DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
2. 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.
3. 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.
8. Claims 49-54, 62 and 92-93 are rejected under 35 U.S.C. 103 as being unpatentable over Graham et al (Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RAD-Seq), Molecular Ecology Resources, 15, 1304-1315, published 2015-03-16) in view of Booth et al (Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution, Science, 336, 934-937, published 2012-05-18).
Regarding claims 49 and 53, Graham teaches a method of preparing a library of nucleic acids (abstract) comprising: fragmenting DNA with first restriction enzymes (pg 1306, column 1, ¶ 1), ligating adapters to the DNA fragments with a ligase (pg 1306, column 1, ¶ 2), and digesting the resulting mixture of library-adapter and adapter-adapter DNAs with a restriction enzyme that only cleaves the adapter dimers (pg 1306, column 1, ¶ 1). Graham specifically teaches that in this mixture DNA chimeras and adapter dimers are cleaved (pg 1306, column 1, ¶ 1). Graham additionally teaches amplifying the adapter-ligated DNA fragments to produce amplified adapter-ligated DNA fragments (pg 1306, column 1, ¶ 2-3).
Graham does not teach subjecting the adapter-ligated DNA fragments to conditions sufficient to permit distinction between methylated nucleic acid bases and unmethylated nucleic acid bases within the adapter-ligated fragments by sequencing.
However, Booth teaches a method for the base-pair resolution detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in adapter ligated DNA fragments by bisulfite sequencing (abstract and SI pg 3 ¶ 7). This method converts C to U (read as T in sequencing) but leaves 5mC unchanged (i.e., permits the distinction between methylated and unmethylated nucleic acid bases; FIG. 1B)
It would have been obvious to one having ordinary skill in the art to have modified the DNA library preparation taught by Graham with the 5-mC and 5-hmC bisulfite conversion taught by Booth to arrive at the instantly claimed invention with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to make this modification in order to identify epigenetic information present in the library taught by Graham. In addition, it would have been obvious to the ordinary artisan that the known techniques in the cited references could have been combined with predictable results because the known techniques of the cited references predictably result in the processing of adapter-ligated libraries for nucleic acid sequencing experiments.
Regarding claim 50, Graham teaches MspI is used to fragment DNA (pg 1306, column 1, ¶ 1).
Regarding claim 51, Graham teaches that ClaI is used to cleave adapter dimers (pg 1306, column 1, ¶ 1).
Regarding claim 52, Graham teaches that their method allows for the restriction digestion and ligation reactions to be performed simultaneously (pg, column 1, 1306 ¶ 1).
Regarding claim 54, Booth teaches that the adapter-ligated fragments are subjected to bisulfite conversion (abstract and SI pg 3 ¶ 7).
Regarding claim 62, Graham teaches that the adapter comprises a GC overhang (see ClaI upper and lower oligos in table S1)
Regarding claim 92, Graham teaches that the amplification reaction (i.e., step d) is performed with a primer that does not bind a junction between the end of the DNA fragment and the adapter, but does not bind a junction between the end of one adapter and the end of another adapter (See table S1 iTru5_01).
Regarding claim 93, Graham teaches digesting the mixture of library-adapter and adapter-adapter DNAs with a restriction enzyme that only cleaves the adapter dimers (pg 1306, column 1, ¶ 1). It is noted that the limitations of claim 93 do not specifically require the cleavage of the “adapter dimer by design” nor does it require any additional method steps not present in claim 49. It is also noted that the adapter EcoRI-1 (Table S1) comprises a DpnI restriction site, therefore EcoRI-1 is an adapter dimer by design and meets all the limitations required by claim 93.
4. Claims 49, 53, 55-58 and 60-61 are rejected under 35 U.S.C. 103 as being unpatentable over Graham et al (Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RAD-Seq), Molecular Ecology Resources, 15, 1304-1315, published 2015-03-16) in view of Liu et al (Bisulfite-free direct detection of 5-metylcytosine and 5-hydroxmethylcytosine at base resolution, Nature Biotechnology, 37, 424-429, published 2019-02-25).
Regarding claims 49 and 53, Graham teaches a method of preparing a library of nucleic acids (abstract) comprising: fragmenting DNA with first restriction enzymes (pg 1306, column 1, ¶ 1), ligating adapters to the DNA fragments with a ligase (pg 1306, column 1, ¶ 2), and digesting the resulting mixture of library-adapter and adapter-adapter DNAs with a restriction enzyme that only cleaves the adapter dimers (pg 1306, column 1, ¶ 1). Graham specifically teaches that in this mixture DNA chimeras and adapter dimers are cleaved (pg 1306, column 1, ¶ 1). Graham additionally teaches amplifying the adapter-ligated DNA fragments to produce amplified adapter-ligated DNA fragments (pg 1306, column 1, ¶ 2-3).
Graham does not teach subjecting the adapter-ligated DNA fragments to conditions sufficient to permit distinction between methylated nucleic acid bases and unmethylated nucleic acid bases within the adapter-ligated fragments by sequencing.
However, Liu teaches a method for the base-resolution detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in adapter ligated DNA fragments by bisulfite sequencing (abstract and methods section – cell-free DNA TAPS).
It would have been obvious to one having ordinary skill in the art to have modified the DNA library preparation taught by Graham with the 5-mC and 5-hmC bisulfite conversion taught by Liu to arrive at the instantly claimed invention with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to make this modification in order to identify epigenetic information present in the library taught by Graham. In addition, it would have been obvious to the ordinary artisan that the known techniques in the cited references could have been combined with predictable results because the known techniques of the cited references predictably result in the processing of adapter-ligated libraries for nucleic acid sequencing experiments.
