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 .
Claim Status
Claims 1-15, 17-28, and 31-34 are pending and under examination. Claim 30 was cancelled prior to examination. Claims 16 and 29 have been cancelled. Claims 1-2, 4, 6, 11, 13, 18, 19, 23, 25, 26, and 31 have been amended. Claims 32-34 are new. Claims 1, 2, 26, and 32 are independent claims.
Response to Arguments
Objections Withdrawn
The objection to the abstract for exceeding 150 words is withdrawn following the applicants’ amendments.
The objection to claims 2, 11, 13, and 29 for informalities are withdrawn following the applicants’ amendments and cancellation of claim 29.
Objections maintained
The specification remains objected to because of the use of improperly demarcated trademarks as cited in the previous office action.
New Objections
Claim 4 is objected to because of the following informalities: spelling errors. Appropriate correction is required.
Rejections Withdrawn
The rejection of claims 4, 6, 16, 18-19, 25, 29 and 31 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention is withdrawn following the applicants’ amendments and cancelation of claims 16 and 29.
The rejection of claims 2, 26-29 under 35 U.S.C. 102(a)(1) as being anticipated by Wu et al., (US 2014/0113839 A1, published Apr. 24, 2014, on IDS) is withdrawn following the applicants’ amendments and cancelation of claim 29.
The rejection of claims 1-14 and 20-29 under 35 U.S.C. 103 as being unpatentable over Wu et al., is withdrawn following the applicants’ amendments and cancellation of claim 29.
The rejection of claims 1-15 and 20-29 under 35 U.S.C. 103 as being unpatentable over Wu et al. in view of Shiraishi et al. is withdrawn following the applicants’ amendments and cancellation of claim 29.
The rejection of claims 1-16 and 20-29 under 35 U.S.C. 103 as being unpatentable over Wu and Shiraishi in view of Sartori et al. is withdrawn following the applicants’ amendments and cancellation of claims 16 and 29.
The rejection of claims 1-29 and 31 under 35 U.S.C. 103 as being unpatentable over Wu, Shiraishi, and Sartori in view of NEB SNAP-Capture Magnetic Beads Product Protocol and SNAP-tag Vector Protocol is withdrawn following the applicants’ amendments and cancellation of claims 16 and 29.
New Rejections
Claim Interpretation
One of ordinary skill in the art would recognize that a substrate in enzymology refers to the molecule upon which an enzyme acts that leads to the formation of products. Therefore, the term “DNA substrate” used in claims 1, 26, 27, and 32, and subsequently in the claims that depend upon them, is interpreted to refer exclusively to the single-stranded DNA (ssDNA) comprising a modified nucleotide prior to cleavage by the ssDNA cleavage enzyme.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 18-20, 31-34 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
In regards to claims 18-19, 31, and 33-34 the specification discloses the use of “SNAP-tag” fusion proteins. As understood in the art, SNAP-tag is a specific engineered variant of O6-alkylguanine-DNA alkyltransferase (AGT) that has been modified to covalently react with benzyl guanine derivatives. However, the specification does not describe or otherwise reasonably convey possession of the broader genus of O6-alkylguanine-DNA alkyltransferases. In particular, the specification does not disclose structural features, functional variants, or representative species of O6-alkylguanine-DNA alkyltransferase beyond SNAP-tag, nor does it indicate that other AGT enzymes would be suitable substitutes. Accordingly, the disclosure of a single engineered species (SNAP-tag) does not provide adequate written description support for the broader genus now recited in the claims.
In regards to claim 20, depends of claim 1, and further recites, “wherein the modified nucleotide is an 8-oxoG. The specification does not describe any enzyme that simultaneously targets 8-oxoG and generates a first ssDNA fragment with 3’-OH and a second ssDNA fragment that contains the modified base, which are recited limitations of claim 1. The specification describes using Tk AGOG to target modified ssDNA containing 8-oxoG, however the use of Tk AGOG does not generate a second ssDNA fragment that maintains the modified nucleotide (see Fig. 5A and pg. 10, 2nd para. of the specification, and pg. 9 of remarks). Accordingly, the specification fails to demonstrate possession of enzymes that both target 8-oxoG modified ssDNA and generate a first ssDNA fragment having a 3’-OH and a second ssDNA fragment having the modified nucleotide.
