Don’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 .
Election/Restrictions
Applicant’s election without traverse of the species elections of copper(II) perchlorate and 2,2,6,6,-tetramethylpiperidine-1-oxyl (Cu(II)/TEMPO) in claims 125, 126, 136, 152, 158, 171, 187, 188, 198, 214, 220, & 233 and of 2-amino-5-methoxybenzoic acid in claims 154 & 216 in the reply filed on 02/06/2026 is acknowledged.
A first office action on the merits of claims 125, 136-155, 171-187, 198-217, 233-248 is set forth herein.
Information Disclosure Statement
The listing of references in the specification in pg. 75-77 is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 152-155, 171-186, & 233-235 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 152, the recitation of “wherein the malononitrile converts 5fC to 5fC-malononitrile adduct (55fC-M adduct), thereby producing a nucleic acid comprising one or more 5fC-M adduct” in lines 11-12 of the claim is unclear if (55fC-M adduct) is meant to refer to a different type of malononitrile adduct than 5fC-M adduct or if this is the result of a typo and should read “wherein the malononitrile converts 5fC to 5fC-malononitrile adduct (5fC-M adduct), thereby producing a nucleic acid comprising one or more 5fC-M adduct”.
Regarding claim 171, the recitation of “wherein the target nucleic acid is contacted with Cu(II)/TEMPO in step (b)” in lines 1-2 of the claim is unclear as claim 152, from which claim 171 depends from, recites “the target nucleic acid” in step (a) of claim 152 and step (b) of claim 152 recites “the nucleic acid comprising one or more 5hmC of step (a)”, therefore it is not clear if “the nucleic acid comprising one or more 5hmC of step (a)” is contacted with Cu(II)/TEMPO in step (b) or if “the target nucleic acid” is contacted with Cu(II)/TEMPO in step (a) in claim 171.
Regarding claim 233, the recitation of “wherein the target nucleic acid is contacted with Cu(II)/TEMPO in step (b)” in lines 1-2 of the claim is unclear as claim 214, from which claim 233 depends from, recites “the target nucleic acid” in step (a) of claim 1214 and step (b) of claim 214 recites “the nucleic acid comprising one or more 5hmC of step (a)”, therefore it is not clear if “the nucleic acid comprising one or more 5hmC of step (a)” is contacted with Cu(II)/TEMPO in step (b) or if “the target nucleic acid” is contacted with Cu(II)/TEMPO in step (a) in claim 233.
Claims 153-155 & 174-186 are rejected due to their dependence on claim 152, claims 172 & 173 are rejected due to their dependence on claim 171, and claims 234 & 235 are rejected due to their dependence on claim 233.
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.
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.
Claim(s) 125, 136, 145, 150, 152, 171, 180, 185, 187, 198, 207, 212, 214, 233, 242, & 247 is/are rejected under 35 U.S.C. 103 as being unpatentable over Matsushita (Matsushita et al.; Chemical Communications, Vol. 53, pages 5756-5759 & S1-S13, May 2017), as cited on the IDS dated 08/04/2023, in view of Zhu (Zhu et al.; Cell Stem Cell, Vol. 20, pages 720-747, March 2017), as cited in the IDS dated 08/04/2023.
Regarding claim 125, Matsushita teaches a method of Cu(II)/TMEPO oxidation comprising contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the target nucleic acid with Cu(II)/TEMPO thereby producing a nucleic acid comprising 5fC) and further detecting T signals at 5hmC sites with a sequencing assay (producing a nucleic acid comprising one or more T and sequencing the nucleic acid comprising one or more T, wherein if a T is detected at a position in the nucleic acid comprising one or more T where a 5hmC was originally present in the target nucleic acid then 5hmC has been detected in the target nucleic acid) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Matsushita does not teach contacting the nucleic acid with one or more 5fC with malononitrile or contacting the nucleic acid comprising one or more 5fC-M adduct with a polymerase.
Zhu teaches a method of single-cell, single-base resolution 5fC-sequencing comprising contacting 5fC with malononitrile to form 5fC-M adduct (contacting the nucleic acid comprising one or more 5fC with malononitrile wherein the malononitrile converts 5fC to 5fC-M adduct, thereby producing one or more 5fC-M adduct) and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing (contacting the nucleic acid comprising 5fC-M adduct with a polymerase, wherein the polymerase converts 5fC-M adduct to T, thereby producing a nucleic acid comprising one or more T and then sequencing the nucleic acid comprising one or more T) (abstract lines 1-12; pg. 721 column 2 1st full paragraph lines 1-11; pg. 721-722 paragraph bridging pg. 721 & 722 lines 1-28; pg. 722 paragraph bridging column 1 & 2 lines 1-27; pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11). Zhu also teaches that treating 5fC with malononitrile enables a high C-to-T conversion rate and that this method shows both high sensitivity and high specificity (pg. 722 paragraph bridging column 1 & 2 lines 1-27).
