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 canceled claims 1-9, 13-17 and 20. Claims 10-12, 18-19 and 21-23 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 Any objection or rejection of record in the previous Office Action, which is not addressed in this action has been withdrawn in light of Applicant’s amendments and/or arguments.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 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.
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 10-11, 18-19 and 21-23 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Translated People’s Republic of China Application Publication CN 109554451 A, published April 02, 2019), cited on the IDS filed December 08, 2021, and Toro et al. (United States Patent 9,677,067 B2, published June 13, 2017), previously cited in the 07/14/2025 Office Action, in view of Adey et al. (U.S. Patent Application Publication US 2018/0355348 A1, published December 13, 20218), previously cited in the 07/14/2025 Office Action. This is a new rejection as necessitated by amendments.
Regarding claim 10, Li teaches a method for determining a methylation state of a DNA sample (Page 6, Last Paragraph). Li teaches constructing a sequencing library based on the DNA sample (Abstract). Li teaches sequencing the sequencing library to obtain sequencing results of the DNA sample (Abstract). Li teaches aligning the sequencing results of a 5'-end and a 3'-end of the DNA sample respectively with a reference genome to determine position information of the 5'-end and the 3'-end (i.e., GET- Genome and Epigenome Tracking, Page 3, Whole Page). Li teaches analyzing a position of the DNA sample by comparison based on the position information of the 5'-end and the 3'-end to determine the methylation state of the DNA sample (Page 3, Last Paragraph). Li teaches using polymerase, dATP, dTTP, dGTP, and methylated dCTP to obtain a hybrid DNA (Page 4, Second to Last Paragraph and Page 3, GET-Step 3). Li teaches a 5'-end sequence of each of the two strands is an original sequence of the DNA sample (Page 6, Last Paragraph). Li teaches all C bases at the 3'-end sequence of each of the two strands are methylated (Figure 1). Li teaches subjecting the hybrid DNA to a bisulfite treatment to obtain a converted hybrid DNA and amplifying the converted hybrid DNA to obtain the sequencing library (Abstract).
Regarding claim 11, Li teaches aligning the sequencing results of a 5'-end and a 3'-end of the DNA sample respectively with a reference genome to determine position information of the 5'-end and the 3'-end (i.e., GET- Genome and Epigenome Tracking, Page 3, Whole Page). and Page 6, Last Paragraph). Li teaches the 3'-end corresponds to multiple candidate positions, the 5'-end corresponds to multiple candidate positions, determining the position information of the 5'-end and the 3'-end based on a common optimal candidate position of the 5'-end and the 3'-end (i.e., GET- Genome and Epigenome Tracking, Page 3, Whole Page). and Page 6, Last Paragraph). Li teaches the 3'-end corresponds to one candidate position, the 5'-end corresponds to multiple candidate positions, and a position adjacent to the candidate position corresponding to the 3'-end is one of the multiple candidate positions corresponding to the 5'-end as well as determining the position information of the 5'-end and the 3'-end based on the candidate position corresponding to the 3'-end (i.e., GET- Genome and Epigenome Tracking, Page 3, Whole Page). and Page 6, Last Paragraph).
Regarding claim 18, Li teaches the method of claim 10 as discussed above.
Regarding claim 19, Li teaches the DNA sample has a length of 100 bp to 1000 bp (Page 3, Fourth Paragraph).
Regarding claim 20, Li teaches constructing a sequencing library based on the DNA sample (Abstract). Li teaches ligating a methylation sequencing adapter to the hybrid DNA and performing the bisulfate treatment to obtain the converted hybrid DNA (Page 5, First Paragraph—Eleventh Paragraph). Li teaches amplifying the converted hybrid DNA using primers (Page 5, Step 8- amplification).
Regarding claim 21, Li teaches the DNA sample is a whole genome DNA sample (Page 3, Last Paragraph).
Regarding claim 22, Li teaches a method for determining a methylation state of a DNA sample (Page 6, Last Paragraph). Li teaches constructing a sequencing library based on the DNA sample (Abstract). Li teaches sequencing the sequencing library to obtain sequencing results of the DNA sample (Abstract). Li teaches aligning the sequencing results of a 5'-end and a 3'-end of the DNA sample respectively with a reference genome to determine position information of the 5'-end and the 3'-end (i.e., GET- Genome and Epigenome Tracking, Page 3, Whole Page). Li teaches analyzing a position of the DNA sample by comparison based on the position information of the 5'-end and the 3'-end to determine the methylation state of the DNA sample (Page 3, Last Paragraph). Li teaches using polymerase, dATP, dTTP, dGTP, and methylated dCTP to obtain a hybrid DNA (Page 4, Second to Last Paragraph and Page 3, GET-Step 3). Li teaches a 5'-end sequence of each of the two strands is an original sequence of the DNA sample (Page 6, Last Paragraph). Li teaches all C bases at the 3'-end sequence of each of the two strands are methylated (Figure 1). Li teaches subjecting the hybrid DNA to a bisulfite treatment to obtain a converted hybrid DNA and amplifying the converted hybrid DNA to obtain the sequencing library (Abstract). Li teaches amplifying the converted hybrid DNA using specific primers to obtain a sequencing library based on a target region of the DNA sample (Page 5, Step 8 amplification). Li teaches the specific primers comprise first specific primers and second specific primers, the first specific primers are located at 5'-ends of the converted hybrid DNA, and the second specific primers are located at 3'-ends of the converted hybrid DNA (Page 5, Step 8 amplification).
