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 .
Applicant’s reply to the February 10, 2026 Office Action, filed May 07, 2026, is acknowledged. Applicant cancels claims 13 and 15. Claims 1-12, 14 and 16-20 are currently pending and under examination.
Any objection or rejection of record in the previous Office Action, which is not addressed
in this action has been withdrawn in light of Applicant’s amendments and/or arguments. This action is Final.
Claim Objections
Claim 14 is objected to because of the following informalities:
In claim 14, line 5, “adapters and reagents capable of ligating the two or more sample-specific adapters”, should read “adapters and reagents for ligating the two or more sample-specific adapters”.
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.
Claims 14-20 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Pan et al. (U.S. Patent Application Publication US 2015/0368694 A1, published December 24, 2015, cited on the IDS filed July 12, 2023. This rejection is maintained and modified as necessitated by amendments.
Regarding claim 14, Pan teaches a kit for multiplexed detection of DNA methylation in pooled DNA libraries comprising micrococcal nuclease and an antibody specific for methylated DNA (Page 2, [0016], Page 5, [0046], Page 3, [0032], Page 7, [0076], Page 17, [0189], Page 18, [0201], Pages 22-23, [0257], Page 26, [0297]-[0298], Pages 28-29, [0330]-[0334], Page 29, [0339] and Pages 31-32, [0364]). Pan teaches two or more sample-specific adapters and reagents capable of ligating the two or more sample-specific adapters to nucleosome-protected fragments (Page 2, [0016], Page 3, [0025], Page 5, [0045]-[0046], Page 6, [0065], Page 8, [0085], Page 9, [0092], Page 7, [0076], Page 9, [0103], Page 15, [0165], Page 17, [0189], Pages 22-23, [0257], Page 26, [0298] and Page 31-32, [0364]).
Regarding claim 16, Pan teaches primers that hybridize with the sample-specific adapters (Page 5, [0045]-[0047], Page 15, [0165] and Page 17, [0187]).
Regarding claim 17, Pan teaches reagents for amplifying the nucleosome-protected fragments (Page 3, [0028]-[0029] and [0032], Page 9, [0092], Page 8, [0085]-[0086], Pages 7-8, [0077] and Page 10, [0109], [0112] and [0114]).
Regarding claim 18, Pan teaches the reagents for amplifying comprise reagents for polymerase chain reaction (PCR) (Page 3, [0031] and [0033] and Page 4, [0038]).
Regarding claim 19, Pan teaches the reagents for PCR comprise a high-fidelity polymerase (Page 21, [0231]).
Regarding claim 20, Pan teaches reagents for next generation sequencing (Page 5, [0047] and Page 25, [0292]).
Pan teaches each and every limitation of claims 14-20, and therefore Pan anticipates claims 14-20.
Claim Rejections - 35 USC § 103
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 1-12 are rejected under 35 U.S.C. 103 as being unpatentable over Pan et al. (U.S. Patent Application Publication US 2015/0368694 A1, published December 24, 2015, cited on the IDS filed July 12, 2023, as applied to claims 14-20 above, in view of Shema-yaacoby et al. (WIPO International Application Publication WO 2017/034970, published March 02, 2017), previously cited in the February 10, 2026 Office Action, as evidenced by Jezek et al. (“Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae”. J Vis Exp. ; (130):57080, published December 29, 2017). This is a new rejection as necessitated by amendments.
