Patent Application 17799084 - KITS AND METHODS FOR EXTRACTING NUCLEIC ACIDS

Title: KITS AND METHODS FOR EXTRACTING NUCLEIC ACIDS FROM COMPLEX SAMPLES KITS AND METHODS FOR EXTRACTING NUCLEIC ACIDS FROM COMPLEX SAMPLES

Application Information

Invention Title: KITS AND METHODS FOR EXTRACTING NUCLEIC ACIDS FROM COMPLEX SAMPLES KITS AND METHODS FOR EXTRACTING NUCLEIC ACIDS FROM COMPLEX SAMPLES
Application Number: 17799084
Submission Date: 2025-04-09T00:00:00.000Z
Effective Filing Date: 2022-08-11T00:00:00.000Z
Filing Date: 2022-08-11T00:00:00.000Z
National Class: 536
National Sub-Class: 025420
Examiner Employee Number: 98516
Art Unit: 1693
Tech Center: 1600

Rejection Summary

102 Rejections: 2
103 Rejections: 7

Cited Patents

No patents were cited in this rejection.

Office Action Text

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 .
Priority
This application, filed August 11, 2022, is a national stage application of PCT/EP2021/053534, filed February 12, 2021, which claims priority to foreign priority application FR2001479, filed February 14, 2020.
Status of the Application
Claims 16-30 are pending and examined on the merits herein.
Claim Interpretation
The term “complex sample” recited in the present claims is interpreted consistent with its definition in the specification (see p. 6), which states that complex sample “relates to a sample which can comprise, in addition to the nucleic acids of interest, other constituents”.
Specification
The title of the application appears to recite the intended title twice. The following title is suggested: “Kits and Methods for Extracting Nucleic Acids From Complex Samples”.
Claim Objections
Claims 16, 20, 25-27, and 29-30 are objected to because of the following informalities:
Claim 16 recites in lines 11-12 “one more syringe(s)”. This is believed to be a typographical error and should read as “one or more syringe(s)”.
Claim 20 recites “selected in the group”. This is believed to be a typographical error and should read as “selected from the group”.
Claim 25 depends from claim 24 and requires step b) is performed when the complex sample is a blood sample. However, claim 24 already requires the complex sample is a biological sample, and the biological sample is blood. To promote clarity in the claims, the examiner suggests removing the limitation “when the complex sample is a blood sample” from claim 25 and amending claim 24 to recite “wherein the biological sample is a blood sample.”
Claims 26-27 depend from claim 24 and recite the limitation “when the complex sample is a blood sample”. However, claim 24 already requires the sample is blood. To promote clarity in the claims, the examiner suggests removing the limitation “when the complex sample is a blood sample” from claims 26-27, and amending claim 24 to recite “wherein the biological sample is a blood sample.”
Claims 29-30 depend from claim 28 and recite the limitation “when the complex sample is selected from group consisting of a feces sample, a saliva sample, a respiratory lavage sample, and a nasopharyngeal secretion sample.” However, claim 28 already requires the sample is selected from the group consisting of feces sample, saliva sample, a product of respiratory
lavage sample, and a nasopharyngeal secretion sample. To promote clarity in the claims, the examiner suggests removing the limitation “when the complex sample is selected from group consisting of a feces sample, a saliva sample, a respiratory lavage sample, and a nasopharyngeal secretion sample.”
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.

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 16-21, 23, 26-27, and 29-30 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 16-20 each recite “selected/chosen from a/the group comprising or consisting of” before reciting a closed Markush group. This is indefinite because it is unclear what other alternatives are intended to be encompassed by the claim.
MPEP 2173.05(h) states: “A Markush grouping is a closed group of alternatives, i.e., the selection is made from a group "consisting of" (rather than "comprising" or "including") the alternative members. Abbott Labs., 334 F.3d at 1280, 67 USPQ2d at 1196. If a Markush grouping requires a material selected from an open list of alternatives (e.g., selected from the group "comprising" or "consisting essentially of" the recited alternatives), the claim should generally be rejected under 35 U.S.C. 112(b) as indefinite because it is unclear what other alternatives are intended to be encompassed by the claim. See In re Kiely, 2022 USPQ2d 532 at 2* (Fed. Cir. 2022).”
For the purposes of expedited prosecution, claims 16-20 are examined as if these claims recite “selected from the group consisting of”.
Similarly, claim 23 recites “selected from the group comprising” before reciting a closed Markush group. This is indefinite because it is unclear what other alternatives are intended to be encompassed by the claim. See MPEP 2173.05(h).
For the purposes of expedited prosecution, claim 23 is examined as if it recites “selected from the group consisting of”.

