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 .
Election/Restrictions
Applicant’s election without traverse of inventive Group I, claims 1-9 in the reply filed on May 23, 2025 is acknowledged.
Claim Status and Action Summary
This action is in response to the papers filed on November 17, 2025
Claims 1-8 are pending in this application.
Claims 9-11 were canceled by applicant.
Claims 1-8 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The objections to the specification have been withdrawn in view of the substitute specification filed on November 17, 2025.
The 112(b) rejections of record have been withdrawn in view of the amendments to the claims.
The 112(d) rejection of record over claim 9 is moot because claim 9 was canceled.
Priority/Effective Filing Date
The present application, filed on March 16, 2022 is a 371 of PCT/CN2019/123892, filed on December 9, 2019 and claims foreign priority to CHINA 201910896457.8, filed on September 20, 2019. A certified copy of the foreign priority document was filed on March 16, 2022, however it is noted that no certified translation of the priority document has been provided. Therefore, the effective filing date of the present application is determined to be December 9, 2019.
Claim Objections
Claims 4 and 5 are objected to because of the following informalities:
Claim 4 contains an apparent typographical error consisting of an absent article. The claim recites “the 3’-end tail region of the specific probe comprises mismatched base” . It appears that the article “a” was omitted between “comprises” and “mismatched base”.
Claim 5 contains an apparent typographical error consisting of an absent article. The claim recites “the single stranded ligase is T4 RNA ligase or thermostable RNA ligase.” “thermostable RNA ligase” is a genus of RNA ligases having the shared property of thermostability. Therefore, it appears that the article “a” was omitted prior to “thermostable RNA ligase”.
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.
Claims 1-8 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
This is a new grounds of rejection necessitated by the amendments to the claims.
Claim 1 recites the claim term “5’ end nucleotides of the specific probe is modified”. It is unclear whether this limitation requires: a) that the one terminal nucleotide at the 5’ end of the specific probe is modified, b) that some unspecified number of nucleotides at the 5’ end of the specific probe are modified (e.g. the last 3 nucleotides at the 5’ end), or c) that some unspecified number of nucleotides at some unspecified location at the 5’ end of the specific probe are modified (e.g. the terminal nucleotide, the third nucleotide from the 5’ end, and the seventh nucleotide from the 5’ end).
Claim 1 recites the claim term “the 3’ end modification group of the specific probe”. There is insufficient antecedent basis for this limitation in the claim.
Claim 6 recites the claim term “adding ssDNA as endonuclease”. In the prior art and specification, ssDNA refers to a single stranded DNA molecule, while endonuclease refers to a protein enzyme with 5’- or 3’- end dependent phosphodiesterase activity (i.e. an enzyme that degrades DNA from a free 5’ or 3’ end). It is unclear whether “ssDNA as endonuclease” is a typographical error for “an ssDNA endonuclease”, as was recited in previous versions of the claims, or if this claim term is meant to require something else.
Claims 2-8 are indefinite because they depend from, and necessarily include the indefinite limitations of, claims 1 and 6.
Claim Rejections - 35 USC § 103
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.
Claims 1-7 are rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al., WO 2019/090621 A1, published May 16, 2019 as evidenced by Agilent, “High-Fidelity PCR Enzymes: Properties and Error Rate Determinations” published May 2019 in view of Chen et al., WO 2018/175258 A1, published September 27, 2018.
This is a new grounds of rejection adapted from the 102(a)(1) rejection of record over Jiang et al. as evidenced by Agilent.
Regarding claim 1, Jiang et al. teaches an enrichment method for gene target regions comprising: (a) amplifying target DNA using a specific “hook probe” that contains sequences that are complementary to the target sequence and sequences that are not complementary to the target sequence, and (b) ligating the specific probe to generate a circular and linear product (Jiang et al., figures 1-2, see below). Jiang et al. further teaches that the 5’ end nucleotide of the hook probe is 5’ phosphorylated and the 3’ end nucleotide of the hook probe is 3’ modified with a blocking group such as: 3’ phosphate, 3’ dideoxynucleotide (i.e. a 3’ hydrogen atom), or a 3’ amino group (Jiang et al., paragraph 0051). Finally, Jiang et al. teaches that the hook probe 3’ end is used as the starting point for a polymerase extension (Jiang et al., paragraph 0094). Jiang et al. teach using “PfuTurbo Cx Hotstart DNA Polymerase” from Agilent, Inc. as the DNA polymerase in the extension reaction (Jiang et al., paragraph 0235). As evidenced by Agilent, PfuTurbo Cx Hotstart DNA Polymerase has 3’-5’ exonuclease activity (i.e. an active substance, a nuclease) (Agilent, page 2, column 1, paragraph 3 and page 4, table 1).
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Jiang et al. do not teach that the blocking group at the 3’ end of the probe is C3-spacer.