Regarding claim 1, Pan teaches a method for multiplexed detection of DNA methylation (Abstract, Page 5, [0046] and Page 17, [0189]). Pan teaches contacting a micrococcal nuclease (MNase) to two or more individual samples comprising genomic DNA (Abstract, Page 3, [0032] and Page 17, [0189]). Pan teaches isolating different size fragments of DNA that correlate to different portions of chromatin and the DNA fragment sizes may be corresponding to mono-nucleosomal DNA and di-nucleosomal DNA as well as the precise level of amplification of each fragment may be determined (Page 10, [0108] and [0112]). Pan teaches purifying the MNase-contacted samples of step (a), whereby DNA samples comprising nucleosome-protected fragments are obtained (Page 3, [0028] and [0032], Page 9, [0092], Page 8, [0085], Pages 7-8, [0077] and Page 10, [0109], [0112] and [0114]). Pan teaches ligating sample-specific adapters to the nucleosome-protected fragments and each of the two or more samples comprises a unique adapter and wherein adapter ligated DNA libraries are generated (Page 2, [0016], Page 3, [0025], Page 5, [0045]-[0046], Page 6, [0065], Page 17, [0189] and Pages 22-23, [0257]). Pan teaches pooling the two or more adapter-ligated DNA libraries to generate a pooled DNA library (Page 5, [0046], Page 7, [0076], Page 8, [0085] and Pages 22-23, [0257]). Pan teaches performing methylated DNA immunoprecipitation on the pooled DNA library of step (d), thereby obtaining a pooled library enriched for methylated DNA (Page 2, [0016], Page 5, [0046], Page 6, [0065] and Pages 28-29, [0330]). Pan teaches amplifying the DNA of step (e) using primers having specificity for the unique adaptors (Page 4, [0038], Page 5, [0046], and Pages 22-23, [0257]). Pan teaches sequencing the amplified sample to detect DNA methylation (Page 3, [0032], Page 4, [0038], Page 5, [0046] and Page 29, [0339]).
Regarding claim 2, Pan teaches analyzing sequences obtained in step (g) to identify, if present, aberrant DNA methylation in genomic DNA from the two or more individual samples (Page 2, [0014]-[0015], Page 3, [0025], Page 5, [0046], Page 6, [0065] and Page 14, [152]).
Regarding claim 3, Pan teaches the pooled DNA library comprises genomic DNA from 2 to 15 individual sources (Page 3, [0025], Page 5, [0046], Page 6, [0065], Page 8, [0082], Page 14, [152] and Page 32, [0367]).
Regarding claim 4, Pan teaches the pooled DNA library comprises at least 5 ng genomic DNA of each individual source (e.g., Less than 1,000 cells of gDNA equates to about 6 ng of gDNA as well as less than 10,000 cells of gDNA equates to about 60 ng of gDNA therefore at least 5 ng, Abstract, Page 5, [0053], Page 19, [0216] and Page 20, [0223]).
Regarding claim 5, Pan teaches the pooled DNA library comprises at least 10 ng genomic DNA of each individual source (e.g., Less than 10,000 cells of gDNA equates to about 60 ng of gDNA therefore at least 10 ng, Abstract, Page 5, [0053], Page 19, [0216] and Page 20, [0223]).
Regarding claim 6, Pan teaches the pooled DNA library comprises less than 100 ng genomic DNA of each individual source (Less than 10,000 cells of gDNA equates to about 60 ng of gDNA (e.g., Less than 10,000 cells of gDNA equates to about 60 ng of gDNA therefore at least 10 ng, Abstract, Page 5, [0053], Page 19, [0216] and Page 20, [0223]).
Regarding claim 7, Pan teaches amplifying comprises quantitative polymerase chain reaction (qPCR) (Page 29, [0339]).
Regarding claim 8, Pan teaches sequencing comprises next generation sequencing (Page 5, [0047] and Page 25, [0292]).
Regarding claim 9, Pan teaches the method has specificity greater than 70% (Page 3, [0025] and [0032]-[0033], Page 5, [0046], Page 7, [0074], Pages 7-8, [0077], Page 31, [0358] and Figs. 18B and 19A-C).
Regarding claim 10, Pan teaches the method has specificity greater than 90% (Page 3, [0025] and [0032]-[0033], Page 5, [0046], Page 7, [0074], Pages 7-8, [0077], Page 31, [0358] and Figs. 18B and 19A-C).
