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
The present application, filed November 29, 2022, is a national stage application of PCT/CN2021/097213, filed May 31, 2021, and claims priority to foreign priority application CN202010488101.3, filed June 1, 2020.
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 February 3, 2026 has been entered.
Status of the Application
Applicant’s communication, received February 3, 2026, wherein claims 1, 6-8, and 16 are amended and claims 10 and 17 are canceled, is acknowledged.
Claims 1-9, 16, and 18 are pending and examined on the merits herein.
Withdrawn Rejections
Applicant’s amendment, received February 3, 2026, with respect to the rejection of claim 16 under 35 USC § 112(b) as indefinite, has been fully considered and found to be persuasive to remove the rejection because claim 16 is amended to recite the nucleic acid extraction process according to claim 2, which is not indefinite. Therefore the rejection is withdrawn.
Applicant’s amendment, received February 3, 2026, with respect to the rejection of claims 7-8 under 35 USC § 102 as anticipated by Montesclaros, has been fully considered and found to be persuasive to remove the rejection because claim 7 is amended to require the quaternary ammonium salt is added to a binding solution, and wherein the binding solution is isopropanol, absolute ethanol or polyethylene glycol, which Montesclaros does not teach. Therefore the rejection is withdrawn.
Applicant’s amendment, received February 3, 2026, with respect to the rejection of claims 7-9 under 35 USC § 102 as anticipated by Deggerdal, has been fully considered and found to be persuasive to remove the rejection because claim 7 is amended to require the quaternary ammonium salt is added to a binding solution, and wherein the binding solution is isopropanol, absolute ethanol or polyethylene glycol, which the embodiments of Deggerdal cited in the previous rejection do not teach. Therefore the rejection is withdrawn.
Applicant’s amendment, received February 3, 2026, with respect to the rejection of claims 7-10 under 35 USC § 102 as anticipated by Greenfield, has been fully considered and found to be persuasive to remove the rejection because claim 7 is amended to require the quaternary ammonium salt is added to a binding solution, and wherein the binding solution is isopropanol, absolute ethanol or polyethylene glycol, which Greenfield does not teach. Therefore the rejection is withdrawn.
Applicant’s amendment, received February 3, 2026, with respect to the rejection of claims 7-9 under 35 USC § 102 as anticipated by Akhaven-Tafti, has been fully considered and found to be persuasive to remove the rejection because claim 7 is amended to require the quaternary ammonium salt is added to a binding solution, and wherein the binding solution is isopropanol, absolute ethanol or polyethylene glycol, which Akhaven-Tafti does not teach. Therefore the rejection is withdrawn.
Claim Interpretation
The limitation “for neutralizing the negative charge of a nucleic acid and facilitating binding of the nucleic acid to an adsorbing material and a binding solution” recited in claim 7 is interpreted herein as an intended use of the nucleic acid extraction reagent recited in claim 7.
MPEP 2111.02 (at II) states: “During examination, statements in the preamble reciting the purpose or intended use of the claimed invention must be evaluated to determine whether or not the recited purpose or intended use results in a structural difference (or, in the case of process claims, manipulative difference) between the claimed invention and the prior art. If so, the recitation serves to limit the claim. …To satisfy an intended use limitation which is limiting, a prior art structure which is capable of performing the intended use as recited in the preamble meets the claim.”
In this instance, claim 7 is satisfied by a composition comprising a compound of Formula I is added to a solution of isopropanol, absolute ethanol, or propylene glycol, even if that composition is not used “for neutralizing the negative charge of a nucleic acid and facilitating binding of the nucleic acid to an adsorbing material and a binding solution.”
Claim Objections
Claims 1, 7, and 8 are objected to because of the following informalities:
The structures of Formula I shown in claims 1 and 7 are of sufficiently low resolution that they are difficult to interpret. Please replace these structures with higher resolution images.
In addition, claim 8 claims the nucleic acid extraction reagent according to claim 7, but references the quaternary ammonium salt having the structure as shown in Formula I of claim 1. Therefore, claim 8 appears to depend from claim 7 and claim 1. To promote clarity in the claims, and because claim 7 also recites the quaternary ammonium salt having the structure as shown in
Formula I, please amend claim 8 to depend from claim 7 and reference the quaternary ammonium salt of Formula I of claim 7.
Appropriate correction is required.
The following are modified grounds of rejection in response to Applicant’s amendments received February 3, 2026.
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.
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 1-6, 16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Montesclaros (Publication no. WO 2005007895 A1; of record).
Montesclaros teaches and claims a method for obtaining nucleic acid from a biological sample and binding the nucleic acid to a solid phase, comprising: combining the sample with at least one chaotrope and at least one zwitterionic compound to form a combination; and exposing the combination to at least one solid phase to bind the nucleic acid (p. 51, claim 14), and also further comprising isolating the nucleic acid (p. 51, claim 15). Montesclaros teaches and claims the chaotrope is selected from a group of chaotropes that includes tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium iodide, tetrapropylammonium bromide, tetrapropylammonium chloride, and tetrapropylammonium iodide (pp. 52-53, claim 17), each of which satisfies the requirements of formula 1 of claim 1. Moreover, at least tetrabutylammonium bromide, tetrabutylammonium chloride, tetrapropylammonium bromide, and tetrapropylammonium chloride satisfy the requirements of the compound of Formula I of claims 2-6 and 16.
In addition, Montesclaros teaches an example in which a protease solution of 1 mg proteinase K 100 mM Tris-HCI, pH 8.0 was combined with 25, 50, or 100 μL whole blood sample in microfuge tubes, incubated at 58 °C for 10 minutes, then 500 μL of chaotrope solution consisting of 5 M GuSCN, 50 mM EDTA pH 8.0, 200 mM Tris-HCI, pH 7.0 and 2% Zwittergent 3-16 was added with mixing (p. 37, [0081], lines 1-9).
