FINAL ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This action is responsive to the Response (including Amendments) filed 16 March 2026. Claims 1, 5-6, 12-13, 18, 20-21, 30-31, 36, and 38 have been amended, claim 23 has been canceled, and claim 85 has been added. All prior rejections of claim 23 are moot in view of the cancelation of that claim. Claims 14-16, 50, 53-54, 70, 72-73, and 75-84 remain withdrawn (see also paragraphs 5-7 below) and claims 1-2, 4-6, 11-13, 17-18, 20-21, 25-31, 33, 36-38, 48, 71, 74, and 85 remain/are under consideration herein. Applicant’s amendments and arguments have been thoroughly reviewed, and have overcome the following objections/rejections set forth in the prior Office action:
The objections to the specification (and it is noted that the Sequence Listing and substitute specification filed 16 March 2026 have also been entered);
The objection to the drawings (and it is noted that the Drawings filed 08 September 2025 have been entered);
The objection to claim 31, in view of Applicant’s clarifying amendments;
Several rejections under 35 USC 112(b) in view of Applicant’s clarifying amendments (although the claims remain indefinite for the reasons given below); and
The rejections of claims under 35 USC 102(a)(1) and 35 USC 103, in view of the amendment of independent claim 1 to require that each primer of “the set of primers” have a structure as is now specified in b) of the claim (although it is noted that the claims remain rejected under 35 USC 103 for the reasons given below).
Claims 1-2, 4-6, 11-13, 17-18, 20-21, 25-31, 33, 36-38, 48, 71, 74, and 85 remain/are rejected for the reasons given below, which include new grounds of rejection necessitated by Applicant’s amendments. Any rejections and/or objections not reiterated in this action have been withdrawn. This action is FINAL.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election/Restrictions
Applicant’s election of the following species in the reply filed on January 8, 2025 is again acknowledged:
For the species of A, “a fully degenerate set of primers wherein XnG is 5’-NNNNNN-3’, and X(n-1)CG is 5’-NNNNCG-3’, wherein N is selected from A, C, T/U, G”;
For the species of B, bisulfite treatment; and
For the species of C, cytosine conversion to uracil.
Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
With further regard to the various species of c), as applicant did not specifically identify a type of cytosine/modified cytosine (or combination of cytosine types) from among the alternatives recited in claims 14-18/50/54, the species of cytosine to uracil has been examined. As the prior art applies against this elected species, further species (and claims limited to/requiring such species) have been withdrawn.
Claims 14-16, 50, 53-54, 70, 72-73, and 75-84 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on January 8, 2025.
Claim Interpretation
Regarding amended independent claim 1 and claims dependent therefrom, it is noted that the recitation “wherein each primer in the set of primers comprises a nucleotide sequence of 5’-XnG-3’ (SEQ ID NO: 19) and/or 5’-X(n-1)CG-3” (SEQ ID NO: 20), wherein G is positioned at the 3’ terminus of the sequence” embraces methods in which each primer in the set comprises either or both of SEQ ID Nos 19 and 20; additionally, while dependent claims 5-6 recite more particular structural requirements for SEQ ID NOS 19 and 20, these claims also embrace primer sets include one or both of the defined primers. Further, given the structural requirement for primers terminating in G (such that all primers must include at least one G) within the context of the claimed method (which requires a prior step of “chemically or enzymatically converting non-target forms of cytosine and/or modified cytosine in target nucleic acid molecules…to non-cytosine residues…”), the new claim limitation “whereby performing second strand synthesis with the set of primers enriches for sequences having a target form of cytosine” simply recites an inherent/necessary result of the performance of second strand synthesis using the defined primers (rather than adding anything that further limits claim scope).