In regards to claim 32, the claim recites “the ssDNA cleavage enzyme is an AGOG,” which encompasses a genus of archaeal 8-oxoguanine DNA glycosylase (AGOG) enzymes. However, the specification fails to reasonably convey to one of ordinary skill in the art that the inventor had possession of this full genus at the time of filing. The specification discloses, at most, a single species, namely Tk AGOG (from Thermococcus kodakarensis), and does not provide a representative number of species across the breadth of AGOG enzymes. Further, the specification does not describe common structural features, conserved sequence motifs, or other identifying characteristics sufficient to define the claimed genus, nor does it provide a structure-function correlation that would allow a person of ordinary skill in the art to recognize which AGOG enzymes across different archaeal species would be suitable for use in the claimed method, particularly given the known diversity of glycosylase enzymes and variability in substrate specificity and cleavage chemistry across AGOG homologs. Accordingly, the disclosure does not demonstrate possession of the claimed genus of AGOG enzymes, and the claim is therefore not supported by an adequate written description.
Furthermore, the claim recites that “the ssDNA cleavage enzyme is an AGOG,” and that the method results in “a first ssDNA fragment having 3’OH”. The claim language reasonably conveys that the recited ssDNA cleavage enzyme (i.e. the AGOG enzyme) is responsible for generating the claimed 3’OH terminus upon cleavage of the ssDNA.
However, the specification does not reasonably convey that AGOG enzymes produce 3’OH terminus upon cleavage. Rather, as understood in the art, AGOG enzymes (including TKO AGOG) are bifunctional DNA glycosylase/AP lyases that cleave DNA via β-elimination, producing 3’-blocked (e.g. a 3’-α,β-unsaturated aldehyde), not a 3’ hydroxyl (see Gehring et al. (of record), Fig. 2 for the mechanism of TKO AGOG ssDNA cleavage). The formation of 3’OH from such intermediates requires the action of an additional enzyme, such as an AP endonuclease (e.g. Endonuclease IV), which is not recited as part of the ssDNA cleavage enzyme.
The specification does not describe or suggest that AGOG enzymes alone are capable of producing the claimed 3’OH. Accordingly, the specification fails to demonstrate possession of the claimed invention, and the claim is therefore not supported by an adequate written description.
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 6, 8, and 23 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.
Claim 6 recites the limitation "the immobilized DNA" in line 2 and “the substrate” in line 3. There is insufficient antecedent basis for these limitation in the claim.
Claim 8 recites the limitation “the ssDNA containing a modified nucleotide proximate to its 5’ end” spanning lines 1 and 2. There is insufficient antecedent basis for these limitations in the claim. Furthermore, the term “proximate” in claim 8 is a relative term which renders the claim indefinite. The term “proximate” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Specifically it is unclear how near to the 5’ end the modified base must be or how near the modified base cleavage must occur.
Claim 23 recites the limitation “the single-stranded oligonucleotide” spanning lines 1 and 2. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 103
Claims 1, 3-4, 8-9, 13-15, 21-22, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Shiraishi et al. (“A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus,” Nucleic Acids Res., 2015, of record).