Regarding claim 11, Pan teaches identifying 5-methylcytosine (Page 8, [0079] and Page 16, [0182]).
Regarding claim 12, Pan teaches the micrococcal nuclease (MNase) reaction of step (a) is maintained at about 55 °C (Page 20, [0219] and Page 21, [0234]).
Pan does not teach or suggest the method can differentiate between 5-methylcytosine and 5-hydroxymethylcytosine. Pan does not teach or suggest the micrococcal nuclease reaction of step (a) results in explicitly about 30% mono-nucleosomal DNA and about 25% di-nucleosomal DNA.
Shema-yaacoby teaches a method for multiplex detection of DNA methylation that can differentiate between 5-methylcytosine and 5-hydroxymethylcytosine using micrococcal nuclease (MNase) (Page 12, [0046], Page 16, [0056], Page 77, [00192] and Example 8). Shema-yaacoby teaches using multiplex split pool analysis (Page 22, [0075] and Page 33, [00110]). Shema-yaacoby teaches contacting two or more samples of genomic DNA with micrococcal nuclease (MNase) (Pages 7-8, [0029]-[0031], Page 22, [0075], Pages 28-29, [0099] and Page 77, [0192]). Shema-yaacoby teaches purifying the MNase-contacted samples and the DNA sample comprises nucleosome protected fragments (Page 4, [0018], Pages 7-8, [0029], Page 76, [00188] and Pages 7-8, [0029]-[0031]). Shema-yaacoby teaches the DNA fragments may be analyzed to determine the fractions (which could be converted into percentages) containing mono-nucleosomes and di-nucleosomes (Page 28, [0096]). Shema-yaacoby teaches optimizing the samples (Page 79, [00198]). Shema-yaacoby teaches using these methods provides the advantage of counting the modified nucleosomes and calculate the percentage of nucleosomes which carry the modifications or do not carry the modifications or a combination of different modifications as well as labeling and detection of single nucleosomes which can advantageously allow the characterization of the combinatorial pattern of histone modifications on a single nucleosome, which can provide significant utility in the study of epigenetics (Pages 61-62, [00149]-[00153]).
Jezek teaches chromatin Immunoprecipitation (chIP) of methylated histones (Abstract and Title). Jezek teaches using micrococcal nuclease (MNase) used to digest the chromatin into soluble fragments (Page 4, Last Paragraph and Fig. 2). Jezek teaches an MNase titration should be performed to determine the most suitable concentration to achieve primarily mono-nucleosomes with a smaller amount of di-nucleosomes in the soluble chromatin fraction (Page 4, Last Paragraph). Jezek teaches one key component of the chIP protocol is optimizing the concentration of micrococcal nuclease (MNase) used to digest the chromatin into soluble fragments (Page 4, Last Paragraph).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the teachings of Pan with the teachings of Shema-yaacoby, differentiating between 5-methylcytosine and 5-hydroxymethylcytosine and using a micrococcal nuclease reaction resulting in about 30% mono-nucleosomal DNA and about 25% di-nucleosomal DNA since it has been held that where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art and not considered inventive. (In re Aller, 105 USPQ 233 (CCPA 1955)). More particularly, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. Routine optimization is not considered inventive and no evidence has been presented that the selection of optimizing conditions performed was other than routine, that the products resulting from the optimization have any unexpected properties, or that the results should be considered unexpected in any way as compared to the closest prior art. The percentages of claim 1 are considered a result-effective variable (See MPEP 2144.05. II. B.). There is a Motivation to Optimize Result-Effective Variables; therefore, as these percentages can be optimized, the requirement of claim 1 is not considered inventive. Moreover, Jezek discloses an MNase titration should be performed to determine the most suitable concentration to achieve primarily mono-nucleosomes with a smaller amount of di-nucleosomes in the soluble chromatin fraction for chIP protocols and one key component of the chIP protocol is optimizing the concentration of micrococcal nuclease (MNase) used to digest the chromatin into soluble fragments (Page 4, Last Paragraph). Therefore it would have been routine to optimize contacting the MNase to two or more individual samples to result in in about 30% mono-nucleosomal DNA and about 25% di-nucleosomal DNA. Additional motivation for modifying the teachings of Pan with the teachings of Shema-yaacoby would be using these methods allows for the advantage of counting the modified nucleosomes and calculate the percentage of nucleosomes which carry the modifications or do not carry the modifications or a combination of different modifications as well as labeling and detection of single nucleosomes which allows for the characterization of the combinatorial pattern of histone modifications on a single nucleosome, which can provide significant utility in the study of epigenetics as taught by Shema-yaacoby (Pages 61-62, [00149]-[00153]).
