DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on March 26, 2026 has been entered. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any rejections or objections not reiterated herein have been withdrawn.
Claims 1-22 are currently pending and have been examined herein.
Claim Rejections - 35 USC § 112
3. The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-22 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.
Claims 1-22 are rejected over the recitation of the phrase “the primary DNA sequence” in claims 1 and 3. There is insufficient antecedent basis for this limitation in the claims.
Claim Rejections - 35 USC § 103
4. 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.
5. Claims 1-10 and 12-22 are rejected under 35 U.S.C. 103 as being unpatentable over Liachko (WO 2019/005763 Pub 1/3/2019) in view of Kato (Journal of Biological Methods 2017 Vol 4(3) e79) and Jovanovich (US 2013/0203634 Pub 8/8/2013).
Regarding Claims 1 and 3 Liachko teaches a method for determining or detecting the presence of one or more nucleic acids in association with a target nucleic acid (para 0036). Liachko teaches that the associations can be between nucleic acid sequences or genes on multiple chromosomes (para 0048). The method comprises:
a) providing a cell in which elements within one or more DNA molecules that are in close proximity are cross-linked (paras 0041, 0045-0048);
b) lysing the cell and fragmenting the DNA molecules within the cell (para 0041);
c) proximity ligating the one or more fragmented DNA molecules (para 0042);
d) reversing the crosslinks in the ligated DNA molecules (para 0043);
e) sequencing the ligated DNA molecules (para 0060); and
f) analyzing the sequencing data to detect interactions between elements within the one or more DNA molecules within the cell (para 0073).
Regarding Claim 4 Liachko teaches a method comprising a step of cross-linking the DNA molecules and/or DNA-interacting proteins (paras 0041, 0045-0048).
Regarding Claim 5 Liachko teaches a method wherein cross-linking is achieved by treating the cells with formaldehyde (para 0041, 0045).
Regarding Claim 6 Liachko teaches that the crosslinked nucleic acids are fragmented using a restriction enzyme or an endonuclease. Liachko teaches that in some cases, fragmentation of the cross linked junctions generates overhanging or sticky DNA ends. The overhanging or sticky ends can be filled in or made blunt (para 0041, 0049).
Regarding Claims 7-9 Liachko teaches that after the crosslinks are reversed, the proximity junctions comprising a query sequence can be selected by PCR. Liachko teaches an embodiment where selection entails the use of PCR wherein the first and/or second primers have sequencing adaptor sequences appended. The presence of the sequencing adapter sequence can enable rapid creation of a sequencing library. The sequencing library can subsequently be loaded into a sequencer and sequenced (para 0060).
Regarding Claim 10 Liachko teaches that after the crosslinks are reversed, the proximity junctions can be enriched prior to purification and detection (para 0054).
Regarding Claims 12-14 Liachko teaches the proximity junctions can be enriched prior to purification and detection. In one embodiment, biotin labels are appended to the proximity junctions as described herein and hybridized to or bound to substrates coated or coupled to avidin or streptavidin moieties such as, for example, affinity beads. Subsequently, non-bound complexes are washed or separated away from the biotin/avidin (streptavidin) complexes. The biotin selection can serve to remove significant amounts of background DNA prior to the selection or purification of the proximity junctions (para 0054, 0056).
Regarding Claim 15 Liachko teaches a method wherein the sequencing is performed by a nanopore-based method (para 0060).
Regarding Claim 16 Liachko teaches that cross linking creates cross linked junctions between nearby nucleic acids within the same cell. Liachko teaches in some cases, the junctions can be between nucleic acid sequences or genes on multiple chromosomes (para 0048).
Regarding Claim 17 Liachko teaches a method wherein the step of analyzing the sequencing data comprises identifying concatenated sequences from different elements within the one or more DNA molecules thereby detecting interacting elements of one or more DNA elements (para 0073).
Regarding Claim 18 Liachko teaches that cross linking creates cross linked junctions between nearby nucleic acids within the same cell. Liachko teaches that some junctions can be between nucleic acid or gene sequences on the same nucleic acid molecule cases (para 0048). Herein the genes are considered to be “loci” within a chromosome.
Regarding Claims 19-21 Liachko teaches to test this method, a library of proximity junctions derived from a mixed population of microbial cells (i.e., Bacillus subtilis, and Escherichia coli) were generated using the methods herein (para 0068). Thus Liachko teaches a method wherein the cells are from a mixture of two prokaryotic microorganisms.
It is noted that Liachko teaches that a mixed population of intact Bacillus subtilis, and Escherichia coli cells were treated with a cross-linker (1% formaldehyde for 20 minutes at room temperature). Subsequently, the cross-linked chromatin was purified from the mixed microbial cell population by a combination of physical (bead beating) and chemical (1% Triton X100 followed by 1% SDS; heated to 65° C.), followed by pelleting of cross-linked material and washing with tris-buffered saline. The DNA within the cross-linked chromatin was fragmented by restriction enzyme digest (para 0068).
However Liachko does not teach a method comprising simultaneously lysing the cell and mechanically fragmenting the DNA molecules within the cell (clm 1). Liachko does not teach a method wherein the cells are lysed and the DNA molecules are fragmented by bead beating (clm 2). Liachko does not teach a method wherein mechanically fragmenting the DNA within the cell occurs by bead beating (clm 3). Liachko does not teach a method wherein the mechanical fragmenting in (b) does not comprise the use of a restriction enzyme (clm 22).