Regarding claim 16, the phrase "preferably" renders the claims indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d). For the purposes of expedited prosecution, the narrower limitations regarding the lysis buffer and filter that follow the term “preferably” are not being considered.
Because claims 17-21 depend from claim 16 and fail to cure these deficiencies, claims 17-21 are also indefinite.
Regarding claims 23, 26-27, and 29-30, the phrase "preferably" renders the claims indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d). For the purposes of expedited prosecution, the narrower limitations that follow the term “preferably” in these claims are not being considered.
Claims 24-25 and 28 depend from claim 23 and require a biological sample. Because these claims require a biological sample, the scope of the claimed invention in claims 24-25 and 28 is clear.

The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.

The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.

Claim 27 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 27 depends from claim 24 and requires the final concentration of potassium salt in the reaction mixture is ranging from about 10 mM to about 500 mM. However, claim 24 depends from claim 23, which depends from claim 22, which already requires the final concentration of potassium salt in the reaction mixture is ranging from about 10 mM to about 500 mM. Therefore, claim 27 fails to further limit the subject matter of the claim upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.

Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –

(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.

(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.

Claims 22-23 are rejected under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) as being anticipated by Kemp (U.S. pre-grant publication no. US 20150225713 A1; cited in IDS received December 14, 2022).
Claim 22 is drawn to a method for extracting nucleic acids from a complex sample, comprising the steps of a) through e) recited in the claim, and claim 23 requires the complex sample is a biological sample, environmental sample, or food sample.
Kemp teaches methods for nucleic acid purification. Kemp teaches that in one preferred embodiment for isolating plasmid DNA, methods of nucleic acid isolation using the present invention comprise (a) suspension of pelleted bacterial culture in TE buffer (50 mM Tris-Cl, pH 8.0, 10 mM EDTA) (Solution Pl); (b) lysis of cells for a specified amount of time depending on the scale of the prep using a common lysis buffer, e.g., Solution P2 (200 mM NaOH, 1 % SDS); (c) neutralization of the alkaline lysis solution and precipitation of genomic DNA and proteins using a common neutralization buffer, (Solution P3) that contains about 1-3 M potassium acetate and about 200 μg/mL RNAse A; clearance of the precipitated genomic DNA and proteins using a filtration method, such as Whatman cellulose paper, silica, glass fiber, membranes or other filters (p. 11, [0084], lines 4-18), and mixing the cleared lysate with a phase separation buffer before isolation using a mineral matrix (p. 11, [0084], lines 24-26).
Kemp teaches that each solution added in the above steps are added in equal volume. Therefore, the SDS has a final concentration of 0.5%, and 1 M solution of potassium acetate has a final concentration of ~0.33 M. These concentrations satisfy the limitations of steps a) and c) in claim 22. In addition, a bacterial culture is interpreted as a biological sample as recited in claim 23.
Thus Kemp anticipates claims 22-23.