Regarding claim 55-57 and 60, Liu teaches that adapter ligated fragments are oxidized with mTet1CD and then reduced with pyridine borane (methods – cell-free DNA TAPS; pg 425 columns 1-2 and FIG 1).
Regarding claims 58 and 61, Liu teaches that 5-hmC is converted to 5-formylCytosine using potassium perruthenate (FIG 1 and pg 425, column 1, ¶ 2) and that 5-hmC can be labeled with glucose by β-glucosyltransferase to protect it from TET oxidation (FIG 1 and pg 425, column 1, ¶ 2).
5. Claim 59 is rejected under 35 U.S.C. 103 as being unpatentable over Graham et al (Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RAD-Seq), Molecular Ecology Resources, 15, 1304-1315, published 2015-03-16) in view of Liu et al (Bisulfite-free direct detection of 5-metylcytosine and 5-hydroxmethylcytosine at base resolution, Nature Biotechnology, 37, 424-429, published 2019-02-25) as applied to claim 56 above, and further in view of Schutsky et al (Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase, Nature Biotechnology, 36(11), 1083-1090, published 2019-10-08).
Regarding claim 59, the method of claim 56 is discussed above and incorporated fully here.
Neither Graham nor Liu teach the reduction or deamination of oxidative reaction products with APOBEC.
However, Schutsky teaches a method wherein unmethylated cytosine (C) and 5-methylcytosine (5-mC) are discriminated from ox-mC adducts using APOBEC3A (i.e., an APOBEC protein, pg 1084, column 1, ¶ 1; FIG 1).
It would have been obvious to one having ordinary skill in the art to have simply substituted the borane reduction taught by Liu with APOBEC3A as taught by Schutsky to arrive at the instantly claimed invention with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to make this substitution in order to replace chemical reagents in the sequencing workflow with enzymatic alternatives, which are generally known to be safer and more environmentally friendly reagents. In addition, it would have been obvious to the ordinary artisan that the known techniques of the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the reductive deamination of select modified or unmodified cytosine bases.
6. Claim 91 is rejected under 35 U.S.C. 103 as being unpatentable over Graham et al (Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RAD-Seq), Molecular Ecology Resources, 15, 1304-1315, published 2015-03-16) in view of Booth et al (Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution, Science, 336, 934-937, published 2012-05-18) and further in view of Hunkapiller et al (US Patent No. US 6258359 B1, published 2001-07-10).
Regarding claim 91, the method of claim 49 is discussed fully above and incorporated here.
Neither Graham nor Booth teach amplifying adapter-ligated fragments using a primer that binds a junction between the end of the DNA fragment and the adapter, but does not bind a junction between the end of one adapter and the end of another adapter.
However, Hunkapiller teaches the amplification of a subset of an adapter-modified restriction fragments (i.e., adapter-ligated DNA fragments) using a “selective primer” that is complementary to the junctions between the adapter and restriction fragments (FIG 7 and description; pg 11, column 2, lines 46-65).
It would have been obvious to one having ordinary skill in the art to have replaced the ClaI primer taught by Graham with a “selective primer,” as taught by Hunkapiller, to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution because Graham states that only the phosphorylated primers can self-ligate to form dimers (pg 1306, column 1, ¶ 1) therefore a primer selective for the fragment-adapter junction would not bind the ClaI adapter-adapter junction. This would provide the additional advantage of another layer of adapter-dimer discrimination during library preparation. A person having ordinary skill in the art would have recognized that the known techniques of the cited methods could have been combined with predictable results because the known techniques of the cited methods predictably result in the amplification of adapter-ligated restriction fragments.
7. Claim 94 is rejected under 35 U.S.C. 103 as being unpatentable over Graham et al (Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RAD-Seq), Molecular Ecology Resources, 15, 1304-1315, published 2015-03-16).
Regarding claim 94, the method of claim 49 is discussed fully above and incorporated here.
Graham does not specifically teach that PCR amplification of the adapter-ligated fragments produces amplified adapter dimers that are degraded by one or more restriction enzymes.
However, Graham does teach the digestion of adapter dimers by restriction enzyme digestion (pg 1206, column 1, ¶ 1). It is noted that claim 94 does not specifically require that the “third one or more” restriction enzymes are different than the “second one or more” restriction enzymes from claim 49. One having ordinary skill in the art would have known that enzymatic degradation steps rarely go 100% to completion, therefore it would have been obvious to the ordinary artisan to have performed the digestion steps described by Graham as many times as desired or needed, after any of the claimed method steps, in order to reduce the amount of adapter dimer contamination before sequencing. It is further noted that the MPEP states in § 2144.04 that a mere duplication of steps has no patentable significance unless a new an unexpected result is produced.
Response to Arguments
8. Applicant's arguments filed 11 August 2025 have been fully considered but they are not persuasive.
All previous rejections not repeated in this Office Action have been overcome by amendments to the claims.
Applicant argues that Graham does not teach digesting the mixture of adapter-ligated DNA fragments and adapter dimers, however, as discussed in the rejection above Graham clearly states that their strategy “allows for the restriction digestion and ligation to be performed simultaneously… because DNA chimeras and adapter dimers are cleaved.” Applicant’s amendment adding step d to independent claim 49 overcomes the previous rejection under 35 U.S.C. 102, however this amendment is rejected under 35 U.S.C. 103 as discussed fully above and incorporated here.
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.
Conclusion
9. No claims are allowed.
10. Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN ELLIS YOUNG whose telephone number is (703)756-5397. The examiner can normally be reached M-F 0730 - 1700.
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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/JULIET C SWITZER/Primary Examiner, Art Unit 1682