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 .
Claims Status
Claims 1-9, 39, & 41-47 filed on 01/22/2026 are pending. The cancellation of claim 10 without prejudice in the reply filed 01/22/2026 is acknowledged. All the amendments and arguments have been thoroughly reviewed but are deemed insufficient to place this application in condition for allowance. The following rejections are either newly applied, as necessitated by amendment, or are reiterated. They constitute the complete set being presently applied to the instant application. Response to Applicant’s argument follow. This action is FINAL.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Any rejection not reiterated is hereby withdrawn in view of the amendments to the claims.
Claim Rejections - 35 USC § 103
Claim(s) 1-9, 39, 41-45, & 47 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al.; Nature Biotechnology Letters, Vol. 37, pages 424-434, February 25th, 2019), as cited on the IDS dated 03/28/2023, in view of Zeng (Zeng et al.; Journal of the American Chemical Society, Vol. 140, pages 13190-13194, October 2018), as cited on the IDS dated 03/28/2023.
Regarding claim 1, Liu teaches a bisulfite-free method for detection of 5-hydroxymethylcytosine (5hmC) in which genomic DNA (source nucleic acid) is oxidized with TET enzymes or potassium perruthenate (KRuO4) (a metal oxide) to oxidize 5hmC to 5fC (produce an oxidized nucleic acid) and then then contacting the oxidized 5fC (oxidized nucleic acid) with pyridine and pic-borane (borane reducing agents) to convert 5fC to dihydrouracil (DHU) (contacting the oxidized nucleic acid with a borane reducing agent to produce a processed nucleic acid wherein the bases in the nucleic acid are converted to DHU in the processed nucleic acid) (abstract lines 6-11; pg. 424-425 paragraph bridging pg. 424 & pg. 425 lines 14-22; pg. 425 paragraph bridging column 1 & 2 lines 1-5 & 10-27; pg. 425-426 paragraph bridging pg. 425 & pg. 426 lines 4-26; pg. 430 column 2 2nd full paragraph lines 1-7; pg. 430 column 2 3rd full paragraph lines 1-7; pg. 430 column 2 4th full paragraph lines 1-4; pg. 430 6th full paragraph lines 1-5). Liu also teaches DNA was oxidized with mTet1CD twice (an oxidizing agent) and then reduced with pyridine borane (one or more additional steps of contacting the oxidized nucleic acid with oxidizing agent) (pg. 431 column 1 4th full paragraph lines 5-6).
Liu does not teach that the 5hmC bases are oxidized with a metal (VI) oxo complex or that the one or more additional steps of contacting the oxidized nucleic acid with a metal (VI) complex.
Zeng teaches a method for bisulfite-free analysis of 5-hydroxymethylcytosine (5hmC) in which genomic DNA 5hmC (source nucleic acid) is oxidized with potassium ruthenate (K2RuO4), a ruthenium (+6) oxidant (metal (VI) oxo complex) to convert 5hmC to 5fC (pg. 13191 column 1 1st full paragraph lines 1-10). In addition, Zeng teaches that potassium ruthenate (K2RuO4) is more oxidative that the reported oxidant potassium perruthenate (KRuO4) and that while potassium perruthenate (KRuO4) caused severe DNA degradation, potassium ruthenate (K2RuO4)-mediated oxidation is very mild but also complete and can efficiently convert 5hmC to 5fC (pg. 13191 column 1 1st full paragraph lines 1-14).
Liu and Zeng are considered to be analogous to the claimed invention because they are all in the same field of bisulfite-free analysis of 5hmC. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method oxidizing 5hmC with TET enzymes or potassium perruthenate (KRuO4) (a metal oxide) to oxidize 5hmC to 5fC and to have modified the method oxidizing 5hmC with TET enzymes twice to oxidize 5hmC to 5fC before reducing with pyridine borane in Liu to incorporate the use of potassium ruthenate (K2RuO4) to oxidize 5hmC to 5fC as taught in Zeng because Zeng teaches that doing so would provide a method that can completely and efficiently oxidize and convert 5hmC to 5fC in a milder method that does not cause severe DNA degradation.
Regarding claim 2, Liu teaches the genomic DNA is from mouse embryonic stem cells (source nucleic acid is a mammalian nucleic acid) (pg. 425-426 paragraph bridging pg. 425 & pg. 426 lines 15-16). Zeng teaches that the genomic DNA is from human embryonic stem cells (source nucleic acid is a mammalian nucleic acid) (pg. 13191 column 1 1st full paragraph lines 10-13).