Response to Arguments
Applicant’s arguments and amendments filed May 07, 2026, with respect to the
Rejections under 35 U.S.C. § 112(b) and 103 have been fully considered and are persuasive. Therefore, this rejection has been withdrawn.
Applicant’s arguments and amendments filed May 07, 2026, with respect to the rejections of claims 14-20 under 35 U.S.C. § 102 have been fully considered, but they are not persuasive.
Applicant asserts “Pan does not teach adapters and reagents for ligating adapters to nucleosome-protected fragments, particularly multiple adapters for pooled DNA libraries. Pan discloses antibodies that bind methylated DNA fragments and micrococcal nuclease. However, they are used for different methods and are not combined in the same way as in the claims. Thus, Pan does not disclose each and every element of amended claims 14-20 combined in the same way as recited in the claims”.
As discussed above, Pan discloses using nucleosome protected fragments in that Pan teaches preparing the DNA used in the method by “avoiding disturbing the nuclear structure … protect the native structure of the nucleus (i.e., nucleosome)… to prevent random shearing … when working with …single cells (Page 9, [0092]). Pan discloses methylated regions of the DNA are protected (Page 15, [0165]), as well as using DNA “protected by compact or closed chromatin… [that] will be selectively amplified” (Page 10, [0109]), and “large DNA fragments protected by closed chromatin, are selectively amplified and/or physically separated and recovered”, (Page 9, [0103]). Pan discloses “It can be important for gDNA to be recovered for CpG methylation analysis using a gentle procedure to avoid non-specific gDNA shearing and to maintain double-strandedness (i.e., protected). Therefore, genomic DNA is typically accessed under conditions that retain the double strandness of the DNA, and which also enables a restriction endonuclease (RE) digestion, followed by adapter ligation” (Page 11, [0119]). Pan discloses “the fragments may be pooled in combination, … for library construction”, (Page 6, [0065]). Pan additionally discloses “A multiplex design can be integrated into these PCR-based methods for CpG methylation … The key for the multiplex design is a barcoded adapter that with a barcode (a combination of nucleotides) built in that are directly ligated to the … fragment end. After ligation … the samples can be combined or pooled together like one sample for the downstream processes” (Page 5, [0046]). Pan further discloses that adapters “includes a barcode… for each sample, at the adapter end that is directly ligated to the end of the fragments generated from the target DNA… the individual samples with different barcoded adapters are pooled together for the downstream processing after the adapter ligation and before PCR amplification”, (Pages 22-23, [0257]). Pan even further discloses “Adaptors are typically ligated to the ends of the amplicons and used as priming sites and barcodes for sequencing reactions. Multiplexing is also possible. For example, by incorporating different bar codes for different starting material or different assays, large numbers of amplicons from different starting material and/or assays can be amplified and/or sequenced as pool and linked to a specific profile by identifying the bar code during bioinformatics analysis” (Page 26, [0298]) and “Kits [comprising] …reagents [and] ligation of sequencing adaptors to oligonucleotides”, (Pages 28-29, [0330]). Therefore, Pan does in fact disclose two or more sample specific adapters and reagents for ligating adapters to nucleosome-protected fragments, particularly multiple adapters for pooled DNA libraries. As applicant has acknowledged, Pan does disclose antibodies that bind methylated DNA fragments and micrococcal nuclease. Independent claim 14 requires a kit for multiplexed detection of DNA methylation in pooled DNA libraries comprising elements (a) micrococcal nuclease, (b) an antibody specific for methylated DNA and (c) two or more sample-specific adapters and reagents capable of ligating the two or more sample-specific adapters to nucleosome-protected fragments; “comprising” is interpreted as to include/requiring specific parts, members or elements. As discussed above, Pan discloses a kit for multiplexed detection of DNA methylation in pooled DNA libraries and the kit comprises reagents and each of the elements (a)-(c), as well as the elements listed in claims 16-20. Therefore, Pan anticipates claims 14-20.