Matsushita and Zhu are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC and further detecting T signals at 5hmC sites with a sequencing assay in Matsushita to incorporate the contacting of 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the C-to-T conversion with PCR and sequencing as taught in Zhu because Zhu teaches that doing so would provide a method to enable a high C-to-T conversion rate with both high sensitivity and high specificity.
Regarding claim 136, Matsushita teaches contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the target nucleic acid with Cu(II)/TEMPO thereby producing a nucleic acid comprising 5fC) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Regarding claim 145, Matsushita teaches contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (producing a nucleic acid comprising one or more 5fC) and further, after oxidation, contacting the sample comprising one or more 5fC with sodium hydroxide (an additional step between step (a) and (b) comprising contacting the nucleic acid comprising one or more 5fC with NaOH) (pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-1).
Regarding claim 150, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (c) of contacting 5fC-M adduct with a polymerase to convert to T occurs in a reaction mixture comprising a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Regarding claim 152, Matsushita teaches a method of Cu(II)/TMEPO oxidation comprising the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample (contacting the target nucleic acid with TET, wherein TET converts 5mC to 5hmC, thereby producing a nucleic acid with one or more 5hmC), contacting 5hmC DNA (nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the nucleic acid comprising one or more 5hmC with Cu(II)/TEMPO thereby producing a nucleic acid comprising 5fC) and further detecting T signals at 5hmC sites with a sequencing assay (producing a nucleic acid comprising one or more T and sequencing the nucleic acid comprising one or more T, wherein if a T is detected at a position in the nucleic acid comprising one or more T where a 5hmC was originally present in the target nucleic acid then 5hmC has been detected in the target nucleic acid) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Matsushita does not teach contacting the nucleic acid with one or more 5fC with malononitrile or contacting the nucleic acid comprising one or more 5fC-M adduct with a polymerase.
Zhu teaches a method of single-cell, single-base resolution 5fC-sequencing comprising contacting 5fC with malononitrile to form 5fC-M adduct (contacting the nucleic acid comprising one or more 5fC with malononitrile wherein the malononitrile converts 5fC to 5fC-M adduct, thereby producing one or more 5fC-M adduct) and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing (contacting the nucleic acid comprising 5fC-M adduct with a polymerase, wherein the polymerase converts 5fC-M adduct to T, thereby producing a nucleic acid comprising one or more T and then sequencing the nucleic acid comprising one or more T) (abstract lines 1-12; pg. 721 column 2 1st full paragraph lines 1-11; pg. 721-722 paragraph bridging pg. 721 & 722 lines 1-28; pg. 722 paragraph bridging column 1 & 2 lines 1-27; pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11). Zhu also teaches that treating 5fC with malononitrile enables a high C-to-T conversion rate and that this method shows both high sensitivity and high specificity (pg. 722 paragraph bridging column 1 & 2 lines 1-27).
Matsushita and Zhu are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample, contacting 5hmC DNA with Cu(II)/TEMPO to produce 5fC, and further detecting T signals at 5hmC sites with a sequencing assay in Matsushita to incorporate the contacting of 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the C-to-T conversion with PCR and sequencing as taught in Zhu because Zhu teaches that doing so would provide a method to enable a high C-to-T conversion rate with both high sensitivity and high specificity.
Regarding claim 171, Matsushita teaches contacting 5hmC DNA (nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the target nucleic acid with Cu(II)/TEMPO) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Regarding claim 180, Matsushita teaches contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (producing a nucleic acid comprising one or more 5fC) and further, after oxidation, contacting the sample comprising one or more 5fC with sodium hydroxide (an additional step between step (b) and (c) comprising contacting the nucleic acid comprising one or more 5fC with NaOH) (pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-1).
Regarding claim 185, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (d) of contacting 5fC-M adduct with a polymerase to convert to T occurs in a reaction mixture comprising a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Regarding claim 187, Matsushita teaches a method of Cu(II)/TMEPO oxidation comprising contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the target nucleic acid with Cu(II)/TEMPO thereby producing a nucleic acid comprising 5fC) and further detecting T signals at 5hmC sites with a sequencing assay (producing a nucleic acid comprising one or more T and sequencing the nucleic acid comprising one or more T, wherein the 5hmC in a target nucleic acid has been converted to T) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Matsushita does not teach contacting the nucleic acid with one or more 5fC with malononitrile or contacting the nucleic acid comprising one or more 5fC-M adduct with a polymerase.
