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 .
Claim Status and Formal Matters
This action is in response to papers filed 4/16/2025.
Claims 1, 5 have been amended.
Claims 1-7, 11, 13-17, 19-23. 27-30, 32-33, 35, 38-46 are pending.
The instant response is technically non-compliant with 37 CFR 1.121 as claim 33 is identified as previously presented, but is amended. However in order to promote compact prosecution and customer service the instant response will be examined. However, future amendments may not be entered and/or examined.
Applicant’s election without traverse of group I, from claim 3 - nucleotides comprising a chemical group capable of participating in a click chemistry reaction; From claim 4 - the alkyne group; From claim 5 - the alkyne group ;From claim 6- a molecule containing a group capable of participating in click chemistry From claim 7- cleavage domains that contain a sequence that is recognized by a cleavage enzyme. From claim 11- Type IIS restriction endonuclease. From claim 17- phi29 DNA polymerase. From claim 33- a chemical group capable of participating in click chemistry in the reply filed on 6/30/2023 is acknowledged.
Claims 27-30, 32 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 6/30/2023.
Claims 1-7, 11, 13-17, 19-23. 33, 35, 38-46 are being examined.
Priority
The instant application was filed 08/05/2021 is a national stage entry of PCT/EP2020/052868 with an international filing date: 02/05/2020 and claims foreign priority to 1901583.3, filed 02/05/2019
Information Disclosure Statement
The information disclosure statement filed 2/28/2025 fails to comply with the provisions of 37 CFR 1.98(a)(4) because it lacks the appropriate size fee assertion. It has been placed in the application file, but the information referred to therein has not been considered as to the merits.
The listing of references in the specification 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. It is noted the examiner has not compared the IDS submitted to those recited in the specification.
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 1-7, 11, 13-17, 19-23. 33, 35, 38-46rejected 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.
Claim 1 has been amended to provide, “consisting of” as a transitional phrase following the preamble and provides steps (a) to (e). However, claim 2 requires, “wherein the circular DNA molecule is double stranded and wherein the method comprises an additional step of cleaving a single strand of the circular DNA molecule to provide an RCA template, before the RCA reaction is performed.” The response asserts, “support for the amendments can be found throughout the PCT publication (W02020161187A1) at least, for example at page 15, lines 4-9; page 16, lines 24-27; page 3, lines 24-27; page 21, lines 16 to 23, line 3; and page 28, lines 30 to 29, line 17.” Review of the cited portions did not reveal support for consisting of language with the specific steps recited in the amended claims. Thus the amendment appears to introduce new matter.
Claim 1 has been amended to recite, “and wherein the circular DNA molecule contains a domain between the cleavage domains that encodes a primer binding site and/or a single strand cleavage site.” Claim 1 has later been amended to recite, “wherein said enzymatic cleavage results in the release of the complete functionalized single stranded functionalized oligonucleotides that consist of the oligonucleotide sequence without any additional nucleotides from the cleavage domains.” The response asserts, “support for the amendments can be found throughout the PCT publication (W02020161187A1) at least, for example at page 15, lines 4-9; page 16, lines 24-27; page 3, lines 24-27; page 21, lines 16 to 23, line 3; and page 28, lines 30 to 29, line 17.” The cited portions of the specification do not provide support for the combination of the recited elements of the claim as amended. The specification on page 11 recites, “The response asserts, “The functionalized oligonucleotides that are released may consist only of the oligonucleotide sequence (i.e. with no additional nucleotides), or they may comprise one or more additional nucleotides from the cleavage domains that border the oligonucleotide sequences at one or both ends. Thus in some embodiments, the functionalised oligonucleotides may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides from the cleavage domains at one or both ends.” Thus the specification supports no additional nucleotides in the oligonucleotide sequence or additional nucleotides of the cleavage domain, but not both. Further the amendment to recite “primer binding site” is not supported as the specification recites, “primer binding site” 5 times and all 5 times.. Each time “primer biding site is recited it is in the context of “comprise RCA primer binding site.” Thus the amendment to recite, “primer binding site” has broadened the scope of the claim from what the specification as originally filed envisions.
Response to Arguments
This is a new grounds of rejection necessitated by amendment.
