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 .
Response to Amendment
The amendment filed October 14th, 2025 is acknowledged. Regarding the Office Action mailed June 12th, 2025:
The rejections set forth under 35 U.S.C. 112(b) are withdrawn in view of the amendments.
The rejection set forth under 35 U.S.C. 102 is withdrawn in view of the Declaration under 37 C.F.R. 1.130(a).
Maintained or modified rejections are set forth below, as necessitated by the amendments. Responses to arguments, if necessary, follow their respective rejection sections.
Claim Summary
Claims 4, 5, 10, 11, and 26 have been amended. Claims 16-23 have been canceled. Claims 1-15 and 24-27 are pending. Claims 1-15 and 24-27 are under examination and discussed in this Office action.
Claim Rejections - 35 USC § 103 – Modified – Necessitated by Amendment
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-15 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Kind (Genome-wide Maps of Nuclear Lamina Interactions in Single Human Cells, Cell, September 2015, 163, 134-147; previously cited), in view of Streets (Microfluidic single-cell whole-transcriptome sequencing, PNAS, April 2014, 111, 7048-7053; previously cited).
Regarding instant claim 1, Kind teaches on incubating a collection of cells that express at least one protein of interest under conditions that allow the at least one protein of interest to contact a DNA sequence (Page 135, column 2, paragraph 3). Kind also teaches on isolating a single cell from the collection of cells (Page 135, column 2, paragraph 3; Figure 1A), and further amplifying and collecting the DNA comprising the DNA sequence and determining the sequence of the DNA sequence (Page 135, column 2, paragraph 4 to Page 136, column 1, paragraph 1; Figure 1A). The collection of cells were also transformed with a fluorescent reporter system to select cells in specific cell stages via FACS (Page 135, column 2, paragraph 3). Kind further teaches a collection of cells from which a single cell can be isolated and the location of the DNA sequence of interest can be determined (Page 141, column 2, paragraph 4 and 5; Figure 6). Although this collection of cells is different from the collection that is sequenced, both collections use the same Dam-LmnB1 for marking DNA sequences of interest (Page 135, column 2, paragraph 3; Page 141, column 2, paragraph 4 and 5). Furthermore, the first collection shows that even with inclusion of a fluorescent analysis system, like FACS, the cells can still be sequenced to determine the DNA sequence of interest (Page 135, column 2, paragraph 3 to Page 136, column 1, paragraph 1). Therefore, because of the provided methods in Kind, it would be obvious to one of ordinary skill in the art to combine determining the cellular location of the DNA sequence in a single cell with further sequencing the DNA sequence in that cell after determining the DNA sequence location.
Kind does not teach that this method is carried out in a microfluidics device, or that steps (b) and (c) are carried out in separate chambers of that device.
Streets, in a reasonably pertinent field, teaches on performing single cell whole transcriptome sequencing using a microfluidic device that can separate out single cells and perform further operations of the method in different chambers (Figure 1 and caption). Streets also teaches that single cells are imaged with a microscope in the trapping chambers, as well as all other chambers, to determine that single cells have been trapped in each lane of the microfluidics device (Figure S1F).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Kind with the microfluidics device of Streets. Since Streets teaches on single cell processing for sequencing, which is reasonably pertinent to Kind’s single cell method, one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because, as suggested in KSR, 550 U.S. at 418, 82 USPQ2d at 1395 (see MPEP 2141(III)), the combination would amount to applying a known technique to a known device (method, or product) ready for improvement to yield predictable results. Streets teaches that single cells can be processed in a microfluidics device. Streets also teaches that single cells can be imaged before being processed. Furthermore, Kind teaches that single cells can be fluorescently examined before being sequenced. Kind also teaches all the claimed methods of determining a cellular location for a DNA sequence in an isolated single cell, as well as amplifying, collecting, and sequencing a DNA sequence from an isolated single cell. Given all of these known elements, combining them would reasonably amount to applying a known technique to a known device (method, or product) ready for improvement to yield predictable results. In regards to the use of a microfluidics device, Streets offers that microfluidics devices are beneficial for the following reasons: “Performing reactions in parallel nanoliter volumes predefined by photolithography ensures high reproducibility by removing stochastic variation caused by pipetting error and variability in handling associated with bench-top experimentation…It has been shown that performing amplification in nanoliter volumes improves reaction efficiency.” (Page 1, column 2, paragraph 2 through Page 2, column 1, paragraph 1).