Claims 16-23 are rejected under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) as being anticipated by Chen (U.S. pre-grant publication no. US 20090227011 A1; cited in PTO-892).
Claim 16 is drawn to a kit for extracting nucleic acids contained in a complex sample comprising the requirements listed therein. Claim 17 requires the zinc salt is selected from a group consisting of zinc sulfate or zinc chloride, claim 18 requires the copper salt is selected from a group consisting of copper sulfate or copper chloride, claim 19 requires the kit is the potassium salt is selected from the group that includes potassium acetate, claim 20 requires the lysis buffer and/or the buffer comprising a zinc and/or copper salt comprise Tris-HCl, HEPES, and MOPS, and claim 21 claims a method for extracting nucleic acids contained in a complex sample, comprising extracting the nucleic acid using the kit according to claim 16.
Claim 22 is drawn to a method for extracting nucleic acids from a complex sample, comprising the steps of a) through e) recited in the claim, and claim 23 requires the complex sample is a biological sample, environmental sample, or food sample.
Chen teaches novel methods to purify plasmid DNA from host cells (cover page, Abstract, lines 1-2). Chen teaches that in their method, following a standard lysis procedure that releases plasmid DNA from the host cells, two sequential precipitation procedures separate plasmid DNA from essentially all impurities of the host cells (cover page, Abstract, lines 2-5).
Chen teaches that in an embodiment of their method, alkaline lysis of host cells step comprises re-suspending harvested host cells by centrifugation or filtration in a buffer solution of Sol. I (e.g., 50 mM Tris-HCl, 10 mM EDTA, pH 8.0) and then lysing the cells by adding a Sol. II (e.g., 0.2 NaOH and 1 % SDS), further teaching that the exact formulations for Sol. I and Sol. II are not limited (p. 3, [0026], lines 4-10). Chen teaches that following the lysis step, host cell impurities are precipitated from the lysate by adding a first solution comprising salts of at least one monovalent cation and at least one divalent cation, and that in an embodiment, the first solution comprises potassium and manganese ions, and that in an embodiment, the addition of the first solution occurs simultaneously with addition of a neutralization buffer (p. 3, [0028], lines 1-9).
Finally, Chen teaches a process for DNA purification that includes the steps of 1. Re-suspension of the host cells in Sol. I of 50 mM Tris-HCl, 10 mM EDTA, pH 8.0; 2. Lysis of the cells by adding equal volume of Sol. II of 0.2 M NaOH, 1 % SDS; 3. Neutralizing the lysate with equal volume of Sol. III of 2.0 M acetic acid, 0.6 M potassium acetate, 0.4 M manganese chloride, 0.4 M calcium chloride; and 4. Obtaining clarified lysate by centrifugation or filtration (p. 6, [0055]-[0059]). Step 4 of Chen’s method is interpreted as satisfying steps d) and e) of claim 22, and the host cells are interpreted as a biological sample, as recited in claim 23.
Thus Chen anticipates claims 22-23.
Furthermore, Chen teaches kits for the purification of plasmid DNA comprising a Solution A that comprises Tris-HCl, EDTA, NaOH, and SDS and a Solution B that comprises acetic acid, potassium acetate, and manganese chloride (p. 4, [0032], lines 1-5). In an embodiment, Solution A comprises 50 mM Tris-HCl, 10 mM EDTA, 0.2 N NaOH and 1 % SDS and Solution B comprises 2.0 M acetic acid, 0.6 M potassium acetate, 0.4 M calcium chloride, and 0.4 M manganese chloride (p. 4, [0032], lines 7-11). A concentration of 0.4 M is interpreted as about 0.5 M, as required by claim 16.
Chen teaches that the divalent ions can be provided by adding a variety of divalent cation salts, including ZnCl2, CuSO4, NiSO4, CaCl2, or MnCl2, and that these can be present in a concentration of about 0.05 M to about 4 M (p. 4, [0038], lines 1-8).
Finally, Chen teaches that for obtaining clarified lysate via filtration, the transferring
adaptor can be used to combine centrifugation with filtration, and that in this circumstance, the pore size of the filter membrane should be smaller, e.g., not larger than about 20 microns, preferably not larger than 10 microns, and preferably about 5 microns (p. 7, [0073], lines 1-6). In addition, Chen teaches an example wherein cell lysate is filtered through a 5 micron membrane after addition of the addition of solution comprising potassium acetate and MnCl2 (p. 8, [0100]-[0101]).
Therefore, a kit comprising a lysis buffer with SDS between about 1% to about 25%, a buffer comprising a concentration of potassium salt between 0.1 M and 5.0 M, a buffer comprising a concentration of a zinc and/or copper salt ranging from about 0.5 M to about 5.0 M, and a filter a pore diameter ranging from about 1 μm to about 10 μm is anticipated by Chen.
MPEP 2131.02 at III states: “A reference disclosure can anticipate a claim when the reference describes the limitations but "'d[oes] not expressly spell out' the limitations as arranged or combined as in the claim, if a person of skill in the art, reading the reference, would ‘at once envisage’ the claimed arrangement or combination." Kennametal, Inc. v. Ingersoll Cutting Tool Co., 780 F.3d 1376, 1381, 114 USPQ2d 1250, 1254 (Fed. Cir. 2015).” In this instance, in view of the kits expressly taught by Chen, the filters used in specific examples therein, and the suggested divalent metal salts, one of ordinary skill in the art would have ‘at once envisaged’ the claimed combination of kit components recited in claims 16-20 and the method of using said kit for extracting nucleic acids contained in a complex sample recited in claim 21.
Thus Chen anticipates claims 16-21.

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.

The factual inquiries for establishing a background for determining obviousness under 35
U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 16-21 are rejected under 35 U.S.C. 103 as being unpatentable over Chen.
The examiner believes that claims 16-21 are anticipated by Chen. However, for the sake of argument, if a concentration of 0.4 M zinc or copper falls outside the range encompassed by the limitation of about 0.5 M recited in claim 16, then claims 16-21 would have been obvious over Chen.
Claim 16 is drawn to a kit for extracting nucleic acids contained in a complex sample comprising the requirements listed therein. Claim 17 requires the zinc salt is selected from a group consisting of zinc sulfate or zinc chloride, claim 18 requires the copper salt is selected from a group consisting of copper sulfate or copper chloride, claim 19 requires the kit is the potassium salt is selected from the group that includes potassium acetate, claim 20 requires the lysis buffer and/or the buffer comprising a zinc and/or copper salt comprise Tris-HCl, HEPES, and MOPS, and claim 21 claims a method for extracting nucleic acids contained in a complex sample, comprising extracting the nucleic acid using the kit according to claim 16.
Chen teaches as described in the above rejection under 35 U.S.C. 102.
Chen does not expressly teach a kit comprising a buffer comprising a concentration of a zinc and/or copper salt ranging from about 0.5 M to about 5.0 M or a filter having a pore diameter ranging from about 1 to 10 μm, as required by claim 16.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the present application to prepare a kit comprising the components expressly taught as a kit by Chen as well as a buffer comprising a concentration of a zinc and/or copper salt ranging from about 0.5 M to about 5.0 M and a filter having a pore diameter ranging from about 1 to 10 μm. One of ordinary skill in the art would have been motivated to prepare a kit comprising a buffer comprising a concentration of a zinc and/or copper salt ranging from about 0.5 M to about 5.0 M and a filter having a pore diameter ranging from about 1 to 10 μm because Chen teaches ZnCl2 and CuSO4 as alternative divalent metal salts to MnCl2 and teaches these salts may be present in concentrations from about 0.05 M to about 4 M, and additionally teaches examples wherein filters with 5 μm pore sizes were used to obtain clarified lysate. Thus one of ordinary skill in the art would have contemplated modifying the kits expressly taught by Chen with these components, including considering concentrations of ZnCl2 and CuSO4 that fall within the claimed concentration range of 0.5 M to 5.0 M range, which overlaps with the range disclosed by Chen.
Therefore the invention taken as a whole is prima facie obvious.