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 1-7, 11, 13-17, 19-23. 33, 35, 38-46 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 1 has been amended to recite, “ wherein said enzymatic cleavage results in the release of the complete functionalized single stranded functionalized oligonucleotides that consist of the oligonucleotide sequence without any additional nucleotides from the cleavage domains” The claim has previously recites, “and wherein the circular DNA molecule contains a domain between the cleavage domains that encodes a primer binding site and/or a single strand cleavage site.” The specification and claims do not define what is required of a cleavage domain. However dependent claims require the cleavage enzyme is Nb.BsrDim Nt.BspQI, BSMBI or BSaI, each of which recognize multiple nucleotides and cleave outside the biding site. For example BsaI (https://www.neb.com/en-us/products/r0535-bsai?srsltid=AfmBOopGX1aKIUg5wUmsJr92pbgk4YeYGzKFDFHV--08OjHnC8rNl9xB, downloaded 6/12/2025)
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Further, it appears that the cleavage would encompass producing fragments for the hairpin structure and the intervening sequence, which both would be considered functionalized single stranded oligonucleotides. It is unclear how all the single stranded functionalized oligonucleotides would exclude sequences (domains) that are required to be present prior to cleavage. Thus the metes and bounds are unclear what is excluded from the functionalized nucleotide in view of the amendment.
Claim 1 provides “consisting” of language following preamble. Thus appearing to limit the claim from additional steps and/or reagents. However, claim 2 recites, “wherein the method comprises an additional step of cleaving a single strand of the circular DNA molecule to provide an RCA template, before the RCA reaction is performed.” Thus the metes and bounds are unclear if the claims are limited to the steps and reagents of claim 1 or allow additional steps as required by claim 2.
Claim 1 has been amended to recite, “encodes a primer binding site and/or a single stranded cleavage site.” The recitation of “encode” in genetics is generally associated with translation of nucleic acids into proteins. Thus it is unclear what sequences are required “encodes a primer binding site and/or a single stranded cleavage site.” If the intent is the single stranded nucleic acid comprises the recited elements, the claim should be amended to clarify.
Response to Arguments
These are a new grounds of rejection necessitated by amendment.
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.
Claim(s) 1- 7, 11, 13-17, 19-20, 24, 33, 35-37, 39-41 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kool (WO9909216) and Jarvius (WO2015/079042).
While claim 1 has been amended to recite, “wherein said enzymatic cleavage results in the release of single stranded functionalized oligonucleotides that consist of the oligonucleotide sequence without any additional nucleotides from the cleavage domains;,” The specification and claim do not define or provide a standard to differentiate the cleavage domain from any other nucleotide sequences. Thus the broadest reasonable interpretation is any functionalized single stranded oligonucleotide.
Further the claims has been amended to recite, “the circular DNA molecule contains a domain between the cleavage domains that encodes a primer binding site and/or a single strand cleavage site.” The broadest reasonable interpretation of a primer site is any nucleic acid sequence, as a primer can be designed to be complementary to any nucleic acid sequence.
Further the independent claim has been amended to provide consisting of language, which appears to be an attempt to limit the claim to only those steps. However, dependent claim 2 requires an additional step. Thus the broadest reasonable interpretation is claim 1 is not limited to only those steps and reagents.
Claim 1 has been amended to recite, “ wherein said enzymatic cleavage results in the release of the complete functionalized single stranded functionalized oligonucleotides that consist of the oligonucleotide sequence without any additional nucleotides from the cleavage domains” The claim has previously recites, “and wherein the circular DNA molecule contains a domain between the cleavage domains that encodes a primer binding site and/or a single strand cleavage site.” The specification and claims do not define what is required of a cleavage domain. However dependent claims require the cleavage enzyme is Nb.BsrDim Nt.BspQI, BSMBI or BSaI, each of which recognize multiple nucleotides and cleave outside the biding site. Thus it is unclear how part of the single strande nucleic acid produced would not comprise these sequences. Thus the metes and bounds are unclear what is excluded from the functionalized nucleotide in view of the amendment.