Shiraishi is in the field of DNA cleavage enzymes, and teaches a method comprising:(a) combining a single stranded DNA (ssDNA) comprising a modified nucleotide (i.e., deoxyinosine (dI), deoxyuridine (dU), xanthine, or a tetrahydrofuran abasic (AP) site) with a ssDNA cleavage enzyme, namely Endonuclease Q, capable of cleaving the ssDNA at the modified nucleotide (see Fig. 4, Fig. 6 demonstrating cleavage of ssDNA substrates containing modified nucleotides). Shiraishi further teaches that such cleavage generates a first ssDNA fragment having a 3'OH and a second ssDNA fragment having the modified nucleotide as EndoQ cleaves at the lesion site (5’ to the modified nucleotide) to produce a 3’-OH terminus and a 5’-phosphate on the fragment retaining the modified nucleotide, as further evidence by re-ligation experiments demonstrating the presence of 3’-OH and 5’-phosphate termini (see Fig. 4 and Fig. 7). Shiraishi also teach cleaving at least 95% of the ssDNA at the modified nucleotide, as the gel electrophoresis results show near-complete conversion of substrate to cleavage products (see Fig. 4 and Fig. 6). Shiraishi perform the cleavage reaction using an enzyme-to-substrate molar ratio of approximately 2:1 (e.g. 20 nM of EndoQ and 10 nM of ssDNA substrate), but do not explicitly disclose a ratio of less than 1:1. The extent of cleavage is a function of reaction conditions, including enzyme concentration, substrate concentration, and reaction time. As described by Michaelis-Menten kinetics, the reaction rate depends on enzyme and substrate concentrations, and changing enzyme concentration relative to substrate correspondingly can adjust the extent of substrate conversion over time. Shiraishi demonstrates cleaving at least 95% of the ssDNA in 15 minutes. Shiraishi demonstrates greater than 95% cleavage in 15 minutes at an enzyme to DNA ration of 2:1 (20 nM enzyme, 10 nM substrate). Under Michaelis-Menten principles, reaction rate depends on enzyme concentration, and, as a first approximation, reducing the enzyme concentration from 2:1 ratio to a 1:1 ratio would be expected to reduce the reaction rate proportionately, such that a similar extent of cleavage would be expected in approximately 30 minutes, with somewhat longer times expected at ratios below 1:1 (see Lehninger Principles of Biochemistry, 6th Ed., pgs. 202-207, 2013). Thus, adjusting the enzyme-to-substrate ratio while maintaining the claimed level of cleavage would have been a predictable matter of routine optimization of a result-effective variable. See In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Therefore it would have been obvious to one of ordinary skill in the art at the time of filing to adjust the molar ratio of enzyme to DNA substrate, including reducing the ratio to 1:1 or less. Accordingly, it would have been obvious to arrive at the claimed method.
In regards to claim 3, Shiraishi teaches that the cleavage reaction occurs within the claimed time frame, as the disclosed reaction conditions demonstrated cleavage of ssDNA substrates containing modified nucleotides in 15 minutes (see Fig. 4).
In regards to claim 4, Shiraishi teaches that the ssDNA substrate comprises, in a 5’ to 3’ direction, a 5’ end, a modified nucleotide (deoxyinosine), and 3’ end, as evidenced by the disclosed substrates containing an internal modified nucleotide (see Fig. 4A illustrating a ssDNA sequence with dI located between the ends).
In regards to claims 8 and 9, Shiraishi teaches ssDNA substrates comprising a modified nucleotide (dI) and a labeled DNA substrate, including 3’-end labeled ssDNA (e.g. 3’-FITC labeled oligonucleotides) for analysis of cleavage products (see Fig. 4C and associated description).
In regards to claims 13-15, Shiraishi teaches that the ssDNA cleavage enzyme comprises a thermophilic endonuclease, namely Endonuclease Q, derived from thermophilic archaeal organisms including Pyrococcus furiosus and Thermococcus kodakarensis.
In regards to claims 21 and 22, Shiraishi teaches using deoxyinosine, deoxyuridine, and tetrahydrofuran (AP)-containing oligonucleotides (see pg. 2854, left column, “DNA Substrates”).
In regards to claim 26, Shiraishi teaches a composition comprising an artificial mixture of a ssDNA-cleaving archaeal endonuclease, namely Endonuclease Q, and a synthetic DNA substrate comprising a modified nucleotide, as Shiraishi discloses a cleavage assay in which EndoQ from archaeal organisms (e.g. Pyrococcus furiosus and Thermococcus kodakarensis) is combined with synthetic oligonucleotide substrates containing defined modified nucleotides (see Fig. 6, Fig. S2, Table S1, pg. 2854 right col. last 2 para.). Shiraishi further teaches that the ssDNA-cleaving archaeal endonuclease is capable of cleaving the ssDNA at the modified nucleotide, as demonstrated by cleavage of substrate containing dI, dU, dX, and abasic tetrahydrofuran sites (see Fig. 6, Fig. S2, Table S1). Shiraishi discloses performing such reactions at an enzyme-to-substrate molar ratio of approximately 2:1 (e.g. 20 nM enzyme and 10 nM DNA substrate), but do not explicitly disclose a ratio of less than 1:1. However, it would have been obvious to one of ordinary skill in the art to adjust the molar ratio of enzyme to substrate, including reducing the ratio to less than 1:1, as enzyme concentration relative to substrate is a result-effective variable routinely optimized to achieve efficient cleave, with a reasonable expectation of success. See In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Accordingly, it would have been obvious to arrive at the claimed composition of “artificial mixture of a ssDNA- cleaving archaeal endonuclease or glycosylase and a synthetic DNA substrate comprising a modified nucleotide, wherein (i) the ratio of (1) the ssDNA-cleaving archaeal endonuclease or glycosylase to (2) the synthetic DNA substrate is less than a 1:1 molar ratio;(ii) the ssDNA-cleaving archaeal endonuclease or the ssDNA-cleaving archaeal glycosylase is capable of cleaving the ssDNA at the modified nucleotide; and(iii) the modified nucleotide is selected from the group consisting of deoxyuridine, deoxyinosine, 8-oxoG, deoxyxanthosine, and a tetrahydrofuran-type abasic.”