Regarding claim 23, Li teaches constructing a sequencing library based on the DNA sample (Abstract). Li teaches amplifying the captured product to obtain the sequencing library (Abstract).
Li does not teach or suggest digesting the DNA sample with endonuclease to obtain a DNA sample with single-strand nicks. Li does not teach or suggest the hybrid DNA comprises two strands that are reversely complementary to each other. Li does not teach or suggest a 3'-end sequence of each of the two strands is a synthetic sequence. Li does not teach or suggest the endonuclease is Dnase I, Dnase II, or any endonuclease capable of producing the single-strand nicks. Li does not teach or suggest the methylation sequencing adapter comprises a first universal sequence and a second universal sequence. Li does not teach or suggest using universal primers to obtain the sequencing library and the universal primer matches the first universal sequence and the second universal sequence. Li does not teach or suggest hybrid capturing the converted hybrid DNA by using a probe and eluting to obtain a captured product, wherein the probe is configured to hybridize a 3'-end sequence of the converted hybrid DNA.
Toro teaches DNA methylation (Column 28, Lines 65-67). Toro teaches creating a sequencing library (Column 57, Lines 31-43). Toro teaches digesting the DNA sample with endonuclease to obtain a DNA sample with single-strand nicks (Column 5, Line 66—Column 6, Line 54). Toro teaches hybrid DNA (Column 8, Lines 47-67). Toro teaches the hybrid DNA comprises two strands that are reversely complementary to each other (Column 6, Lines 4-52). Toro teaches a 3'-end sequence of each of the two strands is a synthetic sequence (Column 44, Lines 53-67). Toro teaches using a bisulfate treatment (Column 35 Lines 42-44). Toro teaches endonucleases capable of producing the single-strand nicks (Column 34, Lines 3-16). Toro teaches using these methods allows for assembly of large nucleic acid target molecules with a high degree of sequence integrity which would be a powerful tool for basic biological research and biotechnology applications (Column 1, Lines 21-26 and Column 14, Lines 63-65).
Adey teaches whole genome libraries for methylation sequencing (Title and Abstract). Adey teaches performing the bisulfate treatment to obtain the converted hybrid DNA (Pages 7-8, [0066]). Adey teaches ligating a methylation sequencing adapter to the hybrid DNA, wherein the methylation sequencing adapter comprises a first universal sequence and a second universal sequence (Page 2, [0013]-[0014], Page 3, [0016] and Page 8, [0067]-[0068]). Adey teaches using universal primers to obtain the sequencing library and the universal primer matches the first universal sequence and the second universal sequence (Page 2, [0013], Page 3, [0016] and Page 8, [0067]-[0068]). Adey teaches amplifying the converted hybrid DNA using universal primers to obtain the sequencing library (Page 2, [0013], Page 3, [0016] and Page 8, [0067]-[0068]). Adey teaches capturing the converted hybrid DNA by using a probe and eluting to obtain a captured product (Page 1, [0003], Page 4, [0023], Page 11, [0091] and Examples 6-9). Adey teaches the probe is configured to hybridize a 3'-end sequence of the converted hybrid DNA (Page 8, [0068]). Adey teaches amplifying the captured product to obtain the sequencing library (Page 11, [0094]). Adey teaches using these methods characterizes the methylation profiles of large numbers of single cells quickly, accurately and inexpensively (Abstract).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the teachings of Li with the teachings of Toro, digesting the DNA sample with endonuclease to obtain a DNA sample with single-strand nicks, having the hybrid DNA comprising two strands that are reversely complementary to each other as well as a 3'-end sequence of each of the two strands is a synthetic sequence. Using these methods allows for assembly of large nucleic acid target molecules with a high degree of sequence integrity which would be a powerful tool for basic biological research and biotechnology applications as taught by Toro (Column 1, Lines 21-26 and Column 14, Lines 63-65).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to have modified the teachings of Li with the teachings of Adey, ligating a methylation sequencing adapter to the hybrid DNA, wherein the methylation sequencing adapter comprises a first universal sequence and a second universal sequence and using universal primers to obtain the sequencing library, the universal primer matches the first universal sequence and the second universal sequence as well as using a probe and eluting to obtain a captured product. Using these method would allow for characterization of the methylation profiles with large numbers of single cells, quickly, accurately and inexpensively, as taught by Adey (Abstract).