Claim Rejections - 35 USC § 112
THE FOLLOWING INCLUDES NEW GROUNDS OF REJECTION NECESSITATED BY APPLICANT’S AMENDMENTS:
Claims 1-2, 4-6, 11-13; 17, 18, 20-21, 25-31, 33, 36-38, 48, 71, 74, and 85 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 1 and claims dependent therefrom (claims 2, 4-6, 11-13; 17, 18, 20-21, 25-31, 33, 36-38, 48, 71, 74, and 85) while the species of X=N is under consideration herein, in the interest of compact prosecution it is again noted that the limitation “wherein Q is modified base selected from A, C, T/U, G” renders the claims indefinite (as does the use of Q, in dependent claims such as claim 5) as directed to this alternative, because this language states that Q is both a “modified base” but also one of the standard bases (A, C, T/U, G). This language thus does not make clear what types of bases are actually encompassed by the term “Q”, and also creates confusion with regard to what is encompassed by “I” (referring to “an irregular base selected from Q or J”) in claim 1 (as well as dependent claim 5) as directed to the alternative of “Q”. Further clarification is required.
Claims 25-26 are indefinite over the recitations “the primers comprises” a “sample barcode sequence” (claim 25) and “a molecular barcode sequence” (claim 26). As claim 1, from which the claims depend, now recite “each primer in the set of primers comprises” rather than “the primers comprise…”, there are now multiple reasonable interpretations of claims 25-26 that impart different boundaries on what is claimed. Particularly, the claims may be interpreted as requiring the specified further limitation with regard to “each primer in the set of primers”, or alternatively, as merely require the presence of such a primer or primers with the set. Accordingly, further clarification is required to ensure that the boundaries of the claims are clear.
Claim 85 contains the trademark/trade name IlluminaTM and the limitation “IlluminaTM sequence adapter sequences p5 and p7”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe particular products (sequence adapters identified both via use of a trademark and associated terms “p5” and “p7”) and, accordingly, the identification/ description is indefinite. Clarification is therefore required (to provide a fixed and definite description of what is being claimed).
Claim Rejections - 35 USC § 103
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.
THE FOLLOWING INCLUDES NEW GROUNDS OF REJECTION NECESSITATED BY APPLICANT’S AMENDMENTS:
Claim(s) 1-2, 4-6, 11-13, 17-18, 25-31, 33, 36-38, 48, 71, 74, and 85 are rejected under 35 U.S.C. 103 as being unpatentable over Clark et al (Nature Protocols 12(3):534 [2017]; previously cited) in view of Huang et al (Chapter 7 of “Ovarian Cancer: Methods and Protocols” [2013]; cited herein), and with regard to claim 31, as evidenced by Miura et al (Nucleic Acids Research 40(17):e136 [online 30 May 2012]; previously cited).
Clark et al disclose a technique for mapping DNA methylation in single cells, termed “single-cell bisulfite sequencing (scBS-seq)” (see entire reference), further teaching that their method “is based on the post-bisulfite adaptor tagging (PBAT) method developed by Miura and colleagues, in which BS treatment is used to both convert unmethylated cytosine to uracil and fragment DNA before tagging with Illumina sequencing adaptors” (see page 534, right column). Clark et al teach that their method comprises:
-The conversion of unmethylated cytosine (i.e., a preferred “non-target form” of cytosine) to uracil via bisulfite treatment of DNA of lysed single cells, meeting the requirements of a) of independent claim 1, and corresponding to the elected species of B (bisulfite treatment) and C (cytosine conversion to uracil)(see entire reference, particularly Figure 1, page 535 left column; procedure at page 539 bridging to page 540);
-The performance of second strand synthesis on denatured, converted nucleic acid via hybridization of a set of primers as discussed below;
-producing double stranded nucleic acid molecules (see again Figure 1, the Preamplification taught on page 535, Table 2, and the Procedure at page 541 bridging to page 542); and
-Analysis of the resulting double stranded nucleic acid molecules, which analysis encompasses both amplification and sequencing (see again Figure 1, page 535 right column-page 536, left column; Procedure at pages 543-544).
Clark et al thus teach methods meeting all requirements of amended independent claim 1, other than the new requirement that “each primer in the set of primers comprises a nucleotide sequence of 5’-XnG-3’ (SEQ ID NO: 19) and/or 5’-X(n-1)CG-3” (SEQ ID NO: 20), wherein G is positioned at the 3’ terminus of the sequence”;
it is reiterated that (as discussed in the Claim Interpretation above), the use of such a primer set also meets the requirements of the new “whereby” clause at the end of b) of claim 1.