Montesclaros teaches that the solutions were transferred to the wells of the purification tray which included silica particles sandwiched between two polypropylene frits (p. 37, [0081], lines 1-5). Montesclaros teaches the samples were washed once with chaotrope solution, three times with 90% ethyl alcohol, and the nucleic acid was eluted from the bound complexes by adding 100 μL 0.01 N NaOH to each well, followed by 15 mM Tris-HCI, 1 mM EDTA pH 7.0 (p. 38, first paragraph, lines 1-6). Montesclaros teaches the isolated gDNA was collected in a 96-well sample archive tray (p. 38, first paragraph, lines 6-7).
The steps of this method taught by Montesclaros constitute a) lysing with a lysis solution, b) binding to an adsorbing material with a binding solution, c) washing the nucleic acid bound to the adsorbing material with a washing solution, and d) eluting the nucleic acid bound to the adsorbing material using an eluent. In this instance, the protease solution above is reasonably considered a lysis solution and would be expected to release nucleic acids from the sample, and the chaotrope solution is reasonably considered a binding solution. In addition, because Montesclaros teaches isolation of gDNA, the nucleic acid in the method of Montesclaros is a deoxyribonucleic acid, as required by claim 18.
Furthermore, Montesclaros teaches an embodiment in which increasing amounts of chaotrope solution are added to a sample of extracted nucleic acid before binding to a glass fiber tray, washing, eluting, and analyzing the DNA by UV/Vis spectrometry (p. 40, [0087]-[0088]). Montesclaros teaches that increasing chaotrope concentration increases the amount of gDNA isolated in their method (document p. 64, Figure 8).
Montesclaros does not teach a specific embodiment wherein the quaternary ammonium salt recited in the present claim 1 is used as the chaotrope for extracting a nucleic acid from a sample.
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 perform the method of nucleic acid isolation taught by Montesclaros above using a quaternary ammonium salt, such as tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium iodide, tetrapropylammonium bromide, tetrapropylammonium chloride, or tetrapropylammonium iodide, as the chaotrope. One of ordinary skill in the art would have been motivated to perform the method of nucleic acid isolation taught by Montesclaros using a quaternary ammonium salt, such as tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium iodide, tetrapropylammonium bromide, tetrapropylammonium chloride, or tetrapropylammonium iodide, as the chaotrope, because Montesclaros teaches the above method using a chaotrope solution with GuSCN, and further teaches the chaotrope may be selected from a list that includes the tetrabutylammonium and tetrapropylammonium salts recited above. Therefore, one of ordinary skill in the art would have reasonably contemplated substituting the chaotrope GuSCN for tetrabutylammonium and tetrapropylammonium salts because Montesclaros expressly suggests these compounds as chaotropes which may be used in their method of isolating nucleic acids. Moreover, because Montesclaros suggests these tetrabutylammonium and tetrapropylammonium salts as chaotropes, one of ordinary skill in the art would have had a reasonable expectation of successfully isolating nucleic acid using the method of Montesclaros substituting tetrabutylammonium and tetrapropylammonium salts in place of GuSCN.
Regarding the limitation of claim 1 that requires “neutralizing the negative charge of the nucleic acids with an agent for neutralizing the negative charge of the nucleic acids and facilitating binding of one or more of the nucleic acids to the adsorbing material, and binding one or more of the nucleic acids to an adsorbing material using a binding solution;” this is believed to be necessarily present when practicing the method of Montesclaros using a quaternary ammonium salt.
As described above, Montesclaros renders obvious a method that involves adding a chaotrope to facilitate binding of the nucleic acid to an adsorbing material, wherein the chaotrope is one of the quaternary ammonium salts recited above. Because the quaternary ammonium group carries a formal positive charge and the gDNA carries a formal negative charge, the addition of the quaternary ammonium salt to the binding buffer would necessarily have the effect of neutralizing the charge of the nucleic acid, absent evidence to the contrary.
Moreover, as evidenced by the instant specification, a certain concentration of quaternary ammonium salts can neutralize the negative charge of nucleic acids, such that the nucleic acids can bind to the adsorbing material or be precipitated in the presence of polar solvents (ethanol, isopropanol, etc.) by hydrogen bond, hydrophobic interaction and electrostatic interaction (p. 3, lines 25-29).
In addition, because Montesclaros teaches that the chaotrope concentration during DNA binding to the adsorbent material affects the amount of DNA recovered (see document p. 64, Figure 8) and suggests, for example, tetrabutylammonium bromide and tetrabutylammonium chloride salts as chaotropes, one of ordinary skill in the art would have contemplated the addition of said salts for the purposes of facilitating DNA binding to the adsorbent material.
This is further evidenced by the instant specification, which provides that nucleic acid can reversibly bind to the nucleic acid-adsorbing material, glass particle, glass fiber, magnetic bead, diatomite, silica gel, in solution or precipitate in the presence of polar organic solvent, and that high concentrations of chaotropic salts can facilitate the binding of nucleic acids to these materials (p. 1, lines 33-37).
Therefore the limitations wherein the quaternary ammonium salt neutralizes the negative charge of the nucleic acids and facilitates binding of one or more of the nucleic acids to the adsorbing material are believed to be necessarily present when practicing the method obvious over Montesclaros, wherein a quaternary ammonium salt is selected as the chaotrope.
Therefore the invention taken as a whole is prima facie obvious.
Response to Applicant’s arguments: Regarding the previous rejection of claims 1-6 and 16-18, Applicant argues Montesclaros fails to teach or suggest every element of the claims as-amended. Applicant submits that Montesclaros fails to teach a nucleic acid extraction method which involves a step of "neutralizing the negative charge of the nucleic acids and facilitating binding of the nucleic acids to the adsorbing material", such as, magnetic beads, silica membranes, or other solid supports. In the absence of such teaching, those skilled in the art cannot envisage neutralizing the negative charge of nucleic acids; that is, Montesclaros does not provide a motivation of neutralizing the negative charge of nucleic acids. In fact, those skilled in the art would try to preserve the native charge of nucleic acids as much as possible to avoid interference with experimental results. Applicant argues that Montesclaros has clearly disclosed the function of chaotropic salts in the method therein, i.e., a substance that causes disorder in a protein or nucleic acid by altering the secondary, tertiary, or quaternary structure of a protein or a
nucleic acid while leaving the primary structure intact. According to this disclosure of Montesclaros, those skilled in the art would select the concentration of quaternary ammonium salts based on this function thereof.