With regards to claim 1, Kool teaches rolling circle amplification (title, abstract throughout). Kool teaches providing a circular template, performing rolling circle amplification and production of cleaved amplified oligomers (figure 1). Kool teaches the use of functionalized dNTP ( phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, nucleotides with sugar modifications such as 2 '-0- 5 methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2 '-0-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides having base modifications such as C-5 substituted pyrimidines (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, 10 pyridyl-), 7-deazapurines with C-7 substituents (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-,
thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.is the rolling circle amplification (page 23). Kool teaches isolation of cleaved oligomers (page 37, lines 23-26, example 8). Kool teaches running products on a gel (page 48). This is isolating or purifying. Thus Kool teaches the steps of the claim.
Kool teaches, “wherein the single-stranded circular template contains at least one copy of a nucleotide sequence complementary to the oligonucleotide sequence
to be repeated in the oligonucleotide multimer. In a preferred method, a detectably labeled RNA oligonucleotide multimer is synthesized.” (page 8-9), which encompassed by, “wherein the circular DNA molecule contains a domain between the cleavage domains that encodes a primer binding site and/or a single strand cleavage site.”
Kool teaches, “Alternately, a Type-II restriction site can be encoded within a hairpin forming sequence, so that the entire cleavable group will be removed by the cleaving enzyme, leaving only the desired sequence, as in Example 3.”
Further, Jarviius teaches, “Figure 1 shows an embodiment using self-templated digestion (cleavage) without external cleavage oligonucleotides by means of stem-like structures formed within each RCA monomer repeat. Thus each monomer repeat contains two regions which are complementary to one another and may hybridize together to form a hairpin or stem-like structure. The self-complementary, palindromic, regions contain a recognition site for a palindromic restriction enzyme. After cleavage using a palindromic restriction enzyme, monomer units are released from which the loop part of the hairpin structures has been removed.” (page 13) Jarvius teaches, “Additionally, the methods of the invention may be used preparatively to synthesized multiple copies of a desired nucleic acid molecule. In such embodiments, the methods may include a further step of recovering or collecting the second or further RCP, or monomer units generated therefrom.” (page 19-20)
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Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claim to design the circular DNA of Kool to provide hairpins cleavable by enzymes to produce single stranded functionalized oligonucleotides via the method of Kool. The artisan would be motivated to produce functionalized single stranded oligonucleotide. The artisan would have a reasonable expectation of success as the artisan is merely using known methods of designing circular DNA which provide for stem-loop RCA products for further amplification of the signal.
With regards to claim 2, Kool teaches cleavage and replication of double stranded circular DNA (page 3).
With regards to claim 3-5, Kool teaches nucleotides with vinyl groups, alkynyl groups, etc. (page 23).
With regards to claim 6, Kool teaches label dNTP (page 29).
With regards to claim 7, Kool teaches cleave with a restriction enzyme to cleave double stranded product (page 36).
With regards to claim 11, 13-16 Kool teaches type II restriction endonucleases.
With regards to claim 17, Kool teaches phi 29 polymerase (page 4).
With regards to claim 19-20, the circular DNA is nucleic acids and thus each is cleavable and different.
With regards to claim 33, Kool teaches the sequences are 20-30 nucleotides (PAGE 15).
With regards to claims 35-37, Kool teaches, “It is to be understood that the term "nucleotides" as used herein includes, but is not limited to, naturally occurring and/or synthetic nucleotides, nucleotide analogs, and nucleotide derivatives. For example, the term includes naturally occurring DNA or RNA monomers, nucleotides with backbone modifications such as peptide nucleic acid (PNA) (M. Εgholm et al., Nature, 365, 566-568 (1993), incorporated by reference in its entirety), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, nucleotides with sugar modifications such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides having base modifications such as C-5 substituted pyrimidines (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-), 7-deazapurines with C-7 substituents (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.
With regards to claims 39-40, Jarvius teaches, “A RCA template typically may comprise about 20-1000 nucleotides, e.g. 26- 1000, 30-1000, 30-900, 60-900, 40-800, 50-700, 60-600, 70-500, 80-400, 90-300 or 100-200 nucleotides, such as at least 20, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200 or 250 nucleotides. More particularly in the methods of the invention the first RCA template may comprise 20-150, e.g. 20-120, 20-100, 25-150, 25-120, 30-150, 30-120, 30-100, 40-150, 40-120, 40-100 nucleotides.”