Claims 1-15, 20-22, and 24-28 are rejected under 35 U.S.C. 103 as being unpatentable over Shiraishi, as applied to claims 1, 3-4, 8-9, 13-15, 21-22, and 26 above, included here for reasons supra, in further view of Wu et al. (US 2014/0113839 Al, published Apr. 24, 2014 on IDS).
In regards to claims 2 and 5, Shiraishi teaches a method comprising combining a single stranded DNA (ssDNA) comprising a modified nucleotide with (ii) a ssDNA cleavage enzyme capable of cleaving the DNA at the modified nucleotide in the ssDNA to generate after cleavage, a first ssDNA fragment having a 3'OH and a second ssDNA fragment having the modified nucleotide as discussed above in relation to claim 1 (see Shiraishi Fig. 4, Fig. 6 demonstrating cleavage of ssDNA substrates containing modified nucleotides, and Fig. 7 demonstrating end chemistries capable ligation (3’OH and 5’phosphate)). Shiraishi does not explicitly disclose that the ssDNA is immobilized on a substrate or that cleavage releases a fragment from the substrate.
Wu, however, teaches DNA molecules that are immobilized on a substrate (see e.g. Wu [0054]-[0058]) and subjected to enzymatic processing in an assay format, including the use of endonucleases (see e.g. Wu [0068]-[0072]). Wu further teaches that cleavage or processing of such immobilized DNA results in the release of DNA fragments from the substrate (see e.g. Wu [0056]). It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to apply the cleavage method of Shiraishi to the immobilized DNA substrates of Wu as both references are directed to enzymatic processing of DNA and immobilization of DNA on a substrate for analysis represents a well-known and predictable modification to facilitate detection, spatial organization, and assay performance. Accordingly it would have been obvious to combine.
In regards to claim 6, for the reasons set forth above in respect to claims 1 and 5, Shiraishi teaches cleaving ssDNA at a modified nucleotide using EndoQ, wherein cleavage occurs on the 5’ side of the modified nucleotide, thereby generating a fragment that includes the modified nucleotide and nucleotides 3’ to the modified nucleotide (see Shiraishi Fig. 4 (showing the cleavage products), Fig. 7 (showing end chemistries), Fig. 10 (showing representative cutting location using dsDNA model), pg. 2681 left col. 3rd para.). Wu teaches ssDNA immobilized on a substate and subjected to enzymatic processing, including cleavage resulting in release of DNA fragments from the substrate. It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to apply the cleavage method of Shiraishi to the immobilized DNA substates of Wu, thereby cleaving the immobilized DNA and releasing from the substate a fragment comprising the modified nucleotide and nucleotide 3’ to the modified nucleotide, as such combinate represents a predictable use of know DNA cleavage chemistry in a known immobilized assay format.
In regards to claim 7, Wu teaches using reverse transcription steps to generate and amplify ssDNA (see Wu [0114], [0118]) Furthermore, generating ssDNA by reverse transcribing an RNA is a standard procedure in molecular biology. One of ordinary skill in the art would know how to generate ssDNA from RNA using reverse transcriptase.
In regards to claim 10-12, Wu teaches the use of many different types of solid support, including beads, plates, and arrayed along a two-dimensional surface ([0054]-[0058]).
In regards to claims 20-22, Wu teaches the use of 8-oxoG, deoxyuridine, or deoxyinosine as modified bases and cleavage targeting ([0071]-[0072]).