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Translated People’s Republic of China Application Publication CN 109554451 A, published April 02, 2019), cited on the IDS filed December 08, 2021, and Toro et al. (United States Patent 9,677,067 B2, published June 13, 2017), previously cited in the 07/14/2025 Office Action, in view of Adey et al. (U.S. Patent Application Publication US 2018/0355348 A1, published December 13, 20218), previously cited in the 07/14/2025 Office Action, as applied to claims 10-11, 18-19 and 21-23 above and evidenced by Tran et al. (“Objective and Comprehensive Evaluation of Bisulfite Short Read Mapping Tools” Adv Bioinformatics. 2014:472045, published April 15, 2014), previously cited in the 07/14/2025 Office Action. This rejection is modified and maintained as necessitated by amendments.
Regarding claim 12, Li teaches aligning with a reference genome as discussed above.
Li does not teach or suggest the 3'-end is aligned with the reference genome using BWA software, and the 5'-end is aligned with the reference genome using BS-map software.
Tran teaches DNA methylation and bisulfite conversion as well as aligning bisulfite reads to a reference genome (Page 1, Right Column, First Paragraph). Tran teaches using BSMAP and BWA software to map reads to a reference genome is routine (Page 2, Left Column, First Paragraph). Tran teaches that different parameters (i.e., choosing which end to align, 3’ or 5’ end) might impact program performance (Page 2, Left Column, First Paragraph).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date to align the 3’-end with a reference genome using BWA software and aligning the 5’end with a reference genome using BS-map software because Tran teaches that using those two software to map reads is routine in bisulfite sequencing. Additionally, Trans teaches that adjusting parameters might impact program performance, which would allow one to determine which software to use and to choose which specific end would be aligned.
Response to Arguments
Applicant’s arguments and amendments filed October 14, 2025 with respect to the rejections under 35 U.S.C. 101 have been fully considered and are persuasive. Therefore these
rejections have been withdrawn.
Applicant’s arguments and amendments filed October 14, 2025 with respect to the rejections under 35 U.S.C. 103 have been fully considered but are not deemed to be persuasive.
Applicant asserts that amended claim 10, “differ from Li at least by: (a) digesting the DNA sample with endonuclease to obtain a DNA sample with single-strand nicks; (b) said ligating a methylation sequencing adapter comprising a first universal sequence and a second universal sequence to the hybrid DNA is performed prior to the bisulfite treatment, the universal primer matching the first universal sequence and the second universal sequence. Specifically, Li discloses that the 3'-end of the DNA fragment is subjected to a digestion treatment using a DNA exonuclease in step 2). That is, Li teaches away … those skilled in the art would not consider replacing the DNA exonuclease in step 2) with an endonuclease to form a DNA sample with single-strand nicks”. Applicant additionally asserts “that neither Toro nor Adey disclose the above limitations and thus they cannot remedy the deficiencies of Li.
While it is acknowledged that Li does in fact disclose using “preferably an exonuclease”, Li additionally discloses using in general DNA excision of the DNA fragment of the 3 ' tail end by crushing, ultrasonic and enzyme cutting processing (e.g., an endonuclease may be an enzyme capable of an enzyme cutting process, Abstract and Step 2 of Genome and Epigenome Tracking). Moreover the MPEP states “the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed…." In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004)(MPEP 2145(D)(1). Additionally, 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). While it is acknowledged Li does not explicitly disclose use of an endonuclease, as discussed above, Toro is cited for disclosing digesting the DNA sample with endonuclease to obtain a DNA sample with single-strand nicks (Column 5, Line 66—Column 6, Line 54). Toro further discloses using these methods allows for assembly of large nucleic acid target molecules with a high degree of sequence integrity which would be a powerful tool for basic biological research and biotechnology applications (Column 1, Lines 21-26 and Column 14, Lines 63-65). Additionally, as discussed above, Adey, is cited for the disclosure of ligating a methylation sequencing adapter to the hybrid DNA, wherein the methylation sequencing adapter comprises a first universal sequence and a second universal sequence as well as using universal primers to obtain the sequencing library and the universal primer matches the first universal sequence and the second universal sequence. Adey discloses that using these method would allow for characterization of the methylation profiles with large numbers of single cells, quickly, accurately and inexpensively (Abstract).
Therefore, for all these reasons, and those listed above, Li and Toro in view of Adey as well as Li and Toro in view of Adey as evidenced by Tran 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