Zhu teaches a method of single-cell, single-base resolution 5fC-sequencing comprising contacting 5fC with malononitrile to form 5fC-M adduct (contacting the nucleic acid comprising one or more 5fC with malononitrile wherein the malononitrile converts 5fC to 5fC-M adduct, thereby producing one or more 5fC-M adduct) and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing (contacting the nucleic acid comprising 5fC-M adduct with a polymerase, wherein the polymerase converts 5fC-M adduct to T, thereby producing a nucleic acid comprising one or more T) (abstract lines 1-12; pg. 721 column 2 1st full paragraph lines 1-11; pg. 721-722 paragraph bridging pg. 721 & 722 lines 1-28; pg. 722 paragraph bridging column 1 & 2 lines 1-27; pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11). Zhu also teaches that treating 5fC with malononitrile enables a high C-to-T conversion rate and that this method shows both high sensitivity and high specificity (pg. 722 paragraph bridging column 1 & 2 lines 1-27).
Matsushita and Zhu are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC and further detecting T signals at 5hmC sites with a sequencing assay in Matsushita to incorporate the contacting of 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) as taught in Zhu because Zhu teaches that doing so would provide a method to enable a high C-to-T conversion rate with both high sensitivity and high specificity.
Regarding claim 198, Matsushita teaches contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the target nucleic acid with Cu(II)/TEMPO) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Regarding claim 207, Matsushita teaches contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (producing a nucleic acid comprising one or more 5fC) and further, after oxidation, contacting the sample comprising one or more 5fC with sodium hydroxide (an additional step between step (a) and (b) comprising contacting the nucleic acid comprising one or more 5fC with NaOH) (pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-1).
Regarding claim 212, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (c) of contacting 5fC-M adduct with a polymerase to convert to T occurs in a reaction mixture comprising a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Regarding claim 214, Matsushita teaches a method of Cu(II)/TMEPO oxidation comprising the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample (contacting the target nucleic acid with TET, wherein TET converts 5mC to 5hmC, thereby producing a nucleic acid with one or more 5hmC), contacting 5hmC DNA (nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the nucleic acid comprising one or more 5hmC with Cu(II)/TEMPO thereby producing a nucleic acid comprising 5fC) and further detecting T signals at 5hmC sites with a sequencing assay (producing a nucleic acid comprising one or more T and sequencing the nucleic acid comprising one or more T, wherein the 5hmC in a target nucleic acid has been converted to T) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Matsushita does not teach contacting the nucleic acid with one or more 5fC with malononitrile or contacting the nucleic acid comprising one or more 5fC-M adduct with a polymerase.
Zhu teaches a method of single-cell, single-base resolution 5fC-sequencing comprising contacting 5fC with malononitrile to form 5fC-M adduct (contacting the nucleic acid comprising one or more 5fC with malononitrile wherein the malononitrile converts 5fC to 5fC-M adduct, thereby producing one or more 5fC-M adduct) and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing (contacting the nucleic acid comprising 5fC-M adduct with a polymerase, wherein the polymerase converts 5fC-M adduct to T, thereby producing a nucleic acid comprising one or more T) (abstract lines 1-12; pg. 721 column 2 1st full paragraph lines 1-11; pg. 721-722 paragraph bridging pg. 721 & 722 lines 1-28; pg. 722 paragraph bridging column 1 & 2 lines 1-27; pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11). Zhu also teaches that treating 5fC with malononitrile enables a high C-to-T conversion rate and that this method shows both high sensitivity and high specificity (pg. 722 paragraph bridging column 1 & 2 lines 1-27).
Matsushita and Zhu are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample, contacting 5hmC DNA with Cu(II)/TEMPO to produce 5fC, and further detecting T signals at 5hmC sites with a sequencing assay in Matsushita to incorporate the contacting of 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) as taught in Zhu because Zhu teaches that doing so would provide a method to enable a high C-to-T conversion rate with both high sensitivity and high specificity.
Regarding claim 233, Matsushita teaches contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (contacting the target nucleic acid with Cu(II)/TEMPO thereby producing a nucleic acid comprising 5fC) (abstract lines 1-8; pg. 5756-5757 paragraph bridging pg. 5756 & 5757 lines 14-39; pg. 5757 paragraph bridging column 1 & 2 lines 1-6; pg. 5757-5758 paragraph bridging pg. 5757 & 5758 lines 1-5; pg. 5758 paragraph bridging column 1 & 2 lines 4-10; pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-8).
Regarding claim 242, Matsushita teaches contacting 5hmC DNA (target nucleic acid) with Cu(II)/TEMPO to produce 5fC (producing a nucleic acid comprising one or more 5fC) and further, after oxidation, contacting the sample comprising one or more 5fC with sodium hydroxide (an additional step between step (b) and (c) comprising contacting the nucleic acid comprising one or more 5fC with NaOH) (pg. S2 2nd full paragraph lines 1-15; pg. S2 3rd full paragraph lines 1-1).
