DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Status of the Application
Claim(s)1-16 are pending and under examination
The following Office Action is in response to Applicant's communication dated 04/29/2024.
Claim Objections
Claim 6 and 14 objected to because of the following informalities: Claim 6 and 14 recites “ 3′ poly-T 5′ adaptor primer”, which is unclear what the “3’” and “5’” modifiers attach to. The claim does not specify whether the primer comprises a 3′ poly-T and a 5′ adaptor region or whether “3’ adaptor” having poly T and a 5’ feature. Appropriate correction is required.
For purposes of examination only, and to facilitate a complete analysis of the claim, the Examiner interprets the claim as requiring primer comprises a 3′ poly-T region and a 5′ adaptor region. This interpretation is adopted solely for examination and does not resolve the lack of clarity in the claim language.
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.
Claim(s) 7 and 15 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.
Claim(s) 7 and 15 recites “a 3′ blocked N6” which is unclear as to what is blocked and what N6 means structurally. The claims does not make clear whether “3′ blocked” refer to the 3’ terminus of oligonucleotide, the N6 region, or another portion thereof. The specification also failed to define claimed “a 3′ blocked N6” limitation.
For purposes of examination only, and to facilitate a complete analysis of the claim, the Examiner interprets the “a 3′ blocked N6” limitation as requiring oligonucleotide has a chemical block at the 3' end to stop unwanted extension. The term “N6” is interpreted broadly as a six nucleotides region of unspecified sequence.
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 pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Grunenwald et al. and Smith et al.
Claim(s) 1-3, 8-11, and 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Grunenwald et al. (US20100120098A1, Published: May 13th 2010, disclosed in IDS) in view of Smith et al. (Methods. 2009;48(3):226-232)
Regarding claims 1 and 9, Grunenwald discloses method of sequencing for determining DNA comprising: (a) performing in vitro transposition into target DNA molecules with transposase complexes, wherein each transposase complex comprises a double-stranded DNA transposase recognition sequence and a 5′ single-stranded DNA adaptor overhang (e.g. generating sequencing library by incubating target DNA in an in vitro transposition reaction with transposome complexes comprising a transposase and a transposon end composition. The transposon end composition comprises a transferrable strand having a transposase recognition sequence, a strand complements to the transposon end sequence (i.e. double strand), and an additional flanking sequencing tag at 5’ end. Transposition reaction is catalyzed by the transposase, and wherein the target DNA is fragmented to generate a plurality of target DNA fragments and a transferred strand of transposon end composition is joined to the 5′ ends of each of a plurality of the target DNA fragments, to produce a plurality of 5′ tagged target DNA fragments [¶0018 and Fig. 4]. The tag domains of target DNA fragments may comprise appropriate adapter for downstream amplification and sequencing analysis [¶0021])
However, Grunenwald does not disclose the method is a bisulfite sequencing method wherein all cytosine (C) residues of the adaptor/tag are methylated and that the resulting tagged DNA fragments are subjected to bisulfite treatment.
Smith discloses preparing DNA methylation sequencing libraries using adapter containing 5-meC instead of C to prevent deamination during the bisulfite reaction. The DNA labeled fragments then subjected to bisulfite conversion, follow by amplification and sequencing [Abstract, Fig 1.A, and page 228-231].Smith further discloses that bisulfite seq protocols rely on bisulfite conversion, which will change nucleotide sequence and retain converted fragments as single stranded DNA that will pose challenges in adapter ligation, hence it is advantageous for adapters to be installed before the treatment [Page 228].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Grunenwald’s sequencing tag portion of transposon end composition to contain 5-meC instead of Cytosine because Smith explicitly teaches the advantage of installing the adapter before bisulfite treatment and the probe need to be preserved during bisulfite treatment. A skilled artisan would have been motivated to made the modification to allow transposase generated library to be used for bisulfite sequencing and determination of DNA methylation.
Regarding claims 2, 3, 10, and 11 Grunenwald discloses incorporating a second adaptor to the DNA fragments after step (b) and before step (c), wherein the second adaptor is designed to facilitate nucleic acid amplification in step (c). (e.g. In some embodiments, the methods further comprise incubating the plurality of 5′-tagged target DNA fragments with at least one nucleic acid modifying enzyme under conditions wherein a 3′ tag is joined to a 3′ end of the 5′-tagged target DNA fragment to produce a comprising di-tagged target DNA fragments. [¶0018]. Amplifying comprises a polymerase chain reaction using a first and a second oligonucleotide primer, each comprising 3′ end portions, wherein at least the 3′ end portion of the first PCR primer is complementary to the 3′ tag of the di-tagged target DNA fragments [¶0025]. A skilled artisan would have understood that )
Regarding claims 8 and 16, Grunenwald discloses the method further comprises the step of modifying the double-stranded DNA transposase recognition sequence with the single-stranded DNA adaptor overhang before step (a). (e.g. the reference describes the transposon end composition comprises a transferrable strand having a transposase recognition sequence, a strand complements to the transposon end sequence (i.e. double strand), and an additional flanking sequencing tag at 5’ end. The whole complex is assembled before incubation with target DNA [¶0018 and Fig. 4].