Regarding instant claim 2, Kind, in view of Streets, teaches the method of claim 1. Street further teaches wherein the incubating step (a) is carried out in a chamber within one lane of the microfluidic device (Supporting Information, Page 1, column 2, first two paragraphs of Device Design and Operation). It is noted that the same 103 analysis as applied for claim 1 is applied here.
Regarding instant claim 3, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein the DNA sequence comprises a DNA-binding site (Page 135, column 2, paragraph 3).
Regarding instant claim 4, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein the cells have been induced to express the at least one protein of interest (Page 135, column 2, paragraph 3).
Regarding instant claim 5, Kind, in view of Streets, teaches the method of claim 4. Kind further teaches wherein the at least one protein of interest is a recombinant protein and is expressed from an expression vector (Page 135, column 2, paragraph 3; Supplemental Information Page 2, Cell lines).
Regarding instant claim 6, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein the at least one protein of interest is a nuclear lamina protein (Page 134, column 2, paragraph 2: NL=nuclear lamina; Page 135, column 1, paragraph 3).
Regarding instant claim 7, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein the at least one protein of interest has been engineered to modify one or more nucleotides at or near the DNA sequence (Page 135, column 2 to Page 136, column 1: Single-Cell DamID Methodology).
Regarding instant claim 8, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein contacting the DNA sequence by the at least one protein of interest results in a modification to the DNA that is detectable by imaging (Page 141, column 2 to Page 144, column 1: NL Contacts Often Involve Embedding of DNA in the NL).
Regarding instant claim 9, Kind, in view of Streets, teaches the method of claim 8. Kind further teaches wherein the modification is methylation (Page 135, column 2 to Page 136, column 1: Single-Cell DamID Methodology).
Regarding instant claim 10, Kind, in view of Streets, teaches the method of claim 9. Kind further teaches wherein the methylation occurs at or near a sequence comprising GATC (Page 135, column 2 to Page 136, column 1: Single-Cell DamID Methodology).
Regarding instant claim 11, Kind, in view of Streets, teaches the method of claim 7, Kind further teaches wherein the protein of interest is a fusion of the at least one protein of interest and DNA adenine methyltransferase (Dam) (Page 135, column 2, paragraph 3).
Regarding instant claim 12, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein the collection of cells that expresses the at least one protein of interest also expresses at least one imaging protein that binds to methylation sites (Page 141, column 2, paragraph 4 and 5; Figure 6).
Regarding instant claim 13, Kind, in view of Streets, teaches the method of claim 12. Kind further teaches wherein the imaging protein is a fusion of a protein that binds methylated DNA and a green fluorescent protein (GFP) (Page 141, column 2, paragraph 4 and 5; Figure 6).
Regarding instant claim 14, Kind, in view of Streets, teaches the method of claims 13. Kind further teaches wherein the imaging protein is m6a-Tracer (Page 141, column 2, paragraph 4 and 5; Figure 6).
Regarding instant claim 15, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein the cell is a eukaryotic cell (Page 135, column 2, paragraph 2).
Regarding instant claim 24, Kind, in view of Streets, teaches the method of claim 1. Kind further teaches wherein the determining the sequence of the DNA sequence of step (d) allows the identification of an associated gene and/or locus within a genome (Figure 1B).
Regarding instant claim 25, Kind, in view of Streets, teaches the method of claim 1. Streets further teaches wherein the microfluidic device comprises from 1-100 lanes (Figure 1A, 8 lanes). It is noted that the same 103 analysis as applied for claim 1 is applied here.
Regarding instant claim 26, Kind, in view of Streets, teaches the method of claim 1. Streets further teaches wherein each lane of the microfluidic device can carry out steps (b)-(c) in parallel (Page 2, column 1, paragraph 3). It is noted that the same 103 analysis as applied for claim 1 is applied here.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Kind (Genome-wide Maps of Nuclear Lamina Interactions in Single Human Cells, Cell, September 2015, 163, 134-147; previously cited), in view of Streets (Microfluidic single-cell whole-transcriptome sequencing, PNAS, April 2014, 111, 7048-7053; previously cited) and Miyashita (Confocal Microscopy for Intracellular Co-Localization of Proteins, In: Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 261. Humana Press, 2004; previously cited).
