DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . Applicant’s preliminary amendment filed 1/15/2024 is acknowledged. Applicant’s substitute specification filed 1/15/2024 is acknowledged and has been entered.
Priority
This application is a CIP of PCT/CN2022/073322 filed 01/21/2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/24/2023 and 9/27/2023 are acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
The drawings were received on 1/14/2024 is acknowledged. These drawings are found acceptably by the Examiner.
Claim Objections
Claim 1 is objected to because of the following informalities:
(a) The claims 1, 2, 3, 16, 17, 19 and 20 are objected to for the Arabic numerical representation of the method steps (1) – (15) because the claim numbering should be distinct from the numbering of the claimed method steps (see MPEP 1824). It is suggested replacing the numbering of the method steps from Arabic numerical representation to alphabetical or Roman numeral characters. Appropriate correction is required.
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 1-20 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.
(a) The claims 1-20 are indefinite in the claim 1 at the step (3) at the recitation of “in more detail”, because it is unclear if the limitations “in more details” is recited to disclosed that the limitation following the limitation is specific and required or if the limitation following “in more details” is recited to be optional. Likewise, the in the step (3) at lines 5-6 further recites limitations in parentheses, e.g., “(the fragmentation is employed with a methylation – insensitive restriction endonuclease whose recognition sequence is with 50% or more deoxynucleotide composed of C and G)” and lines 8-9 “(the recognition sequence is 4 deoxynucleotides composed of C and G only with at least one CG di-nucleotide)”. It cannot be determined if the limitations are part of the claimed invention or if they are intended to represent a separate method step(s) or if they are something entirely different. Given the ambiguity, the metes and bounds of the claimed limitation cannot be ascertained and clear interpretation of the claims as a whole cannot be ascertained. Clarification is required.
(b) Claims 1, 2, 4, 7, 10, 12 and 20 are indefinite at the recitation of “preferably” and “more preferably” because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
(c) Claim 1 lacks proper antecedent basis for “the complete barcode adapters” in the step (6). (d) It is suggested deleting “the”.
(d) Claims 1-20 is indefinite at the recitation of “preliminary library” as recited in the step (11) of the claim 1 because it is unclear how the limitation “preliminary” defines or limit the terminology “library” in the context of the claims, neither the specification nor claims provide a limiting definition of the term and thus it cannot be determined how the limitation “preliminary library” in the step (11) differs from the “library” as recited in the step (13).
(e) Claims 1-20 are indefinite at the recitation of “the primer comprises a batch index” in the claim 1 because the limitation has not been defined in the claims or specification. Thus, it cannot be determined how the limitation modifies the structure or function of the primer sequence as recited in the claims.
Claim Interpretation
The claims are sufficiently broad and ambiguous for reasons made of record above. Likewise, the claims are extremely wordy and appear to be a translation from a foreign document. Therefore, for the purpose of application of prior art, the claims are being given the broadest reasonable interpretation by the Examiner.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
12. Claim(s) 1-12, 15-16, 18-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Boyle et al., Genome Biology, 13: R92, 1-10, 2012) in view of Schroeder (US 9745614, August 2017).
Regarding claim 1, Boyle et al teach a method for detecting the methylation of CpG in a plurality of samples, (multiplex reduced representation bisulfite sequencing (mRRBS) comprising the following steps: independently lysing the plurality of samples to release respective genomic DNAs (gDNAs); purifying the released gDNAs or proceeding directly to the next step without purifying the released gDNAs (see page 7, section entitle “Genomic DNA Purification”); fragmenting the released gDNAs or purified gDNAs to obtain DNA fragments of different lengths (see page 7, section entitled “MSpI digestion”); ligating DNA fragments of each of the samples to a barcode adapter with a different barcode, respectively (see page 8, section entitled “Multiplex adapter ligation”); pooling DNA fragments of the plurality of the samples that are ligated with a barcode adapter to obtain a DNA fragment pool (page 8, section entitled “Library Pooling and Bisulfite Conversion”); subjecting the pool of DNA fragments to repair of barcode adapters with a DNA polymerase to construct the barcode adapters (see page 7, which teaches “Gap filling and A-tailing”); converting DNA fragments with the complete barcoded adapters, the conversion involving transformation of non-methylated deoxycytidine triphosphate (dCTP) into uridine triphosphate (UTP) and subjecting converted DNA fragments to a first round of polymerase chain reaction (PCR) amplification (see page 5 which teaches that the barcoded sequences are methylated and followed by PCR amplification (see page 5, column 2, last paragraph to page 6, column 1, first paragraph and Table 1, Legend); sequencing the obtained library obtained with the specific next-generation sequencing (Illumina sequencing) platform to obtain methylation data for the pooled plurality of samples; and decoding the methylation data obtained in step through information analysis to obtain methylation patterns of each batch and each sample (see page 8-9, section entitled “sequencing” and Figures and Table 1)( see also entire document). Boyle et al teaches multiple rounds of amplification in a multiplex format using 96-well plate (see
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page 2 and 3 see also supplementary Figure 1 below).