Applicant further argues that Montesclaros did not recognize that quaternary ammonium salts could have the function of "neutralizing the negative charge of the nucleic acids and
facilitating binding of the nucleic acids to the adsorbing material"; otherwise, they would
have explicitly disclosed it and would have directly performed the step of binding without using quaternary ammonium salts to disorder the nucleic acids. The applicant wishes to emphasize that according to the description of this application, those skilled in the art would know that the quaternary ammonium salts in the binding solution of this application will not denature nucleic acids.
Applicant argues that according to the function of chaotropic salts as disclosed in Montesclaros, such salts should be added before the step of binding and cannot be added to the binding solution. In contrast, the examples of this application (such as, Examples 5 and 6) have clearly demonstrated that quaternary ammonium salts can be added during the binding step and exert the effects of facilitating binding. The method of this application is markedly different from that of Montesclaros in that: in the method of Montesclaros chaotropic salts clearly cannot be added during the step of binding; while in the method of this application, since quaternary ammonium salts are not required for disordering nucleic acids, they need not be added before the step of binding. Thus, adding the quaternary ammonium salt to the binding solution as currently claimed would change the fundamental principle of operation of Montesclaros. Therefore, one of
ordinary skill in the art would not add the quaternary ammonium salt to the binding solution as currently claimed.
Applicant argues that while it is true that quaternary ammonium salts carry a positive charge and may incidentally neutralize, but not necessarily neutralize, the negative charge of nucleic acids in the method of Montesclaros; nevertheless, without any relevant indication to this
effect in Montesclaros, those skilled in the art could not have known that such neutralization would promote nucleic acid binding; in fact, they even cannot notice this. Moreover, as mentioned above, those skilled in the art would try to control the concentration of quaternary ammonium salt in the method of Montesclaros to avoid neutralizing the negative charge of nucleic acids, thereby minimizing potential interference with nucleic acid extraction.
Finally, Applicant argues that the use a quaternary ammonium salt with a structure as shown in Formula I in nucleic acid extraction, in place of the commonly used guanidine salt,
wherein in the presence of a binding solution of polar organic solvent, the quaternary ammonium salt could make the nucleic acid molecules be adsorbed to the materials such as silica beads, magnetic beads, diatomite or silica gel, or be precipitated, and thus can effectively improve the efficiency of nucleic acid purification and recovery; the quaternary ammonium salt can be used for plasmid DNA extraction, viral genome extraction, bacterial genome extraction, or animal, plant and human genome DNA extraction.
Applicant includes a declaration from Dr. Bi Wanli, stating that the "nucleic acids extracted using this method perform exceptionally well in downstream PCR and sequencing, with significantly higher recovery efficiency compared to traditional methods" (see Page 1 of declaration). The declaration further states that this application is the first to use quaternary ammonium salts as both phase-separating salts and charge-neutralizing agents during the nucleic acid extraction workflow. This allows for nucleic acid extraction with "no requirement for organic solvents", "rapid low-temperature lysis and phase separation", and "high-purity, high yield nucleic acid extraction suitable for complex samples" (see declaration, page 1).
Applicant’s arguments have been fully considered but they are not found persuasive. Regarding Applicant’s arguments that Montesclaros did not recognize that quaternary ammonium salts could have the function of "neutralizing the negative charge of the nucleic acids and facilitating binding of the nucleic acids to the adsorbing material" and otherwise, they would
have explicitly disclosed it and would have directly performed the step of binding without using quaternary ammonium salts to disorder the nucleic acids, the examiner maintains that because Montesclaros teaches that the final concentration of chaotrope present in the sample before binding to the solid adsorbent material affects the recovery of gDNA from said sample (document p. 64, Figure 8), one of ordinary skill in the art would have recognized that the concentration of chaotrope may affect retention of nucleic acid on the solid support, which is taken as an acknowledgement that the chaotrope facilitates binding to the adsorbing material. Regarding the denaturation of nucleic acids, it is unclear how this argument affects the present rejection, because Montesclaros suggests the above quaternary ammonium salts for use in their method, and thus the effects of those salts on nucleic acid structure would have been recognized when making said suggestion.
Regarding Applicant’s argument about neutralization of DNA using the quaternary ammonium salt, as recited above, the examiner believes that the limitations wherein the quaternary ammonium salt neutralizes the negative charge of the nucleic acids and facilitates binding of one or more of the nucleic acids to the adsorbing material are believed to be necessarily present when practicing the method of Montesclaros. Because Montesclaros provides motivation to practice their method with quaternary ammonium salts, one of ordinary skill in the art would have contemplated their use in the method of Montesclaros. Although the motivation to practice the method of Montesclaros with quaternary ammonium salts may not be the same as the motivation recited in claim 1, because the method of Montesclaros renders obvious mixing the quaternary ammonium salt with the DNA sample prior to binding, one of ordinary skill in the art would have considered this step. As evidenced by the specification, such a step would have necessarily neutralized the charge of DNA.
The examiner is unclear what is meant by the phrase “incidentally neutralize, but necessarily neutralize” recited in Applicant’s arguments. As stated, even if motivation to add the quaternary ammonium salt were different than Applicant’s motivation, because the addition of a quaternary ammonium salt is suggested as a chaotrope by Montesclaros, one of ordinary skill in the art would have contemplated the guidance of Montesclaros and considered practicing their method with the quaternary ammonium salt suggested by Montesclaros.
Regarding Applicant’s claim of unexpected or superior results, a claim of unexpected results must include a comparison with the closest prior art. In this instance, the declaration from Dr. Bi Wanli states that “nucleic acids extracted using this method perform exceptionally well in downstream PCR and sequencing, with significantly higher recovery efficiency compared to
traditional methods,” but does not point to evidence showing that the claimed method using quaternary ammonium salts for nucleic acid extraction is superior to the method of using guanidinium salts. Applicant is encouraged to provide specific evidence demonstrating that their claimed method offered a significantly higher recovery efficiency compared to traditional the closest prior art.