Applicant’s arguments and amendments filed May 07, 2026, with respect to the
rejections of claims 1-13 under 35 U.S.C. § 102 have been fully considered and are persuasive in part. Therefore, this rejection has been withdrawn.
Upon further consideration, new grounds of rejections under 35 U.S.C. § 103 are made in view of Applicant’s amendments.
As discussed above, Pan teaches isolating different size fragments of DNA that correlate to different portions of chromatin and the DNA fragment sizes may be corresponding to mono-nucleosomal DNA and di-nucleosomal DNA as well as the precise level of amplification of each fragment may be determined (Page 10, [0108] and [0112]). Additionally Shema-yaacoby teaches the DNA fragments may be analyzed to determine the fractions (which could be converted into percentages) containing mono-nucleosomes and di-nucleosomes as well as optimizing the samples. Shema-yaacoby teaches using these methods provides the advantage of counting the modified nucleosomes and calculate the percentage of nucleosomes which carry the modifications or do not carry the modifications or a combination of different modifications as well as labeling and detection of single nucleosomes which can advantageously allow the characterization of the combinatorial pattern of histone modifications on a single nucleosome, which can provide significant utility in the study of epigenetics (Pages 61-62, [00149]-[00153]). Additionally, newly cite Jezek discloses in chIP protocols an MNase titration should be performed to determine the most suitable concentration to achieve primarily mono-nucleosomes with a smaller amount of di-nucleosomes in the soluble chromatin fraction (30% mono-nucleosomes and 25% di-nucleosomes) and one key component of the chIP protocol is optimizing the concentration of micrococcal nuclease (MNase) used to digest the chromatin into soluble fragments. It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the teachings of Pan with the teachings of Shema-yaacoby, differentiating between 5-methylcytosine and 5-hydroxymethylcytosine and using a micrococcal nuclease reaction resulting in about 30% mono-nucleosomal DNA and about 25% di-nucleosomal DNA since it has been held that where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art and not considered inventive. (In re Aller, 105 USPQ 233 (CCPA 1955)). More particularly, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. Routine optimization is not considered inventive and no evidence has been presented that the selection of optimizing conditions performed was other than routine, that the products resulting from the optimization have any unexpected properties, or that the results should be considered unexpected in any way as compared to the closest prior art. The percentages of claim 1 are considered a result-effective variable (See MPEP 2144.05. II. B.). There is a Motivation to Optimize Result-Effective Variables; therefore, as these percentages can be optimized, the requirement of claim 1 is not considered inventive. Therefore it would have been routine to optimize contacting the MNase to two or more individual samples to optimize the results in about 30% mono-nucleosomal DNA and about 25% di-nucleosomal DNA.
Therefore, for these reasons and those listed above, Pan and Pan in view of Shema-yaacoby are deemed to render the instant invention obvious.
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 DANIELLE PARISI whose telephone number is (571)272-8025. The examiner can normally be reached Mon - Friday 7:30-5:00 Eastern with alternate Fridays off.
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/JESSICA D PARISI/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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