Claims 22-23 are rejected under 35 U.S.C. 103 as being unpatentable over Arad (Arad, U. Biotechniques 1998, vol. 24, pp. 760-762; cited in PTO-892) in view of Kemp.
Claim 22 is drawn to a method for extracting nucleic acids from a complex sample, comprising the steps of a) through e) recited in the claim and claim 23 requires the complex sample is a biological sample, environmental sample, food sample.
As described above, Kemp anticipates claims 22-23 with a method directed to isolation of DNA from bacterial cultures. However, the presently claimed method directed to isolation of DNA from mammalian cells would have been obvious over Arad in view of Kemp.
Arad teaches a method of purification of extrachromosomal DNA from mammalian cells (p. 761, Title). Arad teaches that some methods for DNA extraction are laborious and time consuming, and that more rapid methods using anion-exchange chromatography or silicon-based resins use the alkaline lysis procedure that was originally developed for extracting plasmid DNA from bacterial cells (p. 761, left column, lines 5-14).
Arad teaches that after being treated with trypsin and washed with PBS, cells are resuspended in 250 μL 50 mM Tris-HCl, pH 7.5, 10 mM EDTA containing 100 mg/mL RNase A and lysed by the addition of 250 μL 1.2% sodium dodecyl sulfate (SDS) (p. 761, middle column, second paragraph, lines 4-8). The final concentration of SDS in this sample is approximately 0.6%.
Arad teaches that after 5 minutes at ambient temperature (p. 761, middle column, second paragraph, lines 9-11), cellular debris and chromosomal DNA are precipitated by the addition of 350 μL 3M CsCl, 1M potassium acetate and 0.67M acetic acid (p. 761, right column, lines 9-13). The final concentration of potassium acetate in this sample, after addition to the mixture above, is approximately 410 mM.
Arad further teaches that after precipitation of cellular debris and chromosomal DNA, the tubes are mixed, placed on ice for 15 minutes, centrifuged for 15 min, and the supernatant is loaded onto a silica gel membrane column (p. 761, right column, lines 13-17).
Arad does not teach the filtering the reaction mixture from step c), as recited in claim 22 step d).
Kemp teaches as described in the above rejection under 35 U.S.C. 103.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the present application to modify the method of Arad and apply a filtration step for the removal of precipitated cell debris, as taught by Kemp for the purification of plasmid DNA from bacterial cultures. One of ordinary skill in the art would have been motivated to modify the method of Arad and apply a filtration step for the removal of precipitated cell debris because Arad expressly teaches their method as a modification to traditional lysis performed on bacteria, and in view of the similarities in steps between the method of Arad and method of Kemp, one of ordinary skill in the art would have recognized that filtration is an alternative method to remove precipitated cell debris, as taught by Kemp. In this instance, the rationale “simple substitution of one known element for another to obtain predictable results” would apply. Because Arad teaches their method as applied to mammalian cells which satisfies all limitations of claims 22 and 23 except for the required filtration step to remove cell debris, and because Kemp teaches a similar method applied to bacterial plasmid isolation that uses steps, including the required filtration step, one of ordinary skill in the art would have contemplated the filtration step in place of centrifugation in the method of Arad for isolating extrachromosomal DNA from mammalian cells.
Therefore the invention taken as a whole is prima facie obvious.