The examiner has identified an example in the specification wherein which trace genomic DNA is extracted under conditions comprising 40% isopropanol and 1.6 M tetraethylammonium bromide or 1.6 M guanidine hydrochloride (p. 10, lines 43-45). The specification concludes that the results showed that tetraethylammonium bromide could significantly improve the extraction efficiency for trace DNA, and the extraction efficiency of tetraethylammonium bromide is much higher than that of guanidine hydrochloride at the same concentration, indicating that tetrabutylammonium bromide could effectively extract and purify trace genomic DNA (p. 11, lines 31-36), with results of this experiment are shown in Figure 5.
Figure 5 appears to show a set of chromatograms with larger chromatogram signals for the conditions comprising tetraethylammonium bromide compared with guanidine hydrochloride. However, both the x axis and the y axis value of these plots are difficult to interpret given the resolution of the figure. If the examiner’s interpretation is correct, clear evidence (for example, a plot showing relative yield of DNA based on the total integration of each chromatogram) of a higher extraction yield with tetraethylammonium bromide is achieved compared to guanidine or guanidinium salt would be persuasive evidence of non-obviousness to overcome the present rejection.
Therefore, for the reasons stated above, the present rejection of claims 1-6, 16, and 18 as obvious over Montesclaros is maintained.
Claims 1-9, 16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Deggerdal (Publication no. WO 2004108741 A1; of record).
Deggerdal discloses and claims a method to isolate and/or concentrate nucleic acids or nucleic acid analogs from an aqueous solution comprising the steps of: (a) providing an aqueous solution containing nucleic acids, (b) adding an aliquot of substance I to (a), (c) adding an aliquot of substance II to (b), and (d) centrifuging the aqueous solution of (c) and discarding the supernatant, wherein an aliquot of substance I and an aliquot of substance II is given separated from each other but at the same time to the aqueous solution of (a) (p. 16, claim 1). Deggerdal claims that substance I is chosen from a group of negatively charged ionic detergents (p. 16, claim 2) and wherein substance I is lithium dodecyl sulfate (LiDS) or sodium dodecyl sulfate (SDS) (p. 16, claim 3). LiDS and SDS are interpreted herein as surfactants. Deggerdal claims that substance II is a chaotropic salt or a mixture of different chaotropic salts (p. 17, claim 7), and claims substance II is selected from a group that includes tetramethylammonium chloride, tetraethylammonium chloride, tetramethyl ammonium iodide, and tetraethyl ammonium iodide (p. 17, claim 8). These compounds satisfy all limitations of the compound of formula I recited in claims 1-6, and the tetramethylammonium chloride and tetraethylammonium chloride satisfy the limitations of the anion of claim 16.
Deggerdal further teaches that the precipitate obtained in step (d) above can be subjected to additional purification steps utilizing standard methods. Deggerdal teaches that in one exemplary embodiment, step (d) is followed by a purification comprising the rough steps of: (e) resuspending the precipitate obtained in step (d) in a buffer containing chaotropic salt(s) and an alcohol, e.g. ethanol, and, subsequently, adding magnetic silica beads, (f) allowing nucleic acids to bind to the magnetic beads, removing the magnetic beads after an appropriate incubation time and discarding the supernatant, (g) exposing the complex of magnetic beads and nucleic acids to one or more washing steps, (h) and eluting the nucleic acids from the magnetic beads (p. 9, line 27 top p. 10, line 10). Deggerdal also claims the nucleic acid is DNA or RNA or a mixture
thereof (p. 18, claim 15).
Deggerdal additionally teaches examples of isolating nucleic acids from cells. As one example, Deggerdal teaches that 1x106 HL60 cells were incubated in 1 ml of an aqueous solution of 2% (w/v) SDS at pH 12.5, and to lyse the cells efficiently, the suspension was incubated for 5 minutes at 90°C. Deggerdal teaches that at room temperature, 1 ml of an aqueous solution of 2 M guanidinium hydrochloride was added. Thereafter, the solution was centrifuged and the precipitate was further purified as described in Example 1 (p. 13, Example, 4, lines 15-21). Example 1 teaches that each precipitate was further purified using the QIAGEN MagAttract® RNA Cell Mini M48 kit according to the manufacturer’s instructions (p. 11, lines 14-16).
The method of nucleic acid extraction taught by Deggerdal above satisfies each method step of present claim 1, involving a) lysing a sample with a lysis solution, and after a precipitation step, b) resuspending nucleic acids in a buffer containing chaotropic salts and an alcohol and binding to magnetic silica beads, c) washing the nucleic acid bound to the adsorbing material with a washing solution, and d) eluting the nucleic acid bound to the adsorbing material using an eluent.
Finally, Deggerdal teaches that nucleic acids are bound to silica surfaces under chaotropic conditions, that is typically 2 M to 8 M of a chaotropic salt, such as guanidinium salts, alone or in combination with EtOH (p. 1, lines 23-26). Deggerdal teaches that, for a chaotropic salt alone, a final concentration of 2 M to 8 M is typically needed to achieve an appropriate nucleic acid binding to a nucleic acid binding solid phase, and if the chaotropic salt is used in combination with an alcohol, e.g. EtOH, the alcohol has typically a final concentration of 30-60% (v/v) to achieve an appropriate binding of the nucleic acids to a nucleic acid binding solid phase (p. 2, lines 14-18).
Deggerdal does not teach a specific embodiment wherein nucleic acids are extracted according to their method using tetramethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium iodide, or tetraethyl ammonium iodide as the chaotropic salt as the chaotrope.
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 perform the method of Deggerdal using one of tetramethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium iodide, or tetraethyl ammonium iodide as the chaotropic salt. One of ordinary skill in the art would have been motivated to perform the method of Deggerdal using one of tetramethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium iodide, or tetraethyl ammonium iodide as the chaotropic salt because Deggerdal teaches a general strategy to isolate nucleic acids using chaotropic salts that satisfy each method step of the present claims 1-6, 16, and 18, and further teaches a group of chaotropic salts that may be used in such a method that includes tetramethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium iodide, or tetraethyl ammonium iodide. Therefore, in view of Deggerdal suggesting their method using chaotropic salts, including a method wherein the chaotropic salts are added to the binding buffer before binding to an adsorbing material, and further suggesting tetramethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium iodide, or tetraethyl ammonium iodide as chaotropic salts, one of ordinary skill in the art would have contemplated said chaotropic salts when practicing the method of Deggerdal.