However, Chen et al. teach methods for preparing nucleic acid libraries comprising ligating adapter oligonucleotides having a 3’-C3-spacer as a 3’ blocking group that prevents extension of the 3’ ends by polymerase activity or by concatenation of adapters through ligation (Chen et al., paragraphs 0017-0028). Furthermore, Chen et al. teach “examples of 3’ blocking groups include: a 3’-spacer C3, a dideoxynucleotide, attachment to a substrate…, alkyl… groups, a thiol, an azide, or an alkyne” (Chen et al., paragraph 0088).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have selected any of: 3’ phosphate, 3’ dideoxynucleotide (i.e. a 3’ hydrogen atom), or a 3’ amino group (Jiang et al., paragraph 0051), a 3’-spacer C3, a dideoxynucleotide, attachment to a substrate…, alkyl… groups, a thiol, an azide, or an alkyne” (Chen et al., paragraph 0088), because Jiang et al. and Chen et al. teach that these exemplary blocking groups prevent extension of an adapter at one or both ends of the nucleic acid molecule (Chen et al., paragraph 0088). Furthermore, in an example, Chen et al. demonstrate an adapter ligation method for constructing an ssDNA library wherein the overall yield of the ssDNA library was >80% when using 3’-C3-spacer as a 3’ blocking group (Chen et al., paragraphs 0131-0135).
The ordinary artisan would therefore have been motivated to select C3-spacer as a particularly effective 3’ blocking group in the method taught by Jiang et al. as evidenced by Agilent, because of the teaching of Chen et al. that a similar ssDNA library generation method captured >80% of input ssDNA (Chen et al., paragraph 0135).
Regarding claim 2, Jiang et al. teach the size range of target fragmented DNA is between about 50 to about 3000 bases (i.e. 25-200 bp) (Jiang et al., paragraph 0063), and amplification of DNA includes DNA polymerase and dNTP (Jiang et al., paragraph 0235).
Regarding claim 3, Jiang et al., as evidenced by Agilent, teaches that the DNA polymerase (PfuTurbo Cx Hotstart DNA Polymerase) has 3’-5’ exonuclease activity.
Regarding claim 4, Jiang et al. teaches the hook probe comprises a universal primer binding site that is matched according to sequencing primers and/or linker sequences of different sequencing platforms (i.e. a universal sequence that can be recognized by a sequencing system) (Jiang et al., paragraph 0046). Jiang further teaches the target-specific sequence (i.e. the 3’ end tail region) may comprise a SNP, an insertion deletion site, etc. (i.e. a mismatched base) (Jiang et al., paragraph 0044). Finally, Jiang teaches that the 5’ end nucleotide of the hook probe comprises a 5’ phosphate (Jiang et al., paragraph 0051).
Regarding claim 5, Jiang et al. teaches the ligase includes a single-stranded ligase and may be T4 RNA ligase or a thermally stable RNA ligase (i.e. a thermostable RNA ligase) (Jiang et al., paragraph 0067).
Regarding claim 6, Jiang et al. teaches the hook probe comprises a uracil in the region not complementary to the target sequence, and further teaches linearizing circular ligation products using “E coli UDG… and “endonuclease VIII” (Jiang et al., paragraph 0048) or “the USER enzyme” (Jiang et al., paragraph 0090). It is noted that a combination of UDG and endonuclease VIII is marketed by NEB as USERTM Enzyme.
Regarding claim 7, Jiang et al. teaches PCR amplifying the ligated product using a universal primer complementary to the hook probe (Jiang et al., paragraph 0081). Jiang et al. further teaches purifying products after ligation (Jiang et al., paragraph 0151).
Claims 1-7 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., WO 2019062289 A1, Published April 4, 2019 in view of Jiang et al., WO 2019/090621 A1, published May 16, 2019 and Chen et al., WO 2018/175258 A1, published September 27, 2018.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 1, Zhang et al. teaches an enrichment method for a gene target region comprising: hybridizing a probe comprising sequence complementary to the target region, sequences that are not complementary to the target region, and a 5’ phosphate modification (Zhang et al., paragraph 0010-0021 and figure 1; see below).
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Zhang et al. teaches that the 5’ end nucleotide of the probe is modified with a 5’ phosphate group (Zhang et al., paragraph 0016), the probe is extended by a polymerase after annealing to the target sequence, and the produced 3’ end is ligated to the 5’ phosphorylated probe, generating a circular product.