Grunenwald et al., Smith et al., and Mckernan et al.
Claim(s) 4 and 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Grunenwald et al. (US20100120098A1, Published: May 13th 2010, disclosed in IDS) in view of Smith et al. (Methods. 2009;48(3):226-232) and Mckernan et al. (US20100120034A1, Published: May 13th 2010).
Regarding claims 4 and 12, Grunenwald and Smith do not disclose incorporating the second adaptor comprises performing nick translation to generate adaptor-flanked DNA fragments in which each strand has both the 5′ methylated adaptor and a 3′ adaptor that is not methylated.
Mckernan discloses preparing DNA constructs for bisulfite base methyl analysis using primer adapters and nick translation reaction. The method involves DNA fragments are circularized to produce a double- stranded circular DNA comprising a nick on one strand. Circular DNA with a nick is then treated with a nick translation generating two tag regions of a mate-pair. Mckernan further teaches first tag sequence may be converted by a methylation conversion agent, while the second tag sequence is not converted by a methylation conversion agent. The result construct comprises two tags, one tag region has these resistant nucleotides, and the other tag region does not. The DNA can be cut open (linearized) before this step and amplified [¶0008-0010]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to employ Mckernan’s nick translation technique when incorporating second adaptor of Grunenwald and Smith method because this dual-tag approach enables protected tag (with methylation) with high complexity as an anchor to map a low-complexity converted tag (without methylation) from the same mate-pair. This helps align difficult sequences, map genomes, and measure methylation levels across mixed cell samples [Mckernan ¶0073]. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Grunenwald et al., Smith et al., and Dahl et al.
Claim(s) 5-7 and 13-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Grunenwald et al. (US20100120098A1, Published: May 13th 2010, disclosed in IDS) in view of Smith et al. (Methods. 2009;48(3):226-232) and Dahl et al. (Genome Res. 2008;18(3):469-476).
Regarding claims 5 and 13, Grunenwald and Smith do not disclose second adapter is added after bisulfite treatment and before amplification.
Dahl discloses that bisulfite treated DNA remains suitable for subsequent incorporation of oligonucleotide sequences, wherein oligonucleotide probes are ligated to bisulfite-treated DNA and the product subsequently amplified by polymerase chain reaction (PCR). The probes carry universal tails to allow amplification of multiple ligation products using a common primer pair. [abstract]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Grunenwald and Smith by incorporating Dahl’s probe/adapter, which carry universal tails to allow amplification, after bisulfite treatment and before amplification because this method is a simple, highly flexible, and inexpensive way to prepare bisulfite treated DNA library for amplification [Dahl’s Discussion section].
Regarding claims 6 and 14, Grunenwald discloses incorporation of adapter comprises the step of incubating 5′-tagged DNA fragments with TdT and a dNTP under conditions and for sufficient time wherein the 5′- and 3′-tagged DNA fragments that have a second tag comprising a homopolymeric DNA tail is synthesized. The homopolymeric tail is used as the priming site for nucleic acid synthesis [¶0150]. Although Grunenwald does not explicitly recite second adaptor is incorporated by adding an adenosine (A) tail to the DNA fragments, and appending a 3′ adaptor to the DNA fragments using a 3′ poly-T 5′ adaptor primer, it would have been prima facie obvious to a person of ordinary skill in the art to understand from Grunenwald disclosure that homopolymeric DNA tail of second tag can be poly A tail, which means the complimentary homopolymeric DNA primer is poly T tail.
Regarding claims 7 and 15, Grunenwald discloses incorporation of adapter comprises allowing the DNA fragments to extend on an oligonucleotide comprising a 3′ blocked N6 and a 5′ adaptor overhang. (e.g. method of terminal tagging comprise the steps of: 1) providing a terminal tagging oligonucleotide comprising a 5′-portion and 3′-portion, wherein the 5′-portion hybridize to the target DNA fragment, the 3′-portion comprise degenerate sequence of 6 random nucleotides, of which, the 3′-terminal nucleotide is blocked so that it is not capable of being extended by the DNA polymerase; hybridize the target DNA with the tagging oligonucleotide; 3) DNA polymerase extend the 3′-termini of DNA using the terminal tagging oligonucleotide as a template [¶0178-0181]).
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
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