Regarding the limitations wherein the quaternary ammonium salt neutralizes the negative charge of the nucleic acids and facilitates binding of one or more of the nucleic acids to the adsorbing material, these properties are believed to be necessarily present when practicing the method of Deggerdal. Because the chaotropic salts taught by Deggerdal are quaternary ammonium salts that carry a formal positive charge, they would necessarily neutralize the negative charge of the nucleic acids, absent evidence to the contrary.
Moreover, as evidenced by the instant specification, a certain concentration of quaternary ammonium salts can neutralize the negative charge of nucleic acids, such that the nucleic acids can bind to the adsorbing material or be precipitated in the presence of polar solvents (ethanol, isopropanol, etc.) by hydrogen bond, hydrophobic interaction and electrostatic interaction (p. 3, lines 25-29).
In addition, because the chaotropes are added to the buffer for binding to the solid support, they serve the purpose of facilitating binding to the adsorbing material, absent evidence to the contrary. Furthermore, because Deggerdal acknowledges that nucleic acids are bound to silica surfaces under chaotropic conditions, such as high concentrations of a chaotropic salt, either alone or in combination with ethanol, one of ordinary skill in the art would have recognized the role of a chaotropic salt in facilitating binding to adsorbent materials such as silica surfaces, and contemplated their use for the purposes of improving binding to a silica adsorbent material.
This is further supported by evidence from the instant specification providing that nucleic acids can reversibly bind to the nucleic acid-adsorbing material, glass particle, glass fiber, magnetic bead, diatomite, silica gel, in solution or precipitate in the presence of polar organic solvent, and that high concentrations of chaotropic salts can facilitate the binding of nucleic acids to these materials (p. 1, lines 33-37).
Therefore the limitations wherein the quaternary ammonium salt neutralizes the negative charge of the nucleic acids and facilitates binding of one or more of the nucleic acids to the adsorbing material are believed to be necessarily present when practicing the method obvious over Deggerdal.
Regarding claim 7, because Deggerdal teaches the above chaotropic salts in a binding solution with an alcohol, such as ethanol, and because Deggerdal teaches ethanol and isopropanol are both known to facilitate nucleic acids precipitate around magnetically attractable beads and teaches concentrations of chaotropic salt and ethanol that may be used for binding nucleic acids to adsorbent materials, one of ordinary skill in the art would have contemplated the alcohol as ethanol or isopropanol in the binding solution of claim 7. In this case, an appropriate amount of nucleic acid extraction reagent may be reasonably added to a defined volume of alcohol, such as absolute ethanol or to isopropanol, to achieve the desired concentration of chaotropic salt and alcohol when binding nucleic acids to the adsorbent material.
Regarding claims 8-9, because Deggerdal teaches the above chaotropic salts, which satisfy the limitations of the compound of formula I of claim 7, for use in nucleic acid extraction, and further teaches their use with a lysis solution comprising the LiDS or SDS, which are surfactants, one of ordinary skill in the art would have reasonably considered a nucleic acid extraction reagent comprising, for example, the chaotropic salts tetramethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium iodide, or tetraethyl ammonium iodide with a lysis solution that includes a surfactant. Moreover, because this nucleic acid extraction reagent is used in a method that includes a washing solution, an eluent, and an adsorbent material (for example, magnetic silica beads), including these components with the nucleic acid extraction reagent of claim 7 and the lysis solution would have been obvious, because including each of these components with the nucleic acid extraction reagent of claim 7 would facilitate practicing the nucleic acid extraction process taught by Deggerdal.
Therefore the invention taken as a whole is prima facie obvious.
Response to Applicant’s arguments: With response to the previous rejection of claims 1-6 and 16-18, Applicant argues that Deggerdal fails to teach a nucleic acid extraction method which involves a step of "neutralizing the negative charge of the nucleic acids and facilitating binding of the nucleic acids to the adsorbing material", such as, magnetic beads, silica membranes, or other solid supports. Applicant argues that in the absence of such teaching, those skilled in the art cannot envisage neutralizing the negative charge of nucleic acids; that is, Deggerdal does not provide a motivation of neutralizing the negative charge of nucleic acids. In fact, those skilled in the art would try to preserve the native charge of nucleic acids as much as possible to avoid interference with experimental results.
Applicant further argues that Deggerdal describes using quaternary ammonium salts to induce precipitation assisting in nucleic acid co-precipitation. The current method does not recite a precipitation step but rather uses the quaternary ammonium salt to induce a change to the charge of the nucleic acids, thereby facilitating the binding of DNA to a solid-phase carrier. That is, the reason for facilitating phase separation is also facilitating binding of DNA to a solid-phase carrier.
Applicant argues that Deggerdal did not recognize that quaternary ammonium salts could have the function of "neutralizing the negative charge of the nucleic acids and facilitating binding of the nucleic acids to the adsorbing material"; otherwise, they would have explicitly disclosed it and would have directly performed the step of binding without using quaternary ammonium salts to disorder the nucleic acids. The applicant wishes to emphasize that according to the description of this application, those skilled in the art would know that the quaternary ammonium salts in the binding solution of this application will not denature nucleic acids.
Applicant’s arguments have been fully considered but they are not found persuasive. Regarding Applicant’s arguments that Deggerdal did not recognize that quaternary ammonium salts could have the function of "neutralizing the negative charge of the nucleic acids and facilitating binding of the nucleic acids to the adsorbing material" and otherwise, they would
have explicitly disclosed it and would have directly performed the step of binding without using quaternary ammonium salts to disorder the nucleic acids, the examiner maintains that because Deggerdal acknowledges the that nucleic acids are bound to silica surfaces under chaotropic conditions, such as high concentrations of a chaotropic salt, either alone or in combination with ethanol, one of ordinary skill in the art would have recognized the role of chaotropic salt in facilitating binding to adsorbent materials such as silica surfaces.