Claims 24 and 26-30 are rejected under 35 U.S.C. 103 as being unpatentable over Arad in view of Kemp and Krupey (Publication no. WO 2001034844 A1; cited in IDS received December 14, 2022).
Claim 24 requires the biological sample is blood. Claims 26 and 27 depend from claim 24 and further limit the concentration of SDS and potassium salt when the complex sample is a blood sample. Claim 28 requires the biological sample is selected from the group consisting of feces sample, saliva sample, a product of respiratory lavage sample, and a nasopharyngeal secretion sample, and claims 29-30 depend from claim 28 and further limit the concentration of SDS and potassium salt.
Arad and Kemp teach as described in the above rejections under 35 U.S.C. 102 and 35 U.S.C. 103.
Arad and Kemp does not teach the sample as blood, as required by claims 24 and 26-27. In addition, the method of Arad does not teach the required potassium salt concentration recited in claim 30.
Krupey teaches a method for isolating DNA from blood, urine, and saliva (cover page, Abstract, lines 1-2) for use in forensic medicine, molecular bioinformatics, and non-invasive diagnostics (p. 44, lines 10-14). Krupey teaches the method as it applies to whole blood involves first lysing the cells and recovering the nucleated cell fraction (i.e., white blood cells) (p. 7, lines 16-18), followed by treating the white blood cell fraction with a surfactant (e.g. 1.0% aqueous sodium dodecyl sulfate, SDS) containing the metal chelator EDTA, adding potassium acetate to neutralize the SDS and to precipitate hemoglobin, and isolating the liquid phase (p. 7, lines 24-28).
More specifically, Krupey teaches that one volume of whole blood is treated with three volumes of a 1.0% w/v of sodium dodecyl sulfate (SDS) in a buffer, such as a 10 mM solution of Tris buffer and l00 mM EDTA pH 8.0, before adding 3 volumes of a 3M solution of potassium acetate to neutralize the SDS and to precipitate the hemoglobin that is present. Krupey teaches the tubes are then centrifuged and the supernatant is recovered (p. 27, lines 20-27).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the present application to apply the method obvious over Arad and Kemp to blood and saliva samples, in view of Arad teaching their method applied to mammalian cells, and Krupey teaching a method with similar conditions, including SDS-mediated lysis and potassium acetate neutralization, applied to blood and saliva samples. Thus one of ordinary skill in the art would have recognized that the method of Arad, modified to use a filtration step for removing precipitated cell debris as taught by Kemp, may be useful for isolating DNA from a blood or saliva sample for the purposes of diagnostic testing, as taught by Krupey.
One of ordinary skill in the art would have had a reasonable expectation of success applying the method obvious over Arad and Kemp to blood and saliva samples because Arad teaches the method as applying to mammalian cells, and Krupey teaches a method with similar conditions, including SDS-mediated lysis and potassium acetate neutralization that may be applied to blood and saliva samples. Thus one of ordinary skill in the art would have had a reasonable expectation that the method obvious over Arad and Kemp may be used to extract DNA from blood and saliva samples.
Regarding the final concentration of potassium acetate recited in claim 30, if the concentration of approximately 410 mM taught by Arad is interpreted as outside the range of about 300 mM, in view Kemp teaching a final concentration of approximately 330 mM potassium acetate, one of ordinary skill in the art would have recognized that a lower concentration of potassium acetate may be sufficient for this step in the method obvious over Arad in view of Kemp. Thus one of ordinary skill in the art would have contemplated a lower concentration of potassium acetate, such as 330 mM, which is interpreted as about 300 mM herein.
MPEP 2144.05 II at A states: “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).”
Therefore the invention taken as a whole is prima facie obvious.

Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Arad in view of Kemp, Krupey, and Chen.
Claim 25 depends from claim 24 and requires performing step b) as recited in claim 22.
Arad, Kemp, and Krupey teaches as described in the above rejections under 35 U.S.C. 102 and 35 U.S.C. 103. Specifically, as stated above, Krupey teaches in their method that one volume of whole blood is treated with three volumes of a 1.0% w/v of sodium dodecyl sulfate (SDS) in a buffer, such as a 10 mM solution of Tris buffer and l00 mM EDTA pH 8.0, before adding 3 volumes of a 3M solution of potassium acetate to neutralize the SDS and to precipitate the hemoglobin that is present. Krupey teaches the tubes are then centrifuged and the supernatant is recovered (p. 27, lines 20-27) (emphasis added).
Arad, Kemp, and Krupey do not teach contacting the mixture from step a) with a buffer comprising a zinc and/or copper salt in the concentrations recited in claim 22 step b), as required by claim 25.
Chen teaches as described in the above rejections under 35 U.S.C. 102 and 35 U.S.C. 103.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the present application to modify the method obvious over Arad in view of Kemp and Krupey by adding a solution comprising ZnCl2 or CuSO4 to facilitate precipitation of host cell impurities from the lysate, as taught by Chen. One of ordinary skill in the art would have been motivated to modify the method obvious over Arad in view of Kemp and Krupey by adding a solution comprising ZnCl2 or CuSO4 to facilitate precipitation of host cell impurities from the lysate in view of Krupey teaching the precipitation of host cell proteins like hemoglobin by adding potassium acetate in their isolation of blood DNA and Chen teaching addition of a divalent cation, such as ZnCl2 or CuSO4, with a monovalent ion such as potassium to precipitate host cell impurities in the lysate. Therefore, one of ordinary skill in the art, in view of Chen, would have considered adding an additional divalent ion, such as zinc or copper, to the monovalent potassium ion taught by Arad, Kemp, and Krupey for the purposes of improving precipitation of host cell impurities in the lysate.
Regarding the concentration of the ZnCl2 or CuSO4 stock solution, because Chen teaches in an embodiment a solution that comprises 0.4 M manganese chloride which is present in a final concentration of approximately 133 mM (p. 6, [0055]-[0059]). Because Chen teaches ZnCl2 or CuSO4 as alternatives to MnCl2, one of ordinary skill in the art would have reasonably considered the same final concentrations of ZnCl2 or CuSO4 as MnCl2. With respect to the concentration of the stock solution, if 0.4 M is considered to fall outside the scope of about 0.5 M as recited in claim 22, because Chen teaches additional concentrations up to about 4 M, one of ordinary skill in the art would have reasonably considered stock solutions that have higher concentrations of ZnCl2 or CuSO4.
Regarding the timing of the addition of the buffer comprising a concentration of a zinc and/or copper salt, Chen teaches addition of both the divalent metal ion (such as zinc or copper) and monovalent metal ion (such as potassium) simultaneously. However, one of ordinary skill in the art would have recognized that the method of Chen would not have been affected by addition of the buffer comprising the zinc and/or copper salt followed immediately by the buffer comprising a concentration of potassium salt, absent evidence to the contrary. Because Chen teaches the role of these metal ions is to facilitate precipitation of cell debris, one of ordinary skill in the art would have expected that each metal ion being present together in solution would be sufficient to precipitate the cell debris, regardless of which metal ion was added first.
Therefore the invention taken as a whole is prima facie obvious.

Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Arad in view of Kemp, Krupey, and Goldsborough (U.S. pre-grant publication no. US 20180058988 A1; cited in PTO-892).
Arad, Kemp, and Krupey teaches as described in the above rejections under 35 U.S.C. 102 and 35 U.S.C. 103.
Arad, Kemp, and Krupey do not teach contacting the mixture from step a) with a buffer comprising a concentration of a zinc and/or copper salt in the concentrations recited in claim 22, as required by claim 25.
Goldsborough teaches methods of stabilizing biomolecules, including RNA and DNA, in biological samples such as blood (cover page, Abstract, lines 1-4). Goldsborough teaches their invention provides a method for stabilizing RNA or DNA in an RNA- or DNA-containing sample by with a deep eutectic containing solvent (DES) to form a stabilized RNA- or DNA-containing composition (p. 3, [0022]-[0023]). Goldsborough teaches their invention provides use of a DES mixture as a stabilizer of exosomes, other types of vesicles, microvesicles, sub-cellular bodies and particles such as micelles, liposomes, endosomes, cell debris, cell membranes, cell nuclei, cytoplasmic components such as an endocytic compartment, golgi, endoplasmic reticulum and/or mitochondria (p. 4, [0041], lines 1-7) (emphasis added). In addition, Goldsborough teaches that their invention provides use of a DES mixture as a stabilizer of pre-purified particles and molecules such as viral particles, bacteriophages, RNA and DNA (p. 4, [0042], lines 1-4). Therefore, the disclosure of Goldsborough is interpreted to include a method of stabilizing DNA and RNA in tissue, stabilizing cell debris, and stabilizing pre-purified RNA and DNA.
Goldsborough further teaches that their invention includes the use of an additive in combination with a DES mixture to improve the properties of the DES mixture, for example, RNA, DNA and/or protein stabilization and/or cell and tissue fixation or another application (p. 13, [0172], lines 1-5). Goldsborough teaches that such additives include those which act as protein precipitants, such as zinc salts like ZnCl2 and ZnSO4 (p. 13, [0172], lines 26-28).
Goldsborough teaches an example wherein the deep eutectic solvent comprising 1:2 choline chloride:trifluoroacetamide was added to an E. coli prior to lysis, and that after an 18 hour incubation, the integrity of the 16S and 23S rRNA was unchanged compared to a fresh bacterial pellet (p. 32, [0293], lines 1-11). Goldsborough further teaches that ZnSO4 may be added to the choline chloride:trifluoroacetamide mixture at 1-33 mM, preferably 33 mM, to improve stabilization (p. 32, [0293], lines 11-15).
In addition, Goldsborough teaches an example wherein Hela cells were pelleted, choline chloride:trifluoroacetamide (1:2 mol:mol) was added to each pellet, then water at either a final concentration of 10 or 15% (vol:vol) was added in the presence or absence of 33 mM Zinc sulphate and 33% (wt:wt) molecular sieves before storage for 18 hours at 37°C (p. 28, [0279], lines 9-12). Goldsborough teaches that the addition of zinc sulphate or zinc sulphate and molecular sieves, can substantially reduce the amount of RNA degradation when water is present (p. 28, [0279], lines 17-21; see results in table 7 on p. 29). Goldsborough teaches that this is particularly useful to improve sample analyte quality when substantial amounts of water are present, such as with larger tissue samples, blood, serum, plasma or plant material (p. 28, [0279], lines 21-26).
It would have been prima facie obvious to apply the method obvious over Arad in view of Kemp and Krupey with the step of adding a combination of choline chloride:trifluoroacetamide and ZnSO4 to preserve the nucleic acid, as taught by Goldsborough. One of ordinary skill in the art would have been motivated to modify the method obvious over Arad in view of Kemp and Krupey with the step of adding a combination of choline chloride:trifluoroacetamide and ZnSO4 to preserve the nucleic acid because Goldsborough teaches biomolecule degradation is a problem when using biomolecules for assay and diagnostic purposes and teaches the combination of choline chloride:trifluoroacetamide and ZnSO4 may improve the stability of said biomolecules. Therefore, one of ordinary skill in the art would have recognized that addition of a combination of choline chloride:trifluoroacetamide and ZnSO4 may be added to the nucleic acid mixture to stabilize the nucleic acid being isolated.
Regarding the timing of the addition of ZnSO4, because Goldsborough teaches that their invention provides a method to stabilize DNA and RNA in tissues, in pre-purified samples, and to stabilize cell debris, one of ordinary skill in the art would have reasonably considered adding the combination of choline chloride:trifluoroacetamide and ZnSO4 before step a), after step a) (as presently claimed), after step c), or after step d). Such timing of the addition would have been obvious to try in view of Goldsborough teaching that their method may be applied to samples at multiple stages of purification, and thus one of ordinary skill in the art would have optimized the method by testing the addition of said combination at each step of the method and determining the optimal timing of the addition for purifying DNA from blood.
Regarding the concentration of ZnSO4, Goldsborough teaches final concentrations that overlap with the presently claimed range. With respect to the stock concentration of ZnSO4, Goldsborough does not teach the stock concentration. However, absent evidence that a concentration 0.5 M ZnSO4 is not possible to prepare, one of ordinary skill in the art would have recognized the concentration of the stock solution is irrelevant to the final concentration, because different volumes of the stock solution may be added to achieve the desired final concentration. In addition, the stock solution may be pre-diluted to achieve a concentration that is convenient to add to the mixture to achieve the desired final concentration of ZnSO4. See MPEP 2144.05 II at A.
Therefore the invention taken as a whole is prima facie obvious.