With regards to claim 41, . Kool teaches the use of functionalized dNTP ( phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, nucleotides with sugar modifications such as 2 '-0- 5 methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2 '-0-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides having base modifications such as C-5 substituted pyrimidines (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, 10 pyridyl-), 7-deazapurines with C-7 substituents (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-,thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.is the rolling circle amplification (page 23).
Response to Arguments
The response traverses the rejection by asserting, “Applicant asserts that neither Kool nor Jarvius disclose a circular DNA molecule containing a domain between the cleavage domains that encodes a primer binding site and/or a single strand cleavage site or the release of single stranded functionalized oligonucleotides that consist of the oligonucleotide sequence without any additional nucleotides from the cleavage domains.” This argument has been thoroughly reviewed but is not considered persuasive as the claim is unclear what is required or excluded by this recitation. Further the prior art of record teaches the use of the same Type IIs restriction enzymes applicant requires in dependent claims. Thus this argument is not persuasive.
The response continues by asserting, “Applicant further asserts that the combination of Kool and Jarvius would have required additional amplification steps. The passage on pages 19-20 of Jarvius teaches that monomer units may be generated from the "second or further RCA". However, as noted above, the claims now exclude a second RCA reaction.” This argument has been thoroughly reviewed but is not considered persuasive as the instant claim 2 requires additional steps, thus so the independent must allow for additional step. Further the Kool in figure 1 teaches the use of a single rolling circle amplification. Finally as consistent with the responses arguments, Jarvius teaches in some embodiments there may be a second RCA step. Jarvius does not teach this is required to produce the single stranded nucleic acid.
The response continues arguing the method would not provide the single stranded functionalized oligonucleotides that consist of the oligonucleotide sequence without any additional nucleotides from the cleavage domain. First as noted in the rejection and the 112 it is unclear what is excluded by this recitation. Further, the art of record suggests the use of the same type IIs restrictions enzymes to produce the oligonucleotides, thus either anticipating or rendering this obvious. Finally it appears that the cleavage would encompass producing fragments for the hairpin structure and the intervening sequence, which both would be considered functionalized single stranded oligonucleotides. It is unclear how all the single stranded functionalized oligonucleotides would exclude sequences (domains) that are required to be present prior to cleavage.
The response continues by arguing, “The claimed method provides a universal system with the configuration of the circular DNA molecule that allows for the same primer binding site and/or single strand cleavage site to be used for the production of any single stranded functionalized oligonucleotides. In contrast, if the methods of Kool and Jarvius were modified to include a primer binding site or single strand cleavage site in the RCA template, these sequences would have formed part of the molecules that are produced and thus sequences of these domains would have needed to be based on the sequence of the desired sequence in order to achieve products that consist of the desired sequence. Moreover, the inclusion of a primer binding site or single strand cleavage site in the final product may have interfered downstream processing of the molecules.” The arguments with respect to downstream processing are confusing as the applicant has attempted to close the method for downstream processing by the use of consisting of language. Further both Jarvius and Kool both teach the use of the primers, thus this argument is confusing as well. Finally either the hairpin structure or the intervening sequence would have to have the primer sequences as the whole circle is replicated in RCP.
The response continues by arguing, “Furthermore, as explained in the response (filed on May 28, 2024) to Office Action mailed on March 28, 2024, the inclusion of one or both of these domains functions to improve the control of the RCA reaction; as the RCA reaction can be initiated outside of the oligonucleotide sequence, no partial oligonucleotides are produced (e.g. no fragments of the desired oligonucleotide are generated by the method). Moreover, the inclusion of a nickase domain also allows the starting DNA circle to be double stranded. This has the effect of improving the uniformity of the oligonucleotides produced by the method, as the starting template may be obtained from sequence-verified plasmid DNA derived from a single bacterial colony.” This argument has been thoroughly reviewed but is not considered persuasive as the this is confusing as it requires additional steps as it depends from claim 2, which the response alleges is not required for the instant invention. Further the response is arguing limitation not in the claim as the claim does not recite a “nickase domain.” Further it is unclear what is required of nickase domain.