Regarding claim 247, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (d) of contacting 5fC-M adduct with a polymerase to convert to T occurs in a reaction mixture comprising a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Claim(s) 137, 138, 172, 173, 199, 200, 234, & 235 is/are rejected under 35 U.S.C. 103 as being unpatentable over Matsushita (Matsushita et al.; Chemical Communications, Vol. 53, pages 5756-5759 & S1-S13, May 2017), as cited on the IDS dated 08/04/2023, and Zhu (Zhu et al.; Cell Stem Cell, Vol. 20, pages 720-747, March 2017), as cited in the IDS dated 08/04/2023 as applied to claims 125, 136, 145, 150, 152, 171, 180, 185, 187, 198, 207, 212, 214, 233, 242, & 247 above, and further in view of Okamoto (WO/2017039002, September 2017), machine translation obtained from Science & Technical Information Center (STIC).
The teachings of Matsushita and Zhu with respect to claims 125, 136, 152, 171, 187, 198, 214, & 233 are discussed above.
Regarding claims 137 & 138, Matsushita teaches incubating the target nucleic acid with Cu(II)/TEMPO (step (a)) for 45 hours at 25°C and for 24 hours at 50°C (pg. S2 1st full paragraph lines 1-8; pg. S2 2nd full paragraph lines 1-15).
Matsushita and Zhu does not teach wherein step (a) occurs in less than 24 hours or 22 hours.
Okamoto teaches oxidation of target 5hmC DNA with a mixture of copper (II) and TEMPO (Cu(II)/TEMPO) for 44.5 hours at 50°C and for 1 to 3 days at room temperature (paragraph [0032] lines 1-5; paragraph [0033] lines 1-4; paragraph [0077] lines 1-6; paragraph [0083] lines 1-3; paragraph [0090] lines 1-6).
Okamoto does not teach wherein step (a) occurs in less than 24 hours or 22 hours, however, optimizing timing and temperature of oxidation in step (a) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Matsushita and Okamoto. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (a) (contacting the target nucleic acid with Cu(II)/TEMPO to convert 5hmC to 5fC) through modifying the temperature of the reaction conditions as taught in Matsushita and Okamoto through routine optimization to save time in the oxidation occurring in step (a).
Regarding claims 172 & 173, Matsushita teaches incubating the target nucleic acid with Cu(II)/TEMPO (step (a)) for 45 hours at 25°C and for 24 hours at 50°C (pg. S2 1st full paragraph lines 1-8; pg. S2 2nd full paragraph lines 1-15).
Matsushita and Zhu does not teach wherein step (b) occurs in less than 24 hours or 22 hours.
Okamoto teaches oxidation of target 5hmC DNA with a mixture of copper (II) and TEMPO (Cu(II)/TEMPO) for 44.5 hours at 50°C and for 1 to 3 days at room temperature (paragraph [0032] lines 1-5; paragraph [0033] lines 1-4; paragraph [0077] lines 1-6; paragraph [0083] lines 1-3; paragraph [0090] lines 1-6).
Okamoto does not teach wherein step (b) occurs in less than 24 hours or 22 hours, however, optimizing timing and temperature of oxidation in step (b) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Matsushita and Okamoto. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (b) (contacting the target nucleic acid with Cu(II)/TEMPO to convert 5hmC to 5fC) through modifying the temperature of the reaction conditions as taught in Matsushita and Okamoto through routine optimization to save time in the oxidation occurring in step (b).
Regarding claims 199 & 200, Matsushita teaches incubating the target nucleic acid with Cu(II)/TEMPO (step (a)) for 45 hours at 25°C and for 24 hours at 50°C (pg. S2 1st full paragraph lines 1-8; pg. S2 2nd full paragraph lines 1-15).
Matsushita and Zhu does not teach wherein step (a) occurs in less than 24 hours or 22 hours.
Okamoto teaches oxidation of target 5hmC DNA with a mixture of copper (II) and TEMPO (Cu(II)/TEMPO) for 44.5 hours at 50°C and for 1 to 3 days at room temperature (paragraph [0032] lines 1-5; paragraph [0033] lines 1-4; paragraph [0077] lines 1-6; paragraph [0083] lines 1-3; paragraph [0090] lines 1-6).
Okamoto does not teach wherein step (a) occurs in less than 24 hours or 22 hours, however, optimizing timing and temperature of oxidation in step (a) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Matsushita and Okamoto. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (a) (contacting the target nucleic acid with Cu(II)/TEMPO to convert 5hmC to 5fC) through modifying the temperature of the reaction conditions as taught in Matsushita and Okamoto through routine optimization to save time in the oxidation occurring in step (a).
Regarding claims 234 & 235, Matsushita teaches incubating the target nucleic acid with Cu(II)/TEMPO (step (a)) for 45 hours at 25°C and for 24 hours at 50°C (pg. S2 1st full paragraph lines 1-8; pg. S2 2nd full paragraph lines 1-15).
Matsushita and Zhu does not teach wherein step (b) occurs in less than 24 hours or 22 hours.