Claims 28-30 are rejected under 35 U.S.C. 103 as being unpatentable over Arad in view of Kemp and Corstjens (Corstjens, P. L. A. M.; et al. Periodontology 2000, 2015, vol. 70, pp. 93-110; cited in PTO-892).
Arad and Kemp teach as described in the above rejection under 35 U.S.C. 103. In addition, Arad specifically states that methods for extracting extrachromosomal DNA from eukaryotic cells are important for many studies such as DNA replication and identification of certain viruses (p. 761, left column, lines 1-5) (emphasis added).
Arad and Kemp do not teach wherein the biological sample is selected from the group consisting of feces sample, saliva sample, a product of respiratory lavage sample, and a nasopharyngeal secretion sample, as required by claims 28-30.
Corstjens teaches that human viral infections and transmission occur through multiple paths, including fecal–oral, ingestion of contaminated food and drinks, sexual contact, exposure to infected blood, exchange of saliva or by aerosols generated by sneezing or coughing, and further teaches common examples of viruses isolated from the oral cavity include rotavirus, norovirus, HIV, hepatitis C virus, herpes simplex viruses 1 and 2, Epstein–Barr virus and influenza viruses (p. 93, left column, lines 1-10).
Corstjens teaches that the herpes simplex virus (HSV) is a double-stranded DNA virus that enters cells through the interaction of viral coat glycoprotein with cellular receptors and is transported to the cell nucleus where its DNA is released into the nucleus and replication is initiated. Corstjens teaches that the linear viral DNA is not integrated in the human genome but remains as an extrachromosomal circular DNA (p. 102, left column, Herpes simplex viruses section, lines 1-8). Corstjens teaches that several studies have described the detection of anti-herpes simplex virus antibodies and nucleic acid in oral samples, including saliva (p. 103, left column, Herpes simplex virus and saliva section, lines 1-8) (emphasis added).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the present application to apply the method obvious over Arad in view of Kemp to isolate viral DNA from saliva, such as HSV viral DNA. One of ordinary skill in the art would have been motivated to apply the method obvious over Arad in view of Kemp to isolate viral DNA from saliva, such as HSV viral DNA, in view of Arad expressly teaching the method to extract extrachromosomal DNA from eukaryotic cells for the identification of certain viruses, and Corstjens teaching that many viruses occupy the oral cavity, including HSV, which remains as an extrachromosomal circular DNA, and has been found in saliva. Thus one of ordinary skill in the art would have recognized that the method obvious over Arad may be used to extract DNA from saliva to identify viruses that may be present in the oral cavity.
Regarding the final concentration of potassium acetate recited in claim 30, if the concentration of approximately 410 mM taught by Arad is interpreted as outside the range of about 300 mM, in view Kemp teaching a final concentration of approximately 330 mM potassium acetate, one of ordinary skill in the art would have recognized that a lower concentration of potassium acetate may be sufficient for this step in the method obvious over Arad in view of Kemp. Thus one of ordinary skill in the art would have contemplated a lower concentration of potassium acetate, such as 330 mM, which is interpreted as about 300 mM herein.
MPEP 2144.05 II at A states: “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).”
Therefore the invention taken as a whole is prima facie obvious.