Regarding claim 2, Boyle teaches wherein the restriction endonuclease capable of forming a cohesive terminus is MspI (see “Materials and Methods” section).
Boyle et al does not expressly teach wherein the method comprises utilizing primer sequence comprising a batch index or any specific structure of the adapter or barcode connected to the primer sequence. Likewise, the reference does not teach long and short double stranded adapter sequence used in the methylation-based method.
Regarding claims 1-12, 15-16, 18-20, Schroeder teaches a method for profiling method status, the method comprising the method further comprises fragmenting the polynucleotides prior to introducing the pool of oligonucleotide sequence to the 3′ ends of the plurality of polynucleotides. In some cases, the fragmenting is performed by contacting the polynucleotides with an enzyme. In some cases, the enzyme is a restriction enzyme, wherein the restriction enzyme is MspI. In some cases, each oligonucleotide sequence in the pool of oligonucleotide sequences is within a strand of a duplexed adaptor. In some cases, the duplexed adaptor is a partial duplexed adaptor comprising a short strand and a long strand. In some cases, the method further comprises sequencing each of the plurality of clusters (column 2-6). Schroeder teaches that wherein the sequencing method encompass sequencing a reduced representation bisulfite sequencing library with a pool of diversity adapters comprising a variable number of D bases (see Figure 9).
In some cases, the methods, compositions, and kits provided herein are useful for generating bisulfite converted nucleic acid libraries with increased sequence diversity at the ends of nucleic acid inserts within the library. The bisulfite converted libraries can be reduced representation bisulfite sequencing (RRBS) libraries. Such libraries can be useful, for example, for determining the methylation status across a genome, or at given genomic loci. The RRBS libraries generated by the methods, compositions, and kits provided herein can be useful for determining the methylation status of CpG islands in polynucleotides derived from whole genome samples. The whole genome samples can be from eukaryotic cells (e.g., mammalian cells). The methods can produce RRBS libraries that can be more efficiently sequenced by current NGS sequencers (e.g., Illumina sequencers), and in some embodiments, without the need for spiking in a genomic, higher-diversity sample nucleic acid (e.g. PhiX control library). The method for generating an RRBS library can comprise enzymatically fragmenting a nucleic acid (polynucleotide) such that a plurality of nucleic acids fragments comprising substantially similar nucleotide compositions at the ends or termini of each fragment are generated. In some cases, the method comprises fragmenting the nucleic acid with a methylation sensitive restriction enzyme such as, for example, MspI. In some cases, a pool of adaptors is ligated to the 5′ ends of each nucleic acid fragment in the plurality of nucleic acid fragments such that each nucleic acid fragment comprises double stranded and the ligation generates a plurality of adaptor-ligated nucleic acid fragments. The pool of adaptors can comprise sequences complementary to primer sequence wherein the adaptor comprises extender sequence or a dinucleotide extender or trinucleotide extender sequence, wherein the extender sequences are chosen such that the base composition differs for the nucleotide base composition at the end of the nucleic fragment to which the extender sequence is appended. The presence of extender sequence in the plurality of adaptor-ligated nucleic acid fragments can increase the ability of NGS sequencers (e.g., Illumina) to successfully identify and discern between neighboring clusters formed by solid-state amplification during sequencing of the clusters (col. 16-18). Schroeder additionally teaches wherein the melting temperature of the adaptor may comprise about, more than, less than or at least 30 degrees Celsius and wherein the temperature can raise about, more than, less than, or at least about 1-10 degrees difference (col. 45, lines 35-45). Schroeder also teaches wherein the adaptor or primer sequences may further comprise of a unique barcode sequence (col. 30, beginning at line 41).
It would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have been motivated to have modified the mRRBS method of Boyle et al to encompass diversity adaptor sequences in the assay as taught by Schroeder. The ordinary artisan would have been motivated to do because Schroeder teaches that diversity adaptors ad discussed above are useful for generating RRBS nucleic acid libraries comprising increased sequence diversity as well as improved sequencing performances.
Conclusion
13. No claims are allowed. However, the claims 13, 14 and 17 have not been rejected under prior art because the prior art does not teach the sequences of SEQ ID NOS: 1-3. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/ Primary Examiner, Art Unit 1681