Zhang et al. does not teach that the 3’ end nucleotide of the probe is substituted. However, Jiang et al. teaches an enrichment method for gene target regions comprising: (a) amplifying target DNA using a specific “hook probe” that contains sequences that are complementary to the target sequence and sequences that are not complementary to the target sequence, and (b) ligating the specific probe to generate a circular and linear product (Jiang et al., figures 1-2, see below). Jiang et al. further teaches that the 5’ end nucleotide of the hook probe is 5’ phosphorylated and the 3’ end nucleotide of the hook probe is 3’ modified with a blocking group including 3’ phosphate, 3’ dideoxynucleotide (i.e. a 3’ hydrogen atom), or a 3’ amino group (Jiang et al., paragraph 0051). Jiang et al. further teaches that the hook probe 3’ end is used as the starting point for a polymerase extension (Jiang et al., paragraph 0094), using “PfuTurbo Cx Hotstart DNA Polymerase” from Agilent, Inc. as the DNA polymerase in the extension reaction (Jiang et al., paragraph 0235).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the probe taught by Zhang et al. to further comprise a 3’-end nucleotide modification that blocks ligation between the 3’ and 5’ ends of the probe prior to hybridization and primer extension by a proofreading polymerase, as taught by Jiang et al.
The ordinary artisan would have been motivated to modify the probe taught by Zhang et al. with the ligation-blocking 3’ end modification taught by Jiang et al. because of the teaching of Jiang et al. that blocking groups at the 5’ and 3’ ends of the hook probe block ligation of the hook probe to unwanted targets (Zhang et al., paragraph 0051).
The ordinary artisan would have been reasonably confident that modifying the probe taught by Jiang et al. to further comprise a 3’ blocking modification, as taught by Zhang et al. would have reduced the frequency at which the padlock probes taught by Jiang et al. underwent premature ligation prior to the elongation step and therefore reduced the occurrence of unwanted side products such as self-ligated, empty padlocks.
The methods taught by Zhang et al. and Jiang et al. do not teach that the 3’ end nucleotide of the hook probe is 3’ modified with a blocking group that is a C3-spacer. Zhang et al. and Jiang et al teach that the 3’ blocking group can be: 3’ phosphate, 3’ dideoxynucleotide (i.e. a 3’ hydrogen atom), or a 3’ amino group (Jiang et al., paragraph 0051).
However, Chen et al. teach methods for preparing nucleic acid libraries comprising ligating adapter oligonucleotides having a 3’-C3-spacer as a 3’ blocking group that prevents extension of the 3’ ends by polymerase activity or by concatenation of adapters through ligation (Chen et al., paragraphs 0017-0028). Furthermore, Chen et al. teach “examples of 3’ blocking groups include: a 3’-spacer C3, a dideoxynucleotide, attachment to a substrate…, alkyl… groups, a thiol, an azide, or an alkyne” (Chen et al., paragraph 0088).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have selected any of: 3’ phosphate, 3’ dideoxynucleotide (i.e. a 3’ hydrogen atom), or a 3’ amino group (Jiang et al., paragraph 0051), a 3’-spacer C3, a dideoxynucleotide, attachment to a substrate…, alkyl… groups, a thiol, an azide, or an alkyne” (Chen et al., paragraph 0088), because Jiang et al. and Chen et al. teach that these exemplary blocking groups prevent extension of an adapter at one or both ends of the nucleic acid molecule (Chen et al., paragraph 0088). Furthermore, in an example, Chen et al. demonstrate an adapter ligation method for constructing an ssDNA library wherein the overall yield of the ssDNA library was >80% when using 3’-C3-spacer as a 3’ blocking group (Chen et al., paragraphs 0131-0135).
The ordinary artisan would therefore have been motivated to select C3-spacer as a particularly effective 3’ blocking group in the method taught by Zhang et al. and Jiang et al., because of the teaching of Chen et al. that a similar ssDNA library generation method captured >80% of input ssDNA (Chen et al., paragraph 0135).
Regarding claim 2, Jiang et al. teach the size range of target fragmented DNA is between about 50 to about 3000 bases (i.e. 25-200 bp) (Jiang et al., paragraph 0063), and amplification of DNA includes DNA polymerase and dNTP (Jiang et al., paragraph 0235).
Regarding claim 3, Jiang et al., as evidenced by Agilent, teaches that the DNA polymerase (PfuTurbo Cx Hotstart DNA Polymerase) has 3’-5’ exonuclease activity.
Regarding claim 4, Zhang et al. teaches the probe further includes Illumina Tag sequences (i.e. a universal sequence that can be recognized by a sequencing system) (Zhang et al., paragraph 0010 and figure 1). Zhang further teaches that the 5’ end of the probe is phosphorylated (Zhang et al., paragraph 0016).
Regarding claim 5, Jiang et al. teaches the ligase includes a single-stranded ligase and may be T4 RNA ligase or a thermally stable RNA ligase (i.e. a thermostable RNA ligase) (Jiang et al., paragraph 0067).
Regarding claim 6, Zhang et al. teaches the non-target annealing segment of the probe comprises uracil, and the method comprises treating the ligated product with USER enzyme to form a linear ligated product (Zhang et al., figure 1 and paragraph 0038).