Claims 16-21 are rejected under 35 U.S.C. 103 as being unpatentable over Kemp in view of Goldsborough and Yang (Yang, Y.; et al. Journal of Biomolecular Techniques 2008, vol. 19, pp. 205-210; cited in PTO-892).
Claim 16 is drawn to a kit for extracting nucleic acids contained in a complex sample comprising the requirements listed therein. Claim 17 requires the zinc salt is selected from a group consisting of zinc sulfate or zinc chloride, claim 18 requires the copper salt is selected from a group consisting of copper sulfate or copper chloride, claim 19 requires the kit is the potassium salt is selected from the group that includes potassium acetate, claim 20 requires the lysis buffer and/or the buffer comprising a zinc and/or copper salt comprise Tris-HCl, HEPES, and MOPS, and claim 21 claims a method for extracting nucleic acids contained in a complex sample, comprising extracting the nucleic acid using the kit according to claim 16.
Kemp and Goldsborough teach as described in the above rejections under 35 U.S.C. 102 and 35 U.S.C. 103.
Kemp and Goldsborough do not teach the filter pore size, as required by claim 16.
Yang teaches as described in the above rejection under 35 U.S.C. 103.
It would have been prima facie obvious to one of ordinary skill in the art to package the components required by the method of DNA extraction obvious over Kemp in view of Goldsborough as a kit. One of ordinary skill in the art would have been motivated to package the components required by the method of DNA extraction obvious over Kemp in view of Goldsborough as a kit in view of Kemp teaching that the method of their invention encompasses kits, and because packaging of said components may reduce the time and likelihood of error for the user performing the method obvious over Kemp in view of Goldsborough.
With respect to the pore size of the filter, because Yang teaches a method of clarifying lysate using a 1 μm membrane syringe filter, one of ordinary skill in the art would have contemplated a 1 μm pore size filter to be used in the method taught by Kemp and included in the kit used for extraction of nucleic acids.
Regarding the concentration of the zinc salt, Goldsborough does not teach the concentration of the stock solution. However, absent evidence that a concentration 0.5 M ZnSO4 is not possible to prepare, one of ordinary skill in the art would have recognized the concentration of the stock solution is irrelevant to the final concentration disclosed by Goldsborough, because a specific volume of the stock solution may be added to achieve the desired final concentration. In addition, one of ordinary skill in the art would have recognized that the stock solution may be pre-diluted to achieve a concentration that is convenient to achieve the desired final concentration of ZnSO4. See MPEP 2144.05 II at A.
Regarding claim 20, because Kemp and Yang teach that cells are resuspended in a buffer comprising Tris-HCl before SDS is added, one of ordinary skill in the art would have recognized that Tris-HCl may be part of the lysis buffer with a reasonable expectation that the lysis buffer will still be able to perform its role in the method of Kemp and Yang. Alternatively, because the method of Kemp and Yang specifies that lysis occurs in a buffer comprising Tris-HCl, the lysis buffer of a kit may reasonably be considered a two component solution of solution P1 comprising Tris-HCl and solution P2 comprising SDS, which together form the lysis buffer.
Regarding claim 21, in view of Kemp teaching a method of extracting nucleic acids from bacterial samples using all components of claim 16 except the buffer comprising a zinc and/or copper salt, one of ordinary skill in the art would have recognized the kit of claim 16, obvious over Kemp in view of Goldsborough and Yang, may be used to extract nucleic acids from a complex sample, such as a sample of bacteria.
The combination of Kemp, Goldsborough, and Yang do not teach a method that requires a copper salt. However, because the copper salt is recited as an alternative to the zinc salt, the method of claim 16 can be practiced without a copper salt. Therefore, claim 18, which only limits the copper salt, is included in this rejection as obvious over Kemp in view of Goldsborough and Yang.
Therefore the invention taken as a whole is prima facie obvious.

Conclusion
No claim is allowed.
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/B.M.B./Examiner, Art Unit 1693
/ERIC OLSON/Primary Examiner, Art Unit 1693