With regard to a primer set meeting the requirements of the claims and the elected species of A, Clark et al teach the use in their preamplification of primers containing Illumina adaptor sequences at the 5’ end and random hexamers at the 3’ end, stating that these primers were “designed so that collectively the oligos should bind to all locations in the genome”, and further that the “composition of the random segment is 25% of each base” (page 535, left column under “Preamplification”). Thus, the primer set of Clark et al includes all possible combinations of the 4 bases (A, C, G, and T) within random hexamers, i.e., the set taught by Clark et al includes at least some primers include a G or CG at the 3’ terminus, and meets the requirement of being “fully degenerate” for the sequence XnG or X(n-1)G; however, Clark et al do not teach a primer set in which G is positioned at the 3’ terminus of each primer, as required by amended claim 1.
Like Clark et al, Huang et al teach a method for analysis of methylation patterns in the genome that relies on bisulfite treatment to prepare target DNA for differentiation of methylated and unmethylated cytosines, followed by primer hybridization and amplification (see entire reference). Clark et al teach that such methods may be improved via primer design, including adjustment of numbers of CGs and non-CG cytosines (page 77, second full paragraph), and Clark et al further teach that “CG sites in primers work best to increase specificity of primer binding to the appropriate template when they are located as close as possible to the 3’ end for both M (CG) and U (TG) primers” (page 81, Note number 8).
In view of the teachings of Clark et al and Huang et al, 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 primer set of Clark et al so as to have limited it to primers terminating in G (inclusive of both -CG-3’ and non-CG-3’), or – for the benefit more specificity, only -CG-3’ (as it is again noted that the claims embrace use of either one or both type of recited primer) - and thereby to have employed primer sets meeting the requirements of the claims (inclusive of dependent claims 2 and 4-6 as directed to the elected species); further, given the structure of the primers in such a primer set, this modification would also necessarily result in a method meeting the limitation “whereby performing second strand synthesis with the set of primers enriches for sequences having a target form of cytosine” (see also the Claim Interpretation above), given the 3’-terminal G(s) present in each primer. An ordinary artisan would have been motivated to have made such a modification by the teachings of Huang et al that such primer modifications improve the results obtained when the goal is differentiating methylated from non-methylated DNA (and particularly via an assay type that relies upon bisulfite conversion following by primer hybridization and extension/amplification/etc.).
With further regard to dependent claim 11, Clark et al disclose multiple procedures for handling of DNA of lysed cells, including a protocol for purification of gDNA from RNA, such that Clark et al disclose methods meeting the requirements of the claim (see Protocol at page 539 and Box 1 on page 538). Regarding dependent claim 12, Clark et al teach size distribution of fragments (i.e., a type of “target nucleic acids”) of greater than 200bp and average size of 400-600 bp, which meet the requirements of the claims (see, e.g., page 544 under “scBS-seq library quality control). Regarding claims 13 and 17-18, the limitations of these claims (which are directed to the elected species of bisulfite and conversion of cytosine to uracil) are addressed above. Regarding claims 25-27, the primers of Clark et al include sequences meeting the requirements of the claim, as illustrated in Table 2 (see also Figure 1 and page 535); it is noted that the primer sequences taught by Clark et al include sequences capable as functioning as adaptors, universal primers, and barcodes. Regarding claims 28-30, Clark et al disclose that their method comprises preamplification using Klenow exo-polymerase (followed by the use of an exonuclease I “to remove any remaining oligos that could otherwise synthesize unwanted adaptor dimer molecules”) (see page 535, including Figure 1, and the Procedure at pages 541-542). Regarding claims 33 and 36, Clark et al disclose incorporation of adapters (which also “provides” primer hybridization sequences), as well as amplification, as noted above (again see Figure 1, Table 1, page 535, and the Procedure at pages 541-543). Regarding claims 37-38, Clark et al also disclose capture of preamplification products and sequencing to produce sequence reads for analysis; again see Figure 1, page 535 (right column), Procedure at pages 542-544). Regarding claim 48, Clark et al disclose a procedure as claimed as discussed above; again see Figure 1, page 535; Procedure at pages 539-544). Regarding dependent claims 71 and 74, these claims correspond to the elected species, and each of these limitations is addressed above. With regard to new dependent claim 85, it is reiterated that the claim is indefinite for the reasons given above; further, “adapter sequences” are only one alternative set forth in claim 27, such that they are not required by either claim 27 or its dependent claim 85 (and thus claim 85 is obvious for the same reasons indicated for claim 27 (however, it is also noted that Clark et al disclose the use of Illumina sequencing/Illumina sequencing adaptors; see, e.g., Figure 1 and page 534, right column, last paragraph).