Regarding Applicant’s argument about neutralization of DNA using the quaternary ammonium salt, as recited above, the examiner believes that the limitations wherein the quaternary ammonium salt neutralizes the negative charge of the nucleic acids and facilitates binding of one or more of the nucleic acids to the adsorbing material are believed to be necessarily present when practicing the method obvious over Deggerdal. Because Deggerdal provides motivation to practice their method with quaternary ammonium salts, one of ordinary skill in the art would have contemplated their use in the method of Deggerdal. Although the motivation to practice the method of Deggerdal with quaternary ammonium salts may not be the same as the motivation recited in claim 1 (for example, to neutralize the charge on a nucleic acid). As stated, the method of Deggerdal renders obvious mixing the quaternary ammonium salt with the DNA sample prior to binding to a solid support, and accordingly one of ordinary skill in the art would have considered performing this step. As evidenced by the specification, such a step would have necessarily neutralized the charge of DNA.
Regarding Applicant’s argument that Applicant further argues that Deggerdal describes using quaternary ammonium salts to induce precipitation assisting in nucleic acid co-precipitation, and the current method does not recite a precipitation step but rather uses the quaternary ammonium salt to induce a change to the charge of the nucleic acids, thereby facilitating the binding of DNA to a solid-phase carrier, as stated, although the motivation to add a quaternary ammonium salt for the purposes of binding in the present application may differ from that of Deggerdal, the addition of the quaternary ammonium salt necessarily neutralizes the charge of the nucleic acid.
Regarding Applicant’s claim of unexpected or superior results, a claim of unexpected results must include a comparison with the closest prior art. In this instance, the declaration from Dr. Bi Wanli states that “nucleic acids extracted using this method perform exceptionally well in downstream PCR and sequencing, with significantly higher recovery efficiency compared to
traditional methods,” but does not point to specifically to evidence showing that the claimed method using quaternary ammonium salts for nucleic acid extraction is superior to the method of using guanidinium salts. Applicant is encouraged to provide specific evidence demonstrating that their claimed method offered a significantly higher recovery efficiency compared to traditional the closest prior art.
The examiner has identified an example in the specification wherein which trace genomic DNA is extracted under conditions comprising 40% isopropanol and 1.6 M tetraethylammonium bromide or 1.6 M guanidine hydrochloride (p. 10, lines 43-45). The specification concludes that the results showed that tetraethylammonium bromide could significantly improve the extraction efficiency for trace DNA, and the extraction efficiency of tetraethylammonium bromide is much higher than that of guanidine hydrochloride at the same concentration, indicating that tetrabutylammonium bromide could effectively extract and purify trace genomic DNA (p. 11, lines 31-36), with results of this experiment are shown in Figure 5.
Figure 5 appears to show a set of chromatograms with larger chromatogram signals for the conditions comprising tetraethylammonium bromide compared with guanidine hydrochloride. However, both the x axis and the y axis value of these plots are difficult to interpret given the resolution of the figure. If the examiner’s interpretation is correct, clear evidence (for example, a plot showing relative yield of DNA based on the total integration of each chromatogram) of a higher extraction yield with tetraethylammonium bromide is achieved compared to guanidine hydrochloride would be persuasive evidence of non-obviousness to overcome the present rejection.
Regarding present claims 7-9, claims 7-10 were previously rejected as unpatentable over Deggerdal. Applicant’s arguments appear to only address the previous anticipation rejection. Accordingly, the rejection of claims 7-9 as obvious over Deggerdal is maintained here.
Therefore, for the reasons described above, the present rejection of claims 1-9, 16, and 18 is maintained.
The following are new rejections in response to Applicant’s amendments received February 3, 2026.
Claim Rejections - 35 USC § 102
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 1-3, 6, 16, and 18 are rejected under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) as being anticipated by Singer (U.S. Patent 8,679,744; cited in PTO-892), as evidenced by Wolfgang (Publication no. DE 102014103107 A1; cited in PTO-892) and Tomanee (Tomanee, P.; et al. Biotechnology and Bioengineering 2004, vol. 88, pp. 52-59; cited in PTO-892).
Wolfgang was published in a language other than English. Both the original document and English language machine translation are cited in the PTO-892 and included with this office action.
Singer teaches and claims a method for isolating and cleaning nucleic acids from a
lysate, comprising:
a) lysing a biological sample containing nucleic acid with a lysis buffer;
b) optionally neutralizing the preparation resulting from the lysis with a neutralisation buffer;
c) mixing a reaction mixture so obtained with one or more compounds of formula (I) with formula Y+R1R2R3R4X- as defined in the claim, and brought into contact with a mineral matrix,
wherein the nucleic acids bind to the mineral matrix in the presence of the one or more compounds of formula I;
d) washing nucleic acids with a wash buffer; and
e) isolating nucleic acids, and wherein the compound of formula (I) promotes binding to the matrix (column 17, claim 1, lines 24-49). Isolating nucleic acids is referred to as eluting in an exemplary method of Singer (see column 8, lines 1-3), and thus is interpreted equivalent to eluting as recited in step d) of present claim 1.
Singer further claims wherein R1 is a higher alkyl residue, R2, R3, and R4 each is a methyl group, and Y is nitrogen (column 17, claim 2, lines 50-52). A compound of formula (I) recited in claim 1 with the structure recited in claim would satisfy the limitations of a compound of Formula I as recited in claim 1.
Singer additionally teaches an example of this method in which 25 ml of a DH5α/pCMVB culture (high-copy plasmid) were precipitated with 2 ml, 2.5 ml and 4 ml respectively of a detergent (4 weight-% in 0.5 M NaCl) or with isopropanol, and uses cetyltrimethylammonium bromide (CTAB) as one compound of formula (I) (column 8, lines 5-57). In addition, the high-copy plasmid is an example of deoxyribonucleic acid, as required by claim 18.
This example of practicing the method claimed by Singer with CTAB satisfies the limitations of claims 1-3, 16, and 18.