However Kato teaches using bead beating for bacterial cell lysis and DNA fragmentation. Kato teaches that bead-beating simultaneously and rapidly produces bacterial lysis and genomic DNA fragmentation, with no special equipment required to obtain large DNA fragments (page 2, col 2, Fig 1).
Additionally Jovanovich teaches that the OmniLyse and bead beating methods shear nucleic acids as cells or tissues are lysed (para 0191).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Liachko by using bead beating to simultaneously lyse cells and fragment DNA as suggested by Kato and Jovanovich. One of skill in the art would have been motivated to use bead beating because Kato teaches that bead beating is a rapid, simple, affordable way to simultaneously lyse cells and fragment DNA. Further one of skill in the art would have been motivated to use bead beating because as demonstrated by Jovanovich it was known in the art that this method would shear nucleic acids as cells or tissues are lysed (para 0191). Finally it would have been obvious to fragment the DNA molecules within the cell using mechanical means (such as bead beating) without using a restriction enzyme since both Kato and Jovanovich demonstrate that bead beating methods are capable of producing nucleic acid fragments that are useful for downstream processes.
6. Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Liachko (WO 2019/005763 Pub 1/3/2019) in view of Kato (Journal of Biological Methods 2017 Vol 4(3) e79) and Jovanovich (US 2013/0203634 Pub 8/8/2013) as applied to claim 1 and in further view Green (US 2015/0363550 Pub 12/17/2015).
The teachings of Liachko in view of Kato and Jovanovich are discussed above.
The combined references do not teach a method further comprising selecting DNA fragments of a desired size after step (b), (c) or (d) (clm 11).
However Green teaches that crosslinked DNA molecules may be subjected to a size selection step. Size selection of the nucleic acids may be performed to cross linked DNA molecules below or above a certain size (para 0147).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Liachko, Kato, and Jovanovich by performing a step of size selection as suggested by Green. One of skill in the art would have been motivated to use size selection for the benefit of removing background DNA of a certain length to thereby improve detection.
Response To Arguments
7. In the response the Applicants traversed the rejection under 35 USC 103 over the combination of Liachko and Kato. In the response the Applicants argue that the proposed modification would render Liachko's method inoperable for its intended purpose of enriching for proximity junctions. They argue that Liachko's method in Example 1, paragraph [0068], critically depends on restriction enzyme digestion. Restriction enzymes cut DNA at specific recognition sequences, generating predictable overhanging ends ("sticky ends"). This predictability is essential to Liachko's workflow. As explained in at least paragraphs [0049] and [0068] of Liachko, restriction enzyme digestion generates overhangs that the Klenow fragment of E. coli DNA polymerase I needs in order to perform the fill-in reaction. The Klenow fill-in step incorporates biotinylated nucleotides which are subsequently relied upon in biotin-streptavidin capture assays to selectively enrich for proximity junctions. In particular, Liachko discloses that biotin enrichment "can serve to remove significant amounts of background DNA prior to the selection or purification of the proximity junctions." See Liachko, paragraph [0054]. The Applicants state that in contrast, mechanical fragmentation-such as by bead beating does not generate predictable DNA ends. The instant specification indicates "the disclosed methods provide DNA fragments that have been obtained in a sequence-independent manner" and "the fragmentation steps of the method involving mechanical fragmentation such as bead-beating will not be affected by over- or under-represented restriction enzyme motifs or chemical modifications of DNA." See Specification as filed, p. 12, lines 28-32. This sequence-independent nature of mechanical fragmentation is precisely why it is incompatible with Liachko's restriction enzyme-dependent workflow: mechanical fragmentation produces random breaks rather than the predictable overhangs that Liachko's Klenow fill-in step requires. Therefore, the proposed combination fails to establish a prima facie case of obviousness because the proposed modification of Liachko's method to replace restriction enzyme-based fragmentation with bead beating allegedly disclosed by Kato renders Liachko's method inoperable for its intended purpose of enriching for proximity junctions.
These arguments have been fully considered but are not persuasive. It is noted for the record that the claims are now rejected over the combination of Liachko, Kato, and Jovanovich. Kato teaches using bead beating for bacterial cell lysis and DNA fragmentation. Kato teaches that bead-beating simultaneously and rapidly produces bacterial lysis and genomic DNA fragmentation, with no special equipment required to obtain large DNA fragments (page 2, col 2, Fig 1). Additionally Jovanovich teaches that the OmniLyse or bead beating methods shear nucleic acids as cells or tissues are lysed (para 0191). The skilled artisan would understand that because the DNA is broken randomly, bead beating creates a mixture of fragmented DNA comprising blunt-ended and sticky-ended fragments. Jovanovich discloses that nucleic acids fragmented by shearing can have their ends repaired. Jovanovich teaches mixing the nucleic acid fragments with a polymerase and a polynucleotide kinase (PNK), to perform end repair on the fragmented nucleic acids (para 0363). The Examiner does not agree that replacing the restriction enzyme-based fragmentation with bead beating would render Liachko's method inoperable for its intended purpose because the sheared ends could still be repaired and biotin could still be incorporated.
Regarding Claim 11 the Applicants argue that Green does not remedy the deficiencies of
Liachko and Kato that have been described above.
This argument has been fully considered but is not persuasive. The Applicants arguments regarding what is missing in Liachko and Kato have been fully addressed above. The response to Applicants arguments, as set forth above, applies equally to the present ground of rejection.
8. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Shen can be reached on 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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