Okamoto teaches oxidation of target 5hmC DNA with a mixture of copper (II) and TEMPO (Cu(II)/TEMPO) for 44.5 hours at 50°C and for 1 to 3 days at room temperature (paragraph [0032] lines 1-5; paragraph [0033] lines 1-4; paragraph [0077] lines 1-6; paragraph [0083] lines 1-3; paragraph [0090] lines 1-6).
Okamoto does not teach wherein step (b) occurs in less than 24 hours or 22 hours, however, optimizing timing and temperature of oxidation in step (b) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Matsushita and Okamoto. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (b) (contacting the target nucleic acid with Cu(II)/TEMPO to convert 5hmC to 5fC) through modifying the temperature of the reaction conditions as taught in Matsushita and Okamoto through routine optimization to save time in the oxidation occurring in step (b).
Claim(s) 139-144, 146-149, 174-179, 181-184, 201-206, 208-211, 236-241, & 243-246 is/are rejected under 35 U.S.C. 103 as being unpatentable over Matsushita (Matsushita et al.; Chemical Communications, Vol. 53, pages 5756-5759 & S1-S13, May 2017), as cited on the IDS dated 08/04/2023, and Zhu (Zhu et al.; Cell Stem Cell, Vol. 20, pages 720-747, March 2017), as cited in the IDS dated 08/04/2023 as applied to claims 125, 136, 145, 150, 152, 171, 180, 185, 187, 198, 207, 212, 214, 233, 242, & 247 above, and further in view of Yi (CN106957350A, July 2017), machine translation obtained from Science & Technical Information Center (STIC).
The teachings of Matsushita and Zhu with respect to claims 125, 145, 152, 180, 187, 207, 214, & 242 are discussed above.
Regarding claims 139 & 140, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (b)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein step (b) occurs at around or at 60°C.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 60°C (step(b) occurs at or around 60°C (see claims 139 & 140)) (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0101] lines 1-3). In addition, Yi teaches that treatment with malononitrile enables labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples (paragraph [0031] lines 8-16).
Matsushita, Zhu, and Yi are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C in Zhu to incorporate the contacting of 5fC with malononitrile to obtain 5fC-M comprising a reaction temperature of 60°C as taught in Yi because Yi teaches that doing so would provide a method to enable labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples.
Regarding claim 141, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution (step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (paragraph [0048] lines 1-5).
Regarding claim 142, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which some of the buffers tested comprises 20mM of Tris (the buffer of step (b) comprises Tris) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Matsushita and Zhu does not teach that the buffer comprises 25 mM Tris.
Yi a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction (the buffer of step (b) comprises Tris) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0120] lines 1-5).
Yi does not teach wherein the buffer comprises 25 mM Tris, however, optimizing concentration of the buffer in the reaction mixture in step (b) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the concentration of Tris buffer in the reaction mixture in step (b) as taught in Zhu and Yi due to the routine nature of reaction condition optimization.
Regarding claim 143, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which one of the buffers tested comprises a pH of 8 (the buffer of step (b) is at a pH of around 8) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction comprising a preferable buffer solution with a pH of 8 (the buffer of step (b) is at a pH of around 8) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0065] lines 1-5; paragraph [0120] lines 1-5).
Regarding claim 144, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (b)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (b)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours, however, optimizing timing and temperature of oxidation in step (b) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (b) (contacting the nucleic acid comprising one or more 5fC with malononitrile for 1.5 hours) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (b).
Regarding claims 146-149, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (b)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour (see claims 146 & 147) or for about 30 minutes (see claims 148 & 149).
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (b)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour or about 30 minutes, however, optimizing timing and temperature of oxidation in step (b) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (b) (contacting the nucleic acid comprising one or more 5fC with malononitrile for an hour or about 30 minutes) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (b).
Regarding claims 174 & 175, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (c)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein step (c) occurs at around or at 60°C.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 60°C (step(c) occurs at or around 60°C (see claims 139 & 140)) (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0101] lines 1-3). In addition, Yi teaches that treatment with malononitrile enables labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples (paragraph [0031] lines 8-16).
Matsushita, Zhu, and Yi are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C in Zhu to incorporate the contacting of 5fC with malononitrile to obtain 5fC-M comprising a reaction temperature of 60°C as taught in Yi because Yi teaches that doing so would provide a method to enable labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples.
Regarding claim 176, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution (step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (paragraph [0048] lines 1-5).
Regarding claim 177, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which some of the buffers tested comprises 20mM of Tris (the buffer of step (c) comprises Tris) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Matsushita and Zhu does not teach that the buffer comprises 25 mM Tris.
Yi a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction (the buffer of step (c) comprises Tris) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0120] lines 1-5).