With further regard to dependent claim 31, this claim recites “wherein each primer in the set is biotinylated” and “the method further comprises capturing double-stranded nucleic acid molecules comprising biotin”. While Clark et al do not reference the use of biotin explicitly, they do disclose having employed the technique of Miura et al for capture using streptavidin-coated magnetic beads (page 535, right column), and – based on the cited disclosure of Miura et al (see entire reference, particularly Figure 1, as well as page 2/9 left column, and page 4/9 left column) - this procedure comprises the incorporation of biotin for use in capture of double-stranded nucleic acids. Thus, while it is noted that Clark et al also disclose a preference for a different capture procedure (stating that the capture technique involving streptavidin-coated beads “was found to be unnecessary”), as they do teach the use of this technique as taught by Miura et al, Clark et al suggest the claimed method as a predictable, functioning alternative, as evidenced by the teachings of Miura et al. An ordinary artisan would thus have recognized the modification to employing biotin and streptavidin-coated beads as the simple substitution of one prior art technique for another to achieve a predictable result; further, an ordinary artisan would have been motivated to have made such a modification in instances when it was convenient to do so (for example, when biotin/streptavidin reagents were readily available).
Claim(s) 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Clark et al in view of Huang et al, as applied to claims 1-2, 4-6, 11-13, 17-18, 25-31, 33, 36-38, 48, 71, 74, and 85, above, and further in view of Thomassin et al (Nucleic Acids Research 32(21):e168 [online 2 Dec 2004]; previously cited).
The teachings of Clark et al and Huang et al are set forth above. Neither Clark et al nor Huang et al teach primers including modifications as required by claims 20-21.
Thomassin et al teach a method called “MethylQuant”, disclosed as providing “accurate quantification of the methylation level of a specific cytosine within a complex genome”, and taught as relying upon “the well-established treatment of genomic DNA with sodium bisulfite” (see entire reference, particularly the Abstract). Thomassin et al state that their method “permits high-throughput quantification of the methylation status of a single specific cytosine in an accurate, sensitive and cost-effective manner” (page 2/9, top of left column), noting that methods for both “global, large-scale” and specific analyses of methylation are widely used in the art (page 1/9, right column). Thomassin et al teach how to perform their methods, including the use of primers containing inosine (pages 2/9-3/9, left column), and teach that the inclusion of inosine prevents the methylation of other cytosines from affecting quantification of a position of interest (page 5/9, right column bridging to 6/9, left column).
In view of the teachings of Thomassin et al, 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 Clark et al in view of Huang et al so as to further analyzed target sequences comprising any specific cytosines of interest using inosine-containing primers as taught by Thomassin et al, and thereby to have employed primers meeting the requirements of the claims. An ordinary artisan would have been motivated to have made such a modification for the benefit of quantifying methylation (as opposed to simply detecting its presence) in an accurate, sensitive, and cost-effective manner, as suggested by Thomassin et al.
It is noted that Applicant’s arguments of 16 March 2026 have been considered to the extent that they may apply to the current rejections, however those arguments were not found persuasive. It is particularly noted that the new claim limitations upon which Applicant’s arguments rely have been addressed in the revised rejections of the claims.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DIANA B JOHANNSEN whose telephone number is (571)272-0744. The examiner can normally be reached Monday-Friday, 7:30 am-3:30 pm EST.
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/DIANA B JOHANNSEN/Primary Examiner, Art Unit 1682
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