Thus the rejection is maintained
Claim(s) 1- 7, 11, 13-17, 19-20, 24, 33, 35-37, 39-40 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kool (WO9909216) and Jarvius (WO2015/079042) as applied to claims 1- 7, 11, 13-17, 19-20, 24, 33, 35-37, 39-40 above, and further in view of El-Sagheer (| Chem. Soc. Rev., 2010, 39, 1388–1405)
Kool and Jarvius do not specifically teach the use of dNTP functionalized with alkynes groups for click chemistry (8011, 2nd column, 1st paragraph).
El-Sagheer teaches, “The best example of this new class of extremely efficient chemical reactions is the CuI catalyzed [3+2] azide–alkyne cycloaddition (CuAAC)reaction.1,2 There is a great deal of interest in developing new synthetic methods to construct chemically modified DNA oligonucleotides (ODNs) for biological and nanotechnological applications and it quickly became apparent to researchers in the nucleic acids field that the CuAAC reaction has great potential. The features of the click ligation reaction that are potentially useful in such applications are: Azides and alkynes can be attached to nucleic acids without greatly disturbing their biophysical properties. Azides and inactivated alkynes are almost entirely unreactive towards the functional groups normally encountered in nature; they react only with each other. The triazole unit is extremely stable, and is not toxic. In this review we describe the use of click chemistry across the nucleic acids field, focusing on synthetic strategies and briefly describing some important practical applications. The basic CuAAC click reaction is shown in Fig. 1..”
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use alkyne modified dNTPs for rolling circle amplification. The artisan would be motivated to use alkyne modified dNTPs to allow for a molecular handle and/or easy addition or attachment to additional elements. The artisan would have reasonable expectation of success as the artisan is merely using known reagents in known assays.
With regards to claims 39-40, Jarvius teaches, “A RCA template typically may comprise about 20-1000 nucleotides, e.g. 26- 1000, 30-1000, 30-900, 60-900, 40-800, 50-700, 60-600, 70-500, 80-400, 90-300 or 100-200 nucleotides, such as at least 20, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200 or 250 nucleotides. More particularly in the methods of the invention the first RCA template may comprise 20-150, e.g. 20-120, 20-100, 25-150, 25-120, 30-150, 30-120, 30-100, 40-150, 40-120, 40-100 nucleotides.”
Response to Arguments
The response traverses the rejection in asserting the teaching of El-Sagheer do not overcome the deficiencies of Kool and Jarvius. These arguments are not persuasive for the reasons of record.
Claim(s) 21-23, 38, 42-44 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kool (WO9909216) and Jarvius (WO2015/079042) as applied to claims 1- 7, 11, 13-17, 19-20, 24, 33, 35-37, 39-40 in view of Duncani(Nature Methods (2013) volume 10, pages 647-652), Sahsavarian (Journal of Immunological Methods407(2014)26–34)
The teaches of Jarvius and Kool are set forth above. Jarvius and Kool render obvious the method of claim 1.
The teachings of Jarvius and Kool do not specifically teach BseGI. BtsCI, Nb.BsrD1, Nt. BspQ1 or BseGI.
However, Duncani teaches, “ Alternatively, the DNA can be amplified by cloning into a phagemid vector and subsequent production of single stranded DNA (ssDNA) via rescue by helper phage (Fig. 1b). Both processes (Supplementary Fig. 1) furnish ssDNA, digestion of which using a type IIs restriction enzyme releases the ODNs, here referred to as ‘MOSIC ODNs’. Using the restriction enzymes BtsCI or BseGI and the hairpin architecture depicted in Figure 1c, sequences of consecutive ODNs can be designed to be independent of each other as no base-pairing between the MOSIC ODNs is required for the digestion..”
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Ducani teaches:
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Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use double stranded templates and BseGI. BtsCI, Nb.BsrD1, Nt. BspQ1 or BseGI.. The artisan would be motivated as Duncani teaches,” hairpin architecture depicted in Figure 1c, sequences of consecutive ODNs can be designed to be independent of each other as no base-pairing between the MOSIC ODNs is required for the digestion.” The artisan would be further motivated as Duncani teaches the method is cheaper than synthetic ODN synthesis (supp fig 13. The artisan would have a reasonable expectation of success as the artisan is merely using know methods and reagents.
Kool and Jarvius do not specifically teach cloning, amplification, excision and circularization.