Regarding CTAB as a chaotropic agent, Wolfgang discloses a method of nucleic acid isolation in which no chaotropic chemicals such as CTAB are needed (English translation, p. 9, fourth paragraph, lines 5-6). Therefore, absent evidence showing that CTAB does not act as a chaotropic salt in the method of Singer, claim 6 is also anticipated by Singer.
Regarding the limitation in step b) of claim 1 that involves neutralizing the negative charge of the nucleic acids with an agent for neutralizing the negative charge of the nucleic acids, as evidence, Tomanee teaches that surfactant molecules such as CTAB ionize in aqueous solution resulting in an ionic micelle CTA+ and a counterion Br-, and that electrostatic interaction between the cationic head group of the micelle and the negatively charged phosphate sites of the DNA are thought to be the primary interaction in binding of cationic surfactant to the DNA. Tomanee teaches this interaction leads to the neutralization of charge on the phosphate group (p. 55, Results and Discussion section, first paragraph, lines 1-9).
Thus Singer, as evidenced by Wolfgang and Tomanee, anticipates claims 1-3, 6, 16, and 18.
Claims 7-9 are rejected under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) as being anticipated by Colpan (US 6,383,393 B1; cited in PTO-892), as evidenced by Tomanee (Tomanee, P.; et al. Biotechnology and Bioengineering 2004, vol. 88, pp. 52-59; cited in PTO-892).
Colpan teaches an example in which nucleic acids are isolated from blood (column 9, lines 11-12). Colpan teaches that to 200 μL of citrate, heparin or EDTA blood are added 200 μL of a 4-8 M solution of a chaotropic salt, optionally an organic solvent, and a 5-100% detergent of NP40, Tween 20, Triton X-100, SDS, or CTAB. Colpan teaches that 200–1000 μg of a protease is added, and the mixture is incubated for 10 min at 70° C to lyse cells. Colpan teaches that 210 μL of a 95-100% alcohol (methanol, ethanol, n-propanol, isopropanol, PEG, Secondary and tertiary, short chain or long-chain alcohols), is then added to the lysed sample and provides highly specific binding conditions for nucleic acids (column 9, lines 12-30).
CTAB is cetyltrimethylammonium bromide, as evidenced by the instant specification (p. 11, line 40). As evidenced by the PubChem entry for CTAB (cited in PTO-892; see structure on p. 4), its structure satisfies the limitations of the compound of Formula I as recited in claim 7.
Therefore, the solution that comprises CTAB and 100% ethanol or isopropanol and is used for binding nucleic acids satisfies the structural limitations of the nucleic acid extraction reagent of claim 7. Colpan teaching the ethanol or isopropanol is added to the lysed sample (which comprises CTAB) is interpreted as equivalent to adding the quaternary ammonium salt to the binding solution, because in each instance, a new solution is formed in which these components are present together.
Colpan further teaches that adsorption of nucleic acids on silica gel will also take place in the presence of anionic or cationic or neutral detergents, such as e.g. SDS, NP40, Tween 20, Triton X-100, CTAB, in combination with chaotropic salts, or that the presence of these detergents will even increase the DNA yield (column 3, lines 23-29) (emphasis added). This is taken as an express teaching that CTAB may be added for facilitating binding of the nucleic acid to an adsorbing material, as recited in claim 7.
Regarding the limitation of claim 7 of neutralizing the negative charge of the nucleic acids with an agent for neutralizing the negative charge of the nucleic acids, as evidence, Tomanee teaches that surfactant molecules such as CTAB ionize in aqueous solution resulting in an ionic micelle CTA+ and a counterion Br-, and that electrostatic interaction between the cationic head group of the micelle and the negatively charged phosphate sites of the DNA are thought to be the primary interaction in binding of cationic surfactant to the DNA. Tomanee teaches this interaction leads to the neutralization of charge on the phosphate group (p. 55, Results and Discussion section, first paragraph, lines 1-9).
Therefore, because Colpan teaches binding of a solution that comprises CTAB and 95-100% of ethanol or isopropanol, the solution that is bound to the solid support in Colpan is a binding solution, as recited in claim 7.
In addition, Colpan teaches their method involves binding by passing the lysate
through the membrane or gel matrix by centrifuging or applying pressure, with reversible binding of the nucleic acids to the membrane fibers or gel particles, washing with a wash solution as described, and eluting with a buffer with a low concentration of salts (column 9, lines 30-40). Therefore, the reagent comprising the quaternary ammonium salt and ethanol or isopropanol taught by Colpan also further comprises lysis solution with protease (as described above, washing solution, eluent, and adsorbing material (for example, a membrane).
Thus Colpan anticipates claims 7-9.
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 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Deggerdal (Publication no. WO 2004108741 A1; of record) in view of Colpan (US 6,383,393 B1; cited in PTO-892).
Regarding claim 7, the examiner believes that Deggerdal provides sufficient motivation to add the compound of Formula I to an isopropanol or absolute ethanol binding solution and generate the nucleic acid extraction reagent of claim 7. However, for the sake of argument, if Deggerdal does not provide sufficient motivation to add the compound of Formula I to an isopropanol or absolute ethanol binding solution because, for example, Deggerdal does not teach a specific embodiment that includes a compound of Formula I added to an isopropanol or absolute ethanol binding solution, then claims 7-9 would have been obvious over Deggerdal in view of Colpan.
Deggerdal teaches as described in the above rejection under 35 U.S.C. 103. In addition, Deggerdal cites WO 95/01359 for teaching that nucleic acids are bound to silica surfaces under chaotropic conditions, that is typically 2 M to 8 M of a chaotropic salt, e.g. guanidinium salts, alone or in combination with EtOH (p. 1, lines 23-26). The publication WO 95/01359 is the WO publication that corresponds with the U.S. patent to Colpan referenced in the present rejection. Therefore, one of ordinary skill in the art would have found the methods taught by Colpan to be reasonably pertinent to Deggerdal.
Deggerdal does not teach wherein the quaternary ammonium salt is added to a binding solution, and wherein the binding solution is isopropanol, as recited in independent claim 7.