Regarding claim 3, Liu teaches the method is bisulfite-free method for detection of 5hmC (the method does not comprise treatment with bisulfite) (abstract lines 1-8).
Regarding claim 4, Liu teaches the 5hmC bases are detected through Sanger sequencing (pg. 425-426 paragraph bridging pg. 425 & pg. 426 lines 6-11).
Regarding claim 5, Liu teaches the false-positive rate of conversion of unmodified C was 0.23% (false positive rate of detection of 5hmC is below 1%) (pg. 426 paragraph bridging columns 1 & 2 lines 8-10 & 19-20; Fig. 3b).
Regarding claim 6, Liu teaches the step of reducing the oxidized DNA with a borane reducing agent is conducted at 70°C and 37°C depending on the borane reducing agent used (one or more of the contacting steps are conducted above a temperature of 4°C) (pg. 430 column 2 3rd full paragraph lines 1-7; pg. 430 column 2 4th full paragraph lines 1-4).
Regarding claim 7, Liu teaches the conversion of 5hmC is 89.1% (efficiency of conversion of 5hmC to DHU is greater than 80%) (pg. 426 paragraph bridging columns 1 & 2 lines 27-29; Fig. 3a).
Regarding claim 8, Liu teaches no affinity enrichment is performed in oxidizing and reducing the 5hmC bases (no affinity enrichment is performed after contacting with a metal complex and before contacting with borane reducing agent) (pg. 430 column 2 2nd full paragraph lines 1-7; pg. 430 column 2 3rd full paragraph lines 1-7; pg. 430 column 2 4th full paragraph lines 1-4; pg. 430 6th full paragraph lines 1-5).
Regarding claim 9, Liu teaches no affinity enrichment is performed in oxidizing and reducing the 5hmC bases (no affinity enrichment is performed) (pg. 430 column 2 2nd full paragraph lines 1-7; pg. 430 column 2 3rd full paragraph lines 1-7; pg. 430 column 2 4th full paragraph lines 1-4; pg. 430 6th full paragraph lines 1-5).
Regarding claim 39, Liu teaches the borane reducing agent is 2-picoline-borane or pyridine borane (pg. 430 column 2 3rd full paragraph lines 1-7; pg. 430 column 2 4th full paragraph lines 1-4).
Regarding claim 41, Liu teaches detecting the oxidized and reduced nucleic acid (processed nucleic acid) with restriction enzyme digestion and Sanger sequencing (chain termination sequencing) (pg. 425-426 paragraph bridging pg. 425 & pg. 426 lines 6-11).
Regarding claim 42, Liu teaches the genomic DNA is from mouse embryonic stem cells (source nucleic acid is provided as part of a sample) (pg. 425-426 paragraph bridging pg. 425 & pg. 426 lines 15-16). Zeng teaches that the genomic DNA is from human embryonic stem cells (source nucleic acid is provided as part of a sample) (pg. 13191 column 1 1st full paragraph lines 10-13).
Regarding claim 43, Zeng teaches that the genomic DNA is from human embryonic stem cells (sample is derived from a human) (pg. 13191 column 1 1st full paragraph lines 10-13).
Regarding claims 44 & 45, Liu teaches the genomic DNA is from mouse embryonic stem cells (source nucleic acid is genomic DNA) (pg. 425-426 paragraph bridging pg. 425 & pg. 426 lines 15-16). Zeng teaches that the genomic DNA is from human embryonic stem cells (source nucleic acid is genomic DNA) (pg. 13191 column 1 1st full paragraph lines 10-13).
Regarding claim 47, Liu teaches a bisulfite-free method for detection of 5-hydroxymethylcytosine (5hmC) in which genomic DNA (source nucleic acid) is oxidized with TET enzymes or potassium perruthenate (KRuO4) (a metal oxide) to oxidize 5hmC to 5fC (produce an oxidized nucleic acid wherein the 5hmC bases in the nucleic acid are converted to 5fC) (abstract lines 6-11; pg. 425 paragraph bridging column 1 & 2 lines 1-5 & 10-27; pg. 430 6th full paragraph lines 1-5). Liu also teaches DNA was oxidized with mTet1CD twice (an oxidizing agent) and then reduced with pyridine borane (one or more additional steps of contacting the oxidized nucleic acid with oxidizing agent) (pg. 431 column 1 4th full paragraph lines 5-6).
Liu does not teach that the 5hmC bases are oxidized with a metal (VI) oxo complex or that the one or more additional steps of contacting the oxidized nucleic acid with a metal (VI) complex.