However, Ducani teaches, “Transformation of E. coli cells with a vector containing the pseudogenes. We inserted the linear CP DNA into a pBluescript SK II(−) for the phage cloning strategy, and we used the pMK-RQ containing the CP for the in vitro amplification. We cloned the 378-nt-long ODN pseudogene into a pDrive cloning
vector (Qiagen, PCR cloning kit). We thawed SCS110 or XL10 gold competent cells (Agilent technologies) on ice. For each reaction, we gently mixed and incubated 100 µl of cells with 1.7 µl of β-mercaptoethanol (1.42 M) for 10 min, swirling gently every
2 min. Then we mixed 50 ng of the vector containing the pseudogene, or pUC18 plasmid as a control, with competent cells and incubated for 30 min on ice. Then we heat-pulsed the reactions in a 42 °C water bath for 45 s and incubated on ice again for 2 min. We then added 0.9 ml of preheated (42 °C) NZY+ broth (10 g of NZ amine (casein hydrolysate), 5 g of yeast extract, 5 g of NaCl in 1 l of water subsequently adjusted to pH 7.5 using NaOH, supplemented with 12.5 ml of 1 M MgCl2, 12.5 ml of 1 M MgSO4 and 20 ml of 20% (w/v) glucose), and we incubated the reactions at 37 °C for 1 h at 250 r.p.m. We plated 100 µl of the transformation mixture on LB agar plates containing kanamycin (50 µg/ml) for pMK-RQ–transformed cells and ampicillin (100 µg/ml) for the pBluescript SK II(−), pDrive cloning vector and pUC18 plasmid. We incubated the plates at 37 °C overnight. The day after, we screened single colonies, from pMK-RQ and pBluescript SK II(−)–transformed cells, for the CP insert by BsmBI digestion, and by BsaI digestion for the 378-nt-long ODN pseudogene. Complete sequence of the 378-nt pseudogene, verified by sequencing, is available in Supplementary Table 2. We transformed E. coli (SCS110) with staple pseudogenes as described above, plating the transformed cells on LB agar plates containing ampicillin (100 µg/ml) and screening single colonies DNA for the pseudogenes. We used XL10 gold cells in all cases except when producing phagemid DNA; then we used SCS110 cells”
Further , Shahsavarian teaches cloning predigested fragments into phagemid vectors, amplifying and use of PCR to amplify colonies (2.8-2.9) Shahsavarian also teaches generation of circular products from predigested fragments.
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to clone a sequence into a vector (plasmid or phagemid), introduce it into bacteria, excise by the same enzymes as the predigestion (or PCR), circularize and perform the method of Kool. The artisan would be motivated as Shahsavarian teaches the method allows for directional cloning and amplification. The artisan would have a reasonable expectation of success as the artisan is merely applying known methods
Response to arguments
The response traverses the rejection in asserting the teaching of El-Sagheer do not overcome the deficiencies of Kool and Jarvius. These arguments are not persuasive for the reasons of record.
Claim(s) 45- 46 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kool (WO9909216) and Jarvius (WO2015/079042) as applied to claims 1- 7, 11, 13-17, 19-20, 24, 33, 35-37, 39-40 in view of Chen (WO2006/063355)
The teaches of Jarvius and Kool are set forth above. Jarvius and Kool render obvious the method of claim 1.
While Kool and Jarvius suggest isolating or purifying the functionalized single stranded oligonucleotides, they do not specifically teach the level of purity of the claims in weight/weight.
However, Chen teaches in example 10, purification of DNA with less than 1 % RNA or Genomic DNA by w/w. (able 3)
Therefore it would have been prima facie to one of ordinary skill in the art prior to the effective filing date of the claims to ensure purification to greater than 99% w/w of the single strand functionalized DNA. The artisan would be motivated to provide the purest nucleic acids, to minimize contaminants which may interfere with downstream use. The artisan would have a reasonable expectation of success as the artisan is merely using known methods to purify nucleic acids.
Response to Arguments
The response traverses the rejection in asserting the teaching of El-Sagheer do not overcome the deficiencies of Kool and Jarvius. These arguments are not persuasive for the reasons of record.
Summary
NO claims are allowed.
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.
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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