Colpan teaches and claims a method for the purification and separation of a nucleic acid mixture by chromatography, comprising the steps of a) adsorbing on a substrate the nucleic acid mixture from an aqueous adsorption solution containing (i) salts effecting a high ionic strength and (ii) 1 to 50% by volume of at least one C1-C5 aliphatic alcohol or polyethylene glycol or at least one C1-C5 aliphatic alcohol and polyethylene glycol wherein said substrate comprises a porous or non-porous mineral substrate selected from the group consisting of silica gel, glass
fibers, quartz fibers, and zeolites; b) optionally washing said substrate with a washing
solution; c) eluting said nucleic acid mixture with a solution having a lower ionic strength than the aqueous adsorption solution, effecting thereby a nucleic-acid fraction; and d) collecting the nucleic-acid fraction (column 12, claim 1, lines 18-36). Colpan further claims the salts in the adsorption solution are chaotropic salts in concentrations of from 1 to 8 M (column 12, claim 2, lines 37-39).
Finally, Colpan teaches embodiments in which a chaotropic salt and isopropanol are used to bind a nucleic acid solution to a solid support. For example, Colpan teaches example 4, in which a PCR amplification reaction is mixed with 500 μL of PB buffer (5 M GuHCl, 30% isopropanol) and added to a silica gel membrane (column 8, lines 42-49).
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 substitute the ethanol used for binding of a nucleic acid to a solid support suggested by Deggerdal with isopropanol. One of ordinary skill in the art would have been motivated to substitute the ethanol used for binding of a nucleic acid to a solid support suggested by Deggerdal with isopropanol because Deggerdal cites Colpan teaching that nucleic acids are bound to silica surfaces under chaotropic conditions, that is typically 2 M to 8 M of a chaotropic salt, e.g. guanidinium salts, alone or in combination with EtOH, and because Colpan teaches embodiments in which a chaotropic salt is used with isopropanol for binding to a silica surface. Therefore, one of ordinary skill in the art would have reasonably considered substituting the ethanol used for binding of a nucleic acid to a solid support as taught by Deggerdal with isopropanol, because each of ethanol and isopropanol would be expected to be effective in promoting binding of nucleic acid in the presence of a chaotropic salt to a solid support.
Therefore the invention taken as a whole is prima facie obvious.
Claims 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Colpan (US 6,383,393 B1; cited in PTO-892), as evidenced by Tomanee (Tomanee, P.; et al. Biotechnology and Bioengineering 2004, vol. 88, pp. 52-59; cited in PTO-892).
The examiner believes that Colpan anticipates claims 7-9, as recited in the above rejection under 35 U.S.C. 102. However, for the sake of argument, if, for example, the number of alcohols recited in the disclosure of Colpan is large enough such that Colpan does not anticipate claims 7-9, then claims 7-9 would have been obvious over Colpan.
Colpan teaches as described in the above rejection under 35 U.S.C. 103.
Colpan does not teach a specific embodiment in which absolute ethanol or isopropanol is present in a binding solution with CTAB.
It would therefore have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the present application to practice the method of Colpan described above (for example, the process of Example 7) with CTAB and absolute ethanol or isopropanol. One of ordinary skill in the art would have been motivated to practice the method of Colpan described above with CTAB and absolute ethanol or isopropanol because Colpan expressly teaches these components used in the lysis and binding steps of nucleic acid extraction. Accordingly, one of ordinary skill in the art would have considered selecting these components when practicing the method of Colpan.
As evidenced by Tomanee, the negatively charged phosphate sites of the DNA are thought to be the primary interaction in binding of cationic surfactant to the DNA, and this interaction leads to the neutralization of charge on the phosphate group (p. 55, Results and Discussion section, first paragraph, lines 1-9).
Therefore the invention taken as a whole is prima facie obvious.
Claims 1-6, 16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Haesendonckx (U.S. pre-grant publication no. US 20230399633 A1; cited in PTO-892) in view of Montesclaros (Publication no. WO 2005007895 A1; of record).
The U.S. patent application associated with Haesendonckx, 17/633408, filed February 7, 2022, is a national stage application of PCT /EP2020/072225, filed August 7, 2020, and claims the benefit of foreign priority application EP19190647.8, filed August 8, 2019. Because the application of Haesendonckx has an earlier effective filing date than the present application, Haesendonckx is eligible as prior art under 35 U.S.C. 102(a)(2).
Haesendonckx teaches and claims a nucleic acid extraction method comprising contacting a liquid biopsy sample with a silica solid support at pH value between 3 and 6 and in the presence of a salt consisting of: a small quaternary organic compound, defined as a quaternary compound consisting of a central positively charged atom with four organic substituents Rl-R4, wherein the number of carbon atoms in each organic substituent Rl-R4 does not exceed 2; and of a bromide or a chloride anion (pp. 10-11, claim 1), and further claims the small quaternary compound is tetramethylammonium chloride (p. 11, claim 4). Tetramethylammonium chloride satisfies all compound limitations of claims 1-5 and 16.
Haesendonckx describes their invention as using a tetramethylammonium (TMN) cation
combined with a weak chaotropic chloride (Cl-) or bromide (Br-) anion (p. 2, [0021], lines 9-15). Therefore, tetramethylammonium chloride is a chaotropic salt, as required by claim 6.
Haesendonckx further claims wherein the nucleic acid is DNA (p. 11, claim 9).
Haesendonckx teaches their method was developed by spiking 100 μL of plasma with 20,000 copies of nucleosomal DNA (nDNA), isolated from whole blood. Haesendonckx teaches the spiked plasma was mixed with 500 μL of the binding buffer composed of 1.2 M tetramethylammonium chloride (TMAC) dissolved in a 0.24 M sodium acetate pH 5 buffer, resulting in a final concentration of 1 M TMAC and 0.2 M sodium acetate when mixed with the plasma sample. Haesendonckx teaches acidic mixture with a total volume of 600 μL was passed over the silica spin column (p. 8, [0081], lines 1-16). Haesendonckx teaches the silica membrane was washed by passing 1000 μL of 90% ethanol over the spin column (p. 8, [0083], lines 1-2), and eluted by rehydrating the silica membrane with water or a Tris-HCI