Zeng teaches a method for bisulfite-free analysis of 5-hydroxymethylcytosine (5hmC) in which genomic DNA 5hmC (source nucleic acid) is oxidized with potassium ruthenate (K2RuO4), a ruthenium (+6) oxidant (metal (VI) oxo complex) to convert 5hmC to 5fC (pg. 13191 column 1 1st full paragraph lines 1-10). In addition, Zeng teaches that potassium ruthenate (K2RuO4) is more oxidative that the reported oxidant potassium perruthenate (KRuO4) and that while potassium perruthenate (KRuO4) caused severe DNA degradation, potassium ruthenate (K2RuO4)-mediated oxidation is very mild but also complete and can efficiently convert 5hmC to 5fC (pg. 13191 column 1 1st full paragraph lines 1-14).
Liu and Zeng are considered to be analogous to the claimed invention because they are all in the same field of bisulfite-free analysis of 5hmC. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method oxidizing 5hmC with TET enzymes or potassium perruthenate (KRuO4) (a metal oxide) to oxidize 5hmC to 5fC and to have modified the method oxidizing 5hmC with TET enzymes twice to oxidize 5hmC to 5fC before reducing with pyridine borane in Liu to incorporate the use of potassium ruthenate (K2RuO4) to oxidize 5hmC to 5fC as taught in Zeng because Zeng teaches that doing so would provide a method that can completely and efficiently oxidize and convert 5hmC to 5fC in a milder method that does not cause severe DNA degradation.
Claim(s) 46 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al.; Nature Biotechnology Letters, Vol. 37, pages 424-434, February 25th, 2019), as cited on the IDS dated 03/28/2023, and Zeng (Zeng et al.; Journal of the American Chemical Society, Vol. 140, pages 13190-13194, October 2018), as cited on the IDS dated 03/28/2023 as applied to claims 1-10, 39, 41, 42-45, & 47 above, and further in view of Song (WO 2019/136413 A1, published July 11th, 2019), as cited on the IDS dated 03/28/2025.
The teachings of Liu and Zeng with respect to claim 1 is discussed above.
Regarding claim 46, Liu and Zeng do not teach that the source nucleic acid is RNA.
Song teaches a method for identifying 5mC or 5hmC in a target nucleic acid comprising providing a nucleic acid sample with the target nucleic acid, converting 5mC and 5hmC to 5caC and/or 5fC by contacting with potassium perruthenate (KRuO4) (oxidizing the sample), and then converting the 5caC and/or 5fC to DHU through reduction with a borane reducing agent to modify the target nucleic acid (processed nucleic acid) in which the nucleic acid can be RNA (paragraph [0013] lines 1-16; paragraph [0014] lines 3-4; paragraph [0074] lines 1-13; paragraph [0075] lines 1-7; paragraph [0076] lines 1-3). In addition, Song teaches this method for determining 5mC and 5hmC nucleic acids is important and valuable in clinical applications for diagnosis and therapy for aberrant hydroxymethylation associated with various diseases and cancer (paragraph [0003] lines 7-11).
Liu, Zeng, and Song are considered to be analogous to the claimed invention because they are all in the same field of bisulfite-free analysis of 5hmC. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method oxidizing and reducing 5hmC (producing a processed nucleic acid) in Liu to incorporate the use of a source nucleic acid of RNA as taught in Song because Song teaches that doing so would provide a method of producing a processed target nucleic acid that would be important in clinical applications for diagnosis and therapy for diseases and cancers characterized by hydroxymethylation.