In regards to claim 24-25, Wu teaches that the polynucleotides are synthesized on the plate (Fig. 2, [0052], and throughout) and teaches that the nucleotides are used as probes binding to specific substrates (Abstract and throughout). Merriam-Webster dictionary defines an aptamer as “a short segment of DNA, RNA, or peptide that binds to a specific molecular target (such as a protein)”. While Wu never call their probes aptamers, one of ordinary skill in the art would recognize that their probes meet these requirements. Furthermore, Wu teaches multiple methods of synthesizing the polynucleotides, all of which are either chemical or enzymatic (Figs. 1 and 2, [0036], [0051]-[0052], and throughout).
In regards to claim 27, for the reasons set forth above, Shiraishi teaches the limitations of claim 26. Shiraishi teaches a composition comprising a mixture of ssDNA-cleaving archaeal endonuclease, namely EndoQ, and a synthetic DNA substrate comprising a modified nucleotide (see e.g. Shiraishi Fig. 4). Shiraishi does not explicitly disclose that the synthetic DNA substrate is immobilized on a solid support. Wu teaches ssDNA immobilized on a substate and subjected to enzymatic processing, including cleavage resulting in release of DNA fragments from the substrate (see e.g. Wu [0054]-[0058]). It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to apply the composition of synthetic DNA of Shiraishi to the immobilized DNA substates of Wu, thereby cleaving the immobilized DNA and releasing from the substate a fragment comprising the modified nucleotide and nucleotide 3’ to the modified nucleotide, as such combinate represents a predictable use of know DNA cleavage chemistry in a known immobilized assay format.
In regards to claim 28, Wu teaches the use of many different types of solid support, including beads, plates, and arrayed along a two-dimensional surface (see e.g. Wu [0054]-[0058]).
Claims 1, 3-4, 8-9, 13-15, 17-19, 21-22, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Shiraishi as applied to claims 1, 3-4, 8-9, 13-15, 21-22, and 26 above, included here for reasons supra, in further view of NEB SNAP-Capture Magnetic Beads Product Protocol (of record) and SNAP-tag Vector Protocol (of record).
Shiraishi teaches all of the limitations of claim 1 for which claims 17-19 depend. Claim 17 further recites that the ssDNA cleavage enzyme comprises a fusion protein. The SNAP-tag literature teaches that enzymes may be engineers as fusion proteins by combining functional protein domains, including 06-alkylguanine-DNA alkyltransferase domains, with other proteins to enable labeling, detection, or functional modification while retaining enzymatic activity. It would have been obvious to one of ordinary skill in the art at the time of filing to modify the ssDNA cleavage enzyme of Shiraishi to comprise a fusion protein, as such fusion constructs represent routine and predictable modifications with a reasonable expectation of success. The SNAP-tag literature teaches that O6-alkvlguanine-DNA alkyltransferase are commonly used as fusion partners in engineered proteins for covalent labeling and functionalization. It would have been obvious to incorporate such an alkyltransferase domain into the fusion protein of claim 17 for these known purposes.
The SNAP-Capture Magnetic Bead Product Protocol demonstrates that O6-alkvlguanine-DNA alkyltransferase enable covalent immobilization of proteins on solid supports, such as benzyluanine-functionalized surfaces. It would have been obvious to immobilize the fusion protein on a solid support as taught by the NEB protocol, as immobilization of enzymes on solid supports is a well-known and predictable technique to facilitate assay performance, spatial organization, and reuse.
Claims 1, 3-4, 8-9, 13-15, 21-23, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Shiraishi as applied to claims 1, 3-4, 8-9, 13-15, 21-22, and 26 above, included here for reasons supra, in further view of Stoeckius et al. (US 2018/0251825 Al, published Sep. 6, 2018).
As discussed above, Shiraishi teaches all of the limitations of claim 1 for which claim 23 depends. Claim 23 further recites “wherein the single stranded oligonucleotide comprises a barcode of randomly generated nucleotides”. Shiraishi teaches a method in which a ssDNA comprising a modified nucleotide is combined with a ssDNA cleavage enzyme, which cleaves the ssDNA at the modified nucleotide to generate the claimed fragments. Shiraishi does not explicitly teach that the ssDNA or another oligonucleotide comprises a barcode of randomly generated nucleotides. Stoeckius however teaches oligonucleotides comprising barcode sequences of randomly generated nucleotides for identification and tracking purposes in molecular assays (see e.g. Stoeckius [0087]). It would have been obvious to one of ordinary skill in the art to incorporate a randomly generated nucleotide barcode into the ssDNA of Shiraishi to enable identification, multiplexing, or tracking of DNA molecules, as such barcoding represents a well-known and predictable modification in nucleic acid assays with a reasonable expectation of success.