Regarding claim 7, Zhang et al. teaches amplifying the ligated product using primers not complementary to the target sequence, wherein the primers are complementary to the Illumina tag sequences on either side of the uracil site (i.e. the primer binds to universal sequences on both sides of the enzyme-digestion site) (Zhang et al., paragraph 0056-0057). Zhang further teaches purification steps after PCR amplification (Zhang et al., paragraph 0071).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., WO 2019062289 A1, Published April 4, 2019 in view of Jiang et al., WO 2019/090621 A1, published May 16, 2019 and Chen et al., WO 2018/175258 A1, published September 27, 2018 as applied to claims 1-7 above, and further in view of Vet et al., (2005). “Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays”. In: Herdewijn, P. (eds) Oligonucleotide Synthesis. Methods in Molecular Biology, vol 288, pp 273-290.
As discussed in the 103 rejection above, Zhang et al. in view of Jiang et al. and Chen et al. teach an enrichment method for gene target regions comprising amplifying fragmented target DNA by: annealing specific probes comprising 5’ and 3’ modifications to the target sequence, wherein the 3’ blocking modification comprises a C3-spacer group, removing the 3’ blocking modification and the extending the 3’ end using a DNA polymerase and the target sequence as a template, and finally ligating the extension product to the second probe having a 5’ end modification. Zhang et al. in view of Jiang et al. and Chen et al. further teach PCR amplifying the ligation product by: (a) linearizing the ligation product containing uracil in a sequence not complementary to the target sequence (and flanked by Illumina “tag sequences”) by treating the product with USER enzyme and (b) using primers that bind to the tag sequences to amplify the linearized ligation product.
Zhang et al. in view of Jiang et al. and Chen et al. do not teach detecting the resulting amplicons using a probe comprising a labeling molecule. Rather, they teach constructing next-generation sequencing (NGS) libraries from the enriched target sequences. However, Vet et al. teaches methods and principles of design for molecular beacons (i.e. a probe comprising a labeling molecule) including: “different steps necessary for establishing a molecular beacon real-time PCR assay: (1) target design, (2) primer design, (3) optimization of the amplification reaction conditions using SYBR Green, (4) molecular beacon design, and (5) molecular beacon synthesis and characterization. The last section provides an example of a multiplex quantitative real-time PCR.” (Vet et al., Abstract). Vet et al. teaches that “Compared with end point quantification methods, real-time amplification assays offer reproducible results and have a much wider dynamic range.” (Vet et al., page 274, paragraph 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method comprising detecting enriched target sequences by sequencing a library prepared from the target sequence amplicons, taught by Zhang et al. in view of Jiang et al. and Chen et al., by substituting the sequencing steps (i.e. end-point detection) taught by Zhang et al. in view of Jiang et al. and Chen et al. with the real-time detection methodology comprising detecting the amplicons with a molecular beacon (i.e. detectably labeled probe, “probe 3”).
The ordinary artisan would have been motivated to substitute the end-point detection steps taught by Zhang et al. in view of Jiang et al. and Chen et al. with the real-time detection methods taught by Vet et al. because of the teaching of Vet et al. that “Compared with end point quantification methods, real-time amplification assays offer reproducible results and have a much wider dynamic range.” (Vet et al., page 274, paragraph 1).
The ordinary artisan would have been reasonably confident that real-time detection of the target amplicons using molecular beacons, designed by the methods taught by Vet et al., would have predictably improved the reproducibility and dynamic range of the nucleic acid enrichment and detection methods taught by Zhang et al. in view of Jiang et al. and Chen et al.
Response to arguments
The response argues that Jiang et al. and Zhang et al. do not teach a C3-Spacer group at the 3’ end of the specific probe, as required by the claims as presently amended.
This limitation has been addressed in the 103 rejections above necessitated by the amendments to the claims.
The response quotes the specification in asserting “a C3 Spacer group has obvious advantages in linear amplification effect”. This argument has been thoroughly reviewed but is not persuasive because as is described in the 103 rejections above, C3-Spacer groups are well-known in the art as blocking groups that advantageously prevent non-specific extension or ligation of particular primers or probes in methods for generating sequencing libraries. Furthermore, the asserted advantages provided by C3-spacer blocking groups would have inherently been present in the prior art wherein C3-spacers were used in similar methods. Therefore, absent evidence supporting unexpected results commensurate in scope with the claims, the assertion that a 3’ C3-spacer group provides unexpected “obvious advantages” that were not present in the prior art that utilized 3’ C3-spacer blocking groups is not persuasive.
Double Patenting
It is noted that an approved terminal disclaimer was filed in copending application 18/969,786 regarding the present claims.
Conclusion
No claim is allowed.
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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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682