Yi does not teach wherein the buffer comprises 25 mM Tris, however, optimizing concentration of the buffer in the reaction mixture in step (b) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the concentration of Tris buffer in the reaction mixture in step (c) as taught in Zhu and Yi due to the routine nature of reaction condition optimization.
Regarding claim 178, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which one of the buffers tested comprises a pH of 8 (the buffer of step (c) is at a pH of around 8) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction comprising a preferable buffer solution with a pH of 8 (the buffer of step (c) is at a pH of around 8) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0065] lines 1-5; paragraph [0120] lines 1-5).
Regarding claim 179, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (c)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (c)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours, however, optimizing timing and temperature of oxidation in step (c) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (c) (contacting the nucleic acid comprising one or more 5fC with malononitrile for 1.5 hours) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (c).
Regarding claims 181-184, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (c)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour (see claims 181 & 182) or for about 30 minutes (see claims 183 & 184).
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (c)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour or about 30 minutes, however, optimizing timing and temperature of oxidation in step (c) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (c) (contacting the nucleic acid comprising one or more 5fC with malononitrile for an hour or about 30 minutes) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (c).
Regarding claims 201 & 202, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (b)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein step (b) occurs at around or at 60°C.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 60°C (step(b) occurs at or around 60°C (see claims 139 & 140)) (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0101] lines 1-3). In addition, Yi teaches that treatment with malononitrile enables labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples (paragraph [0031] lines 8-16).
Matsushita, Zhu, and Yi are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C in Zhu to incorporate the contacting of 5fC with malononitrile to obtain 5fC-M comprising a reaction temperature of 60°C as taught in Yi because Yi teaches that doing so would provide a method to enable labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples.
Regarding claim 203, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution (step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (paragraph [0048] lines 1-5).
Regarding claim 204, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which some of the buffers tested comprises 20mM of Tris (the buffer of step (b) comprises Tris) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Matsushita and Zhu does not teach that the buffer comprises 25 mM Tris.
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction (the buffer of step (b) comprises Tris) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0120] lines 1-5).
Yi does not teach wherein the buffer comprises 25 mM Tris, however, optimizing concentration of the buffer in the reaction mixture in step (b) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the concentration of Tris buffer in the reaction mixture in step (b) as taught in Zhu and Yi due to the routine nature of reaction condition optimization.
Regarding claim 205, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which one of the buffers tested comprises a pH of 8 (the buffer of step (b) is at a pH of around 8) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Yi a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction comprising a preferable buffer solution with a pH of 8 (the buffer of step (b) is at a pH of around 8) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0065] lines 1-5; paragraph [0120] lines 1-5).
Regarding claim 206, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (b)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (b)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours, however, optimizing timing and temperature of oxidation in step (b) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (b) (contacting the nucleic acid comprising one or more 5fC with malononitrile for 1.5 hours) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (b).
Regarding claims 208-211, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (b)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour (see claims 208 & 209) or for about 30 minutes (see claims 210 & 211).
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (b)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour or about 30 minutes, however, optimizing timing and temperature of oxidation in step (b) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (b) (contacting the nucleic acid comprising one or more 5fC with malononitrile for an hour or about 30 minutes) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (b).
Regarding claims 236 & 237, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (c)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein step (c) occurs at around or at 60°C.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 60°C (step (c) occurs at or around 60°C (see claims 139 & 140)) (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0101] lines 1-3). In addition, Yi teaches that treatment with malononitrile enables labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples (paragraph [0031] lines 8-16).
Matsushita, Zhu, and Yi are considered to be analogous to the claimed invention because they are all in the same field of oxidation of target nucleic acids. 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 of contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C in Zhu to incorporate the contacting of 5fC with malononitrile to obtain 5fC-M comprising a reaction temperature of 60°C as taught in Yi because Yi teaches that doing so would provide a method to enable labeling and detecting of 5fC without degrading DNA or RNA making it suitable for various trace sample and precious samples.
Regarding claim 238, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers (step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11).
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution (step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer) (paragraph [0048] lines 1-5).
Regarding claim 239, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which some of the buffers tested comprises 20mM of Tris (the buffer of step (c) comprises Tris) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Matsushita and Zhu does not teach that the buffer comprises 25 mM Tris.
Yi a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction (the buffer of step (c) comprises Tris) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0120] lines 1-5).
Yi does not teach wherein the buffer comprises 25 mM Tris, however, optimizing concentration of the buffer in the reaction mixture in step (b) would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the concentration of Tris buffer in the reaction mixture in step (c) as taught in Zhu and Yi due to the routine nature of reaction condition optimization.
Regarding claim 240, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in which one of the buffers tested comprises a pH of 8 (the buffer of step (c) is at a pH of around 8) (pg. 734 3rd full paragraph lines 1-5; pg. 734 5th full paragraph lines 1-11; pg. 734 7th full paragraph lines 1-3; Table S1).