Claim 32 is rejected under 35 U.S.C 103 as being unpatentable over Sartori et al. ((“Pa -AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases”, Nucleic Acids Research, Volume 32, Issue 22, 15 November 2004, Pages 6531–6539, of record) in view of Endonuclease IV (NEB, https://www.neb.com/en-us/products/m0304-endonuclease-iv, accessed 4/1/2026).
Sartori is in the field of enzymology and teaches methods comprising combining a single-stranded DNA (ssDNA) comprising a modified nucleotide (8-oxoG, see Fig. 1) with a ssDNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide in the ssDNA (Pa-AGOG) to generate a first ssDNA fragment and a second ssDNA fragment wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (1:10) (see Sartori pg. 6535 left col. 2nd para.). Sartori demonstrates that more than 90% of substrate is processed within 15 minutes, and more than 95% is processed within an hour (see Sartori Fig. 4D). It would have been obvious to one of ordinary skill in the art to achieve cleavage of at least 95% of the ssDNA at the modified nucleotide through routine optimization of reaction conditions, including enzyme concentration and reaction time, as such parameters are result-effective variables with a reasonable expectation of success. See In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Sartori does not explicitly disclose that the resulting cleavage produces a fragment having a 3’OH, as AGOG cleavage via β-elimination produces 3’-blocked terminus. However, Sartori discloses that Endonuclease IV can produce 3’OH (see pg. 6535 left col. 2nd para.). It is well known that AP endonucleases such as Endonuclease IV process such 3’-blocked termini to generate a 3’ hydroxyl suitable for downstream processing (see Endonuclease IV product page). AP endonuclease such as Endonuclease IV have been commercially available (e.g. from NEB) as evidenced by their routine use in literature since at least 1997 (see Citation section of Endonuclease IV product page). It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to include an AP endonuclease such as Endonuclease IV in the reaction of Sartori to convert the 3’ blocked terminus generated by AGOG into 3’OH, as this represents a routine and well-understood step in base excision repair pathways, with a reasonable expectation of success.
Claims 32-34 are rejected under 35 U.S.C. 103 as being unpatentable over Sartori in view of Endonuclease IV as applied to claim 32 above and included here for reasons supra, in further view of NEB SNAP-Capture Magnetic Beads Product Protocol (of record) and SNAP-tag Vector Protocol (of record).
Sartori with Endonuclease IV teach the limitations of claim 32 for which claims 33 and 34 depend, however they do not teach that the ssDNA cleavage enzyme further comprises an 06-alkylguanine-DNA alkyltransferase. However, SNAP-tag literature teaches that 06-alkylguanine-DNA alkyltransferases are routinely uses as fusion partners to generate fusion proteins for labeling and detection, and functional modifications of proteins and enzymes, while retaining their underlying activity, and commercial kits to generate the fusion proteins are readily available (see “Expression of SNAP-tag Fusions”). It would have been obvious to one of ordinary skill in the art to modify the ssDNA cleavage enzyme of Sartori to further comprise an 06-alkylguanine-DNA alkyltransferase, as such fusion constructs represent a routine and predictable modification used to facilitate handling, labeling, or functionalization of enzymes, with a reasonable expectation of success.
Claim 34 further recites that the 06-alkylguanine-DNA alkyltransferase is bound to a solid support. SNAP-tag literature further teaches that 06-alkylguanine-DNA alkyltransferase can be used to immobilize proteins on solid supports via covalent attachment to benzylguanine-functionalized surfaces (see “Use SNAP-Capture Magnetic Beads”). It would have been obvious to one of ordinary skill in the art to immobilize the fusion proteins on a solid support as taught by NEB, as immobilization of enzymes on solid supports is well-known and predictable technique to facilitate assay performance, reuse, and spatial organization. Accordingly, it would have been obvious to arrive at the method of claims 33 and 34.
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/ Primary Examiner, Art Unit 1681
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