Response to Arguments
The response traverses the rejection. The response asserts that the second contacting step with a metal (IV) oxo complex is not taught or suggested in Liu or Zeng. The response further asserts that the claims as amended require contacting the DNA twice with a metal (IV) oxo complex as opposed to mTet1CD and that Liu does not teach recontacting the already metal (IV) oxo complex-oxidized DNA with a metal (IV) oxo complex. Further, the response asserts that Zeng teaches that contacting DNA with potassium ruthenate would completely and efficiently oxidize and convert 5hmC to 5fC in a milder method that does not cause severe DNA degradation and that if this were the case a second contacting step as required in the claims as amended would not be needed and thus the combination of Liu and Zeng fails to teach each element of the claims as amended and as a result there is no prima facie case of obviousness. These arguments have been thoroughly reviewed but were not found persuasive. First, in response to applicant's arguments against the references individually, 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). Second, Liu teaches a bisulfite-free method for detection of 5-hydroxymethylcytosine (5hmC) in which genomic DNA (source nucleic acid) is oxidized with TET enzymes or potassium perruthenate (KRuO4) (a metal oxide) to oxidize 5hmC to 5fC (produce an oxidized nucleic acid), in which DNA was oxidized with mTet1CD twice (an oxidizing agent), and then contacting the oxidized 5fC (oxidized nucleic acid) with pyridine and pic-borane (borane reducing agents) to convert 5fC to dihydrouracil (DHU) (abstract lines 6-11; pg. 424-425 paragraph bridging pg. 424 & pg. 425 lines 14-22; pg. 425 paragraph bridging column 1 & 2 lines 1-5 & 10-27; pg. 425-426 paragraph bridging pg. 425 & pg. 426 lines 4-26; pg. 430 column 2 2nd full paragraph lines 1-7; pg. 430 column 2 3rd full paragraph lines 1-7; pg. 430 column 2 4th full paragraph lines 1-4; pg. 430 6th full paragraph lines 1-5). Zeng teaches a method for bisulfite-free analysis of 5-hydroxymethylcytosine (5hmC) in which genomic DNA 5hmC (source nucleic acid) is oxidized with potassium ruthenate (K2RuO4), a ruthenium (+6) oxidant (metal (VI) oxo complex) to convert 5hmC to 5fC (pg. 13191 column 1 1st full paragraph lines 1-10). In addition, Zeng teaches that potassium ruthenate (K2RuO4) is more oxidative that the reported oxidant potassium perruthenate (KRuO4) and that while potassium perruthenate (KRuO4) caused severe DNA degradation, potassium ruthenate (K2RuO4)-mediated oxidation is very mild but also complete and can efficiently convert 5hmC to 5fC (pg. 13191 column 1 1st full paragraph lines 1-14), therefore highlighting that potassium ruthenate (K2RuO4) can more efficiently convert 5hmC to 5fC without causing severe DNA degradation as compared to other oxidizing metals. Therefore, the combination and Liu and Zeng teaches each element of the claims as amended and it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method oxidizing 5hmC with TET enzymes or potassium perruthenate (KRuO4) (a metal oxide) to oxidize 5hmC to 5fC and to have modified the method oxidizing 5hmC with TET enzymes twice to oxidize 5hmC to 5fC before reducing with pyridine borane in Liu to incorporate the use of potassium ruthenate (K2RuO4) to oxidize 5hmC to 5fC as taught in Zeng because Zeng teaches that doing so would provide a method that can completely and efficiently oxidize and convert 5hmC to 5fC in a milder method that does not cause severe DNA degradation.
The response also asserts that the application as filed teaches that multiple rounds of oxidation with a metal (IV) oxo complex, among other things, improves reaction efficiency and provides a more complete conversion which is in direct contrast with Zeng that treatment with potassium ruthenate would completely and efficiently oxidize and convert 5hmC to 5fC in a milder method that does not case severe DNA degradation and thus a person of ordinary skill in the art would have no reasonable expectation that using multiple rounds of oxidation specifically with a metal (IV) oxo complex would result in an improved method compared to the combined prior art references, with inter alia an improved reaction efficiency and a more complete conversion. These arguments have been thoroughly reviewed but were not found persuasive. First, in response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., multiple rounds of oxidation with a metal (IV) oxo complex improves reaction efficiency and a more complete conversion) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Further, Zeng teaches that potassium ruthenate (K2RuO4) is more oxidative that the reported oxidant potassium perruthenate (KRuO4) and that while potassium perruthenate (KRuO4) caused severe DNA degradation, potassium ruthenate (K2RuO4)-mediated oxidation is very mild but also complete and can efficiently convert 5hmC to 5fC (pg. 13191 column 1 1st full paragraph lines 1-14), therefore highlighting that potassium ruthenate (K2RuO4) can more efficiently convert 5hmC to 5fC and offer a more complete conversion without causing severe DNA degradation as compared to other oxidizing metals.
For these reasons, and the reasons already made of record and modified to address the claims as currently amended, the rejections are maintained and applied to the newly amended claims.
Conclusion
Claims 1-9, 39, & 41-47 are rejected.
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 BAILEY C BUCHANAN whose telephone number is (703)756-1315. The examiner can normally be reached Monday-Friday 8:00am-5:00pm ET.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached on (571) 272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/BAILEY BUCHANAN/Examiner, Art Unit 1682
/JEHANNE S SITTON/Primary Examiner, Art Unit 1682
Prosecution Insights