Yi teaches a method of treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising the use of a reaction solution comprising a buffer solution comprising 10 mM of Tris and a buffer solution comprising 100 mM of Tris in which the concentration of the Tris buffer solution can vary the pH of the reaction comprising a preferable buffer solution with a pH of 8 (the buffer of step (c) is at a pH of around 8) (paragraph [0048] lines 1-16; paragraph [0050] lines 1-4; paragraph [0065] lines 1-5; paragraph [0120] lines 1-5).
Regarding claim 241, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (c)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours.
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (c)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours, however, optimizing timing and temperature of oxidation in step (c) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (c) (contacting the nucleic acid comprising one or more 5fC with malononitrile for 1.5 hours) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (c).
Regarding claims 243-246, Zhu teaches contacting 5fC with malononitrile to form 5fC-M adduct in which the sample was incubated with malononitrile at 37°C for 20 hours (step (c)) (pg. 734 3rd full paragraph lines 2-3).
Matsushita and Zhu does not teach wherein the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour (see claims 243 & 244) or for about 30 minutes (see claims 245 & 246).
Yi teaches a method treatment of 5fC with malononitrile to obtain 5fC-M in which a variety of reaction conditions were tested comprising a reaction temperature of 37°C for 20 hours, reaction temperature of 20°C for 48 hours, and a reaction temperature of 60°C for 12 hours (step (c)) in which varying the temperature and time of incubation with malononitrile altered the yield of the labeled 5fC-M product (paragraph [0031] lines 1-9; paragraph [0034] lines 1-3; paragraph [0053] lines 1-14; paragraph [0081] lines 12-13; paragraph [0099] lines 1-3; paragraph [0097] lines paragraph [0101] lines 1-3).
Yi does not teach the nucleic acid comprising one or more 5fC is contacted with malononitrile for about an hour or about 30 minutes, however, optimizing timing and temperature of oxidation in step (c) with malononitrile would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of Zhu and Yi. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the time in which step (c) (contacting the nucleic acid comprising one or more 5fC with malononitrile for an hour or about 30 minutes) through modifying the temperature of the reaction conditions as taught in Zhu and Yi through routine optimization to save time in the oxidation occurring in step (c).
Claim(s) 151, 186, 213, & 248 is/are rejected under 35 U.S.C. 103 as being unpatentable over Matsushita (Matsushita et al.; Chemical Communications, Vol. 53, pages 5756-5759 & S1-S13, May 2017), as cited on the IDS dated 08/04/2023, and Zhu (Zhu et al.; Cell Stem Cell, Vol. 20, pages 720-747, March 2017), as cited in the IDS dated 08/04/2023 as applied to claims 125, 136, 145, 150, 152, 171, 180, 185, 187, 198, 207, 212, 214, 233, 242, & 247 above, and further in view of ThermoFisher (ThermoFisher Scientific; PCR Setup - Six Critical Components to Consider, pages 1-8, June 2017) and GeneLink (GeneLink; PCR Additives & Enhancers, pages 1-12, 2014).
The teachings of Matsushita and Zhu with respect to claims 125, 150, 152, 185, 187, 212, 214, & 246 are discussed above.
Regarding claims 151, 186, 213, & 248, Matsushita and Zhu does not teach the buffer comprising the specific components in claims 151, 186, 213, & 248.
ThermoFisher teaches essential components of a buffer for treating a sample with polymerase for amplification and sequencing comprising 1-10% DMSO, 1-3 M of Betaine, 0.2 mM or higher dATP, 1.5-2.5 mM MgCl2, 15-30 mM ammonium sulfate, and a type of buffer comprising Tris-HCl (pg. 5 1st full paragraph lines 1-3; pg. 5 2nd full paragraph lines 1-3; pg. 6 3rd full paragraph lines 1-3; pg. 7 1st full paragraph lines 1-2; Table 2). ThermoFisher also teaches that these components can be optimized in various concentrations to enable successful amplification and downstream usage depending on application (pg. 7 2nd full paragraph lines 1-6).
ThermoFisher does not teach the buffer is 70 mM of TMAC.
GeneLink teaches essential components of a buffer for treating a sample with polymerase for amplification and sequencing comprising a buffer of 15-100 mM of TMAC (pg. 3 4th full paragraph lines 1-3; pg. 9 table 1). In addition, GeneLink teaches the buffer composition comprises 2 mM dNTP, 25 mM of MgCl2, DMSO, and 5 M of Betaine (pg. 9 table 1). In addition, GeneLink teaches that the addition of TMAC increases the specificity of hybridization (pg. 3 4th full paragraph lines 1-3).
Matsushita, Zhu, ThermoFisher, and GeneLink are considered to be analogous to the claimed invention because they are all in the same field of analysis of nucleic acids with a reaction mixture comprising a buffer. 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 of contacting 5fC with malononitrile to form 5fC-M adduct and then contacting the 5fC-M adduct with polymerase to convert 5fC-M adduct to a Thymine (T) and detect the T with PCR and sequencing in which that sample is contacted with malononitrile and then the 5fC-M adduct to T for sequencing in varying pH and buffers in Zhu to incorporate the use of a buffer composition comprising DMSO, Betaine, dATP, MgCl2, ammonium sulfate, and a type of buffer as taught in ThermoFisher because ThermoFisher teaches that doing so would provide a method to enable successful amplification and downstream usage depending on application and to incorporate the use of TMAC in the buffer composition as taught in GeneLink because GeneLink teaches that the use of TMAC in the buffer composition increases the specificity of hybridization.
In addition, optimizing the concentrations of the components of the buffer would have been prima facie obvious in view of the routine nature of reaction condition optimization taught in the cited prior art of ThermoFisher and GeneLink. As set for in MPEP 2144.05 II A:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
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 modify the concentrations of the buffer components as taught in ThermoFisher and GeneLink due to the routine nature of reaction condition optimization.
Claim(s) 153 & 215 is/are rejected under 35 U.S.C. 103 as being unpatentable over Matsushita (Matsushita et al.; Chemical Communications, Vol. 53, pages 5756-5759 & S1-S13, May 2017), as cited on the IDS dated 08/04/2023, and Zhu (Zhu et al.; Cell Stem Cell, Vol. 20, pages 720-747, March 2017), as cited in the IDS dated 08/04/2023 as applied to claims 125, 136, 145, 150, 152, 171, 180, 185, 187, 198, 207, 212, 214, 233, 242, & 247 above, and further in view of Hu (Hu et al.; Nature Letter, Vol. 527, pages 118-133, November 2015), as evidenced by Danielsen (Danielsen, Gland, & Hayes; Environmental, Science, & Technology, Vol. 39, pages 756-763, October 2004).
The teachings of Matsushita and Zhu with respect to claims 152 & 214 are discussed above.
Regarding claim 153, Matsushita and Zhu does not teach that the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample (step (a)) occurs in a reaction mixture, wherein the reaction mixture comprises an amine catalyst.
Hu teaches oxidation of 5mC to 5hmC with TET proteins comprising mixing various amounts of TET2 with DNA (nucleic acid) in a buffer containing Fe2+ to measure DNA binding affinities for different substrates under catalytic conditions, in which Fe(II) is an amine catalyst in oxidation reactions as evidenced by Danielsen (pg. 759-760 paragraph bridging pg. 759 & 760 lines 6-13 of Danielsen). In addition, Hu teaches that this method facilitates the generation of 5hmC as a potentially stable mark to analyze regulatory functions (abstract lines 1-29).
Matsushita, Zhu, and Hu are considered to be analogous to the claimed invention because they are all in the same field of oxidation analysis of nucleic acids with a reaction mixture comprising a buffer. 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 of the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample (step (a)) occurs in a reaction mixture) in Matsushita to incorporate the use of a buffer comprising an amine catalyst with TET oxidation as taught in Hu because Hu teaches that doing so would provide facilitate the generation of 5hmC as a potentially stable mark to analyze regulatory functions.
Regarding claim 215, Matsushita and Zhu does not teach that the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample (step (a)) occurs in a reaction mixture, wherein the reaction mixture comprises an amine catalyst.
Hu teaches oxidation of 5mC to 5hmC with TET proteins comprising mixing various amounts of TET2 with DNA (nucleic acid) in a buffer containing Fe2+ to measure DNA binding affinities for different substrates under catalytic conditions, in which Fe(II) is an amine catalyst in oxidation reactions as evidenced by Danielsen (pg. 759-760 paragraph bridging pg. 759 & 760 lines 6-13 of Danielsen). In addition, Hu teaches that this method facilitates the generation of 5hmC as a potentially stable mark to analyze regulatory functions (abstract lines 1-29).
Matsushita, Zhu, and Hu are considered to be analogous to the claimed invention because they are all in the same field of oxidation analysis of nucleic acids with a reaction mixture comprising a buffer. 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 of the use of TET proteins to oxidize 5mC to 5hmC in a nucleic acid sample (step (a)) occurs in a reaction mixture) in Matsushita to incorporate the use of a buffer comprising an amine catalyst with TET oxidation as taught in Hu because Hu teaches that doing so would provide facilitate the generation of 5hmC as a potentially stable mark to analyze regulatory functions.
Conclusion
Claims 125, 136-155, 171-187, 198-215, 233-248 are rejected. Claims 216 & 217 are objected to as being dependent from rejected claims.
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/BAILEY BUCHANAN/Examiner, Art Unit 1682
/JEHANNE S SITTON/Primary Examiner, Art Unit 1682
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