or be ligated by) a mutant sequence it is desired to detect. Thus, by isolating target sequences to generate a first RCA template, the opportunity is afforded to generate a first RCA product comprising multiple (complementary) copies of the target sequence. Thus, an increased number of “targets” can be created in the concatemeric first RCA product. Each of these may be bound by a probe, which may be designed to be specific for the mutation it is desired to detect, to initiate a second RCA reaction, and the second RCA product can be detected to detect the mutation.”
With regards to claim 6, Landegren teaches, “Analytes of particular interest may thus include nucleic acid molecules, such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA etc)” (0110)
With regards to claims 10-11, 23Landegren teaches plasma samples (0112)
With regards to claim 13, Landegren teaches hybridization and ligation at 60oC (example 10)
Claim(s) 1-6, 8-11, 13,16-17, 19-23, 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Landegren (US20160289750) and Gong (.RSC Adv.,2018,8, 27375–27381)
With regards to claim 1, Landegren discloses methods and compositions
for performing rolling circle amplification (RCA) reaction for at least two rounds of RCA using padlock probes. A target nucleic acid is hybridized with the complementary 5' and 3' binding sites of a padlock probe. 3' end of the padlock probe is extended with a polymerase using the target nucleic acid sequence as a template to fill a gap between the hybridized ends of the 5' and 3' binding sites. The padlock probe is ligated after a successful hybridisation to the target in order to generate a circular RCA template from which to produce a first DNA product. One or more subsequent padlock probes and their circularization for targeting the multiple repeats of the first RCA product to produce multiple second RCA products from the second circularized padlock probes is disclosed. Multiple rounds of RCA may be performed using the RCA product of previous round hybridized to a padlock probe. The second RCA products are detected with fluorescently labeled oligonucleotides. Multiple different nucleic acid sequences may be detected in a multiplex assay using different padlock probes with unique recognition sequences to produce different RCA products simultaneously. The target nucleic acid can be gDNA or RNA. The sample containing the target nucleic acid can be blood or plasma or other biopsy sample such as spinal fluid, tissue or clinical sample. In situ detection in cell samples or tissue is also disclosed. The target nucleic acid is hybridized with the padlock probe at 60 °C. The gap between the binding regions of the padlock probe can be 4 nucleotides. Microscopy imaging and flow cytometry are disclosed as a detection method for the RCA products. The method can be performed as a homogenous process in solution or on a solid surface with washing steps. “(Figures 1 and 3, Description, paragraphs [0028], [0029], [0030], [0031],
[0032], [0039], [0041], [0042], [0043], [0046], [004 7], [0052], [0067], [0078],
[0086], [0089], [0095], [0102], [0103], [0108], [011 O], [0112], [0129], [0133],
[0162], [0163], [0223], [0214] Example 2)
Landegren while teaching plasma samples, does not teach cell free DNA.
However, Gong teaches, “Recently, liquid biopsies, especially those which contain cell-free DNA samples, provide a convenient way for the detection of genetic mutations. Its advantage lies in the ability to reduce the risk of biopsy through non-invasive sampling and effectively prolonging the survival of patients. At present, an increasing number of drug targets have been developed, which makes it highly attractive to clinicians to be able to detect multiplex genetic mutations in patients in order to achieve the best clinical outcome. Hence, this non-invasive and multiplex detection approach of tumor-related mutations has great promise in clinical cancer therapies. Although numerous efforts have been made towards the development of new technologies to detect genetic mutations, including ARMS,9,10 COBAS,11,12 BEAMing technology and digital PCR, their applications are significantly limited by low sensitivity, false-positive results, complicated operations, low throughput, expensive chips and closed reagent (27376, 1st paragraph result and discussion). Gong teaches detection of mutations in cell free DNA from plasma samples (clinical evaluation of MPRP assay).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use cell free DNA from a plasma sample. The artisan would be motivated to avoid the invasiveness of having to biopsy tumors or tissues. The artisan would have a reasonable expectation of success as the artisan is merely using known samples in known methods.
With regards to claim 3, Landegren teaches The padlock probe is ligated after a successful hybridisation to the target in order to generate a circular RCA template from which to produce a first DNA product. “(Figures 1 and 3, Description, paragraphs [0028], [0029], [0030], [0031], [0032], [0039], [0041], [0042], [0043], [0046], [004 7], [0052], [0067], [0078], [0086], [0089], [0095], [0102], [0103], [0108], [011 O], [0112], [0129], [0133], [0162], [0163], [0223], [0214] Example 2)
With regards to claim 4, 8 Landegren teaches, “[0172] An example of a method to detect rare mutations utilising the sRCA reaction of the present invention may involve isolating DNA from target cells, fragmenting the DNA, isolating a fragment which may contain a target sequence (e.g. mutation) of interest using a “selector” probe which is designed to have two target-specific ends which bind specifically to a target region of interest and allow the target fragment to be circularised, and performing a first RCA reaction using the circularised target fragment as first RCA template. The first RCA product thus generated may be subjected to the sRCA method of the present invention. The probe for the second RCA can be designed to recognise (e.g. bind to, and/or be ligated by) a mutant sequence it is desired to detect. Thus, by isolating target sequences to generate a first RCA template, the opportunity is afforded to generate a first RCA product comprising multiple (complementary) copies of the target sequence. Thus, an increased number of “targets” can be created in the concatemeric first RCA product. Each of these may be bound by a probe, which may be designed to be specific for the mutation it is desired to detect, to initiate a second RCA reaction, and the second RCA product can be detected to detect the mutation.”
With regards to claim 6, Landegren teaches, “Analytes of particular interest may thus include nucleic acid molecules, such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA etc)” (0110)
With regards to claims 10-11, 23Landegren teaches plasma samples (0112)
With regards to claim 13, Landegren teaches hybridization and ligation at 60oC (example 10)
Summary
No claims are allowed.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEVEN C POHNERT PhD whose telephone number is (571)272-3803. The examiner can normally be reached Monday- Friday about 6:00 AM-5:00 PM, every second Friday off.
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/Steven Pohnert/ Primary Examiner, Art Unit 1683
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