Regarding instant claim 27, Kind teaches on incubating a collection of cells that express at least one protein of interest under conditions that allow the at least one protein of interest to contact a DNA sequence comprising a DNA-binding site (Page 135, column 2, paragraph 3). Kind also teaches on isolating a single cell from the collection of cells (Page 135, column 2, paragraph 3), and further amplifying and collecting the DNA comprising the DNA sequence and determining the sequence of the DNA sequence (Page 135, column 2, paragraph 4 to Page 136, column 1, paragraph 1). The collection of cells were also transformed with a fluorescent reporter system to select cells in specific cell stages via FACS (Page 135, column 2, paragraph 3). Kind further teaches a collection of cells from which a single cell can be isolated and the location of the DNA sequence of interest can be determined (Page 141, column 2, paragraph 4 and 5; Figure 6). Although this collection of cells is different from the collection that is sequenced, both collections use the same Dam-LmnB1 for marking DNA sequences of interest (Page 135, column 2, paragraph 3; Page 141, column 2, paragraph 4 and 5). Furthermore, the first collection shows that even with inclusion of a fluorescent analysis system, like FACS, the cells can still be sequenced to determine the DNA sequence of interest (Page 135, column 2, paragraph 3 to Page 136, column 1, paragraph 1). Therefore, because of the provided methods in Kind, it would be obvious to one of ordinary skill in the art to combine determining the cellular location of the DNA sequence in a single cell with further sequencing the DNA sequence in that cell after determining the DNA sequence location, providing contemporaneous imaging and sequence measurement of a protein-DNA interaction.
Kind further teaches wherein the protein of interest is a fusion of the protein of interest and Dam (Page 135, column 2, paragraph 3); wherein the amplifying the DNA sequence of part (c) comprises the steps of (i) lysing the single cell, (ii) digesting DNA, (iii) ligating universal primers, and (iv) PCR amplification (Page 135, column 2, paragraph 4 through Page 136, column 1, paragraph 1; Figure 1A).
Kind does not teach that this method is carried out in a microfluidics device, or that steps (b) and (c) are carried out in separate chambers of that device.
Streets, in a reasonably pertinent field, teaches on performing single cell whole transcriptome sequencing using a microfluidic device that can separate out single cells and perform further operations of the method in different chambers (Figure 1). Streets also teaches that single cells are imaged with a microscope in the trapping chambers, as well as all other chambers, to determine that single cells have been trapped in each lane of the microfluidics device (Figure S1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Kind with the microfluidics device of Streets. Since Streets teaches on single cell processing for sequencing, which is reasonably pertinent to Kind’s single cell method, one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because, as suggested in KSR, 550 U.S. at 418, 82 USPQ2d at 1395 (see MPEP 2141(III)), the combination would amount to applying a known technique to a known device (method, or product) ready for improvement to yield predictable results. Streets teaches that single cells can be processed in a microfluidics device. Streets also teaches that single cells can be imaged before being processed. Furthermore, Kind teaches that single cells can be fluorescently examined before being sequenced. Kind also teaches all the claimed methods of determining a cellular location for a DNA sequence in an isolated single cell, as well as amplifying, collecting, and sequencing a DNA sequence from an isolated single cell. Given all of these known elements, combining them would reasonably amount to applying a known technique to a known device (method, or product) ready for improvement to yield predictable results. In regards to the use of a microfluidics device, Streets offers that microfluidics devices are beneficial for the following reasons: “Performing reactions in parallel nanoliter volumes predefined by photolithography ensures high reproducibility by removing stochastic variation caused by pipetting error and variability in handling associated with bench-top experimentation…It has been shown that performing amplification in nanoliter volumes improves reaction efficiency.” (Page 1, column 2, paragraph 2 through Page 2, column 1, paragraph 1).
Neither Kind nor Street teach wherein the cellular location of step (b) is determined by confocal fluorescent microscopy.
Miyashita, in a reasonably pertinent field, teaches on using confocal laser scanning microscopy for visualization of protein co-localization (Abstract).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method and device of Kind and Streets with confocal microscopy of Miyashita. Since Miyashita teaches that confocal microscopy can be used to visualize protein localization, which is reasonably pertinent to observing where a protein of interest binds to a DNA sequence of Kind and Streets, one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because confocal laser scanning microscopy is the best method for visualizing protein co-localization because of the point scan/pinhole detection system, which allows light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution (Miyashita, Abstract).
Response to Arguments
Applicant's arguments filed October 14th, 2025 have been fully considered but they are not persuasive.
In regards to the claim rejections under U.S.C. 103, the Applicant first summarizes aspects of the Examiner’s rejection in the previous Office Action (Page 7 of the Remarks filed October 14th, 2025). The Applicant alleges that the rejection should be withdrawn because the cited art fails to disclose or suggest the claimed invention and there is no convincing line or reasoning for combining the references (Page 7 of the Remarks filed October 14th, 2025). The Applicant includes a recitation of claim 1, followed by the assertion that Kind does not disclose the integrated approach of “co-determining the cellular location and nucleotide sequence of a DNA that is contacted by a protein of interest in a single cell” (Page 8 of the Remarks filed October 14th, 2025). The Applicant argues that Kind’s experiments are performed in different cell populations, which is contrary to the unified single-cell approach as claimed (Page 8 of the Remarks filed October 14th, 2025).
In response to these arguments, the Examiner directs the Applicant’s attention to the identified aspects of Kind that suggest both determining cellular location of a DNA sequence and amplifying and collecting DNA comprising the DNA sequence may be performed in the same single cell. While Kind does these experiments separately, it is shown that a single cell may be both fluorescently analyzed and then further sequenced to determine a DNA sequence (see claim 1 rejection above). Therefore, it would be obvious that both methods could be performed in the same single cell.
The Applicant restates an aspect of the previous Office Action where the Examiner stated that Kind does not teach the final aspect of claim 1 (Page 8 of the Remarks filed October 14th, 2025). The Applicant then argues that the enzymatic reaction that works in polypropylene tubes/plates like those of Kind do not guarantee that the same enzymatic reaction will work on a microfluidic device (Page 8 of the Remarks filed October 14th, 2025). The Applicant argues that reagents like enzymes may adsorb onto microfluidic channel surfaces because of the high surface-area-to-volume ratios, causing reaction efficiency to plummet (Page 8 of the Remarks filed October 14th, 2025). The Applicant suggests that standard practice adds a mild surfactant, but it cannot be confidently predicted where the surfactant will prevent adsorption or whether adding surfactant will reduce enzyme activity (Page 8 of the Remarks filed October 14th, 2025). Applicant further states that reaction efficiency is paramount to single-cell genomics (Page 8 of the Remarks filed October 14th, 2025).
In response to these arguments, the Examiner would like to notify the Applicant that MPEP 716.01(c) makes clear that “[t]he arguments of counsel cannot take the place of evidence in the record” (In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965)). There is no evidence on the record that states a reaction performed in a polypropylene tube/plate doesn’t guarantee the reaction will work on a microfluidic device, nor any of the other aspects discussed by the Applicant. Furthermore, there is no claim directed towards a particular efficiency of the method. Therefore, even if the reaction efficiency may be lower through applying the methods of Kind to the microfluidic device of Streets, it would still be functional to some degree and applicable to that which is claimed.
The Applicant further argues that the combination of Kind and Streets fails to teach the specific structure of claim 1 because Streets does not disclose or suggest “co-determining the cellular location and nucleotide sequence of a DNA that is contact by a protein of interest”, which the Applicant earlier argued is not taught by Kind (Page 8 of the Remarks filed October 14th, 2025). The Applicant further argues that the motivation to combine Kind and Streets is in appropriate because the references are addressing fundamentally different technical problems (Page 8 of the Remarks filed October 14th, 2025). The Applicant further states that the methods paired by the disclosure are a new integration not suggested by the cited references (Page 8 of the Remarks filed October 14th, 2025). However, given the above response to the arguments regarding Kind, it can be said that Kind does teach the quoted claimed structure and those aspects do not need to be fulfilled by Streets. In addition, Streets is relied upon for its microfluidics device, not to teach the quoted claim structure. Therefore the combination of Kind and Streets is considered appropriate.
In regards to claim 27, the Applicant argues that the limitation “the cellular location of the DNA comprising the DNA-binding site of step (b) is coupled to the sequence of the DNA-binding site of step (d) to provide contemporaneous imaging and sequence measurement of a protein-DNA interaction” is not taught by the cited references (Page 9 of the Remarks filed October 14th, 2025). In response to this argument, the Examiner directs the Applicant’s attention to the identified aspects of Kind that suggest both determining cellular location of a DNA sequence and amplifying and collecting DNA comprising the DNA sequence may be performed in the same single cell, and therefore provide contemporaneous imaging and sequence measurement of a protein-DNA interaction. While Kind does these experiments separately, it is shown that a single cell may be both fluorescently analyzed and then further sequenced to determine a DNA sequence (see claim 27 rejection above). Therefore, it would be obvious that both methods could be performed in the same single cell.
In conclusion, none of the provided arguments are persuasive and the rejections under U.S.C. 103 are maintained and modified where needed due to amendment.
Conclusion
All claims are rejected.
THIS ACTION IS MADE FINAL. 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 Allison E Schloop whose telephone number is (703)756-4597. The examiner can normally be reached Monday-Friday 8:30-5 ET.
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/ALLISON E SCHLOOP/ Examiner, Art Unit 1683
/ANNE M. GUSSOW/ Supervisory Patent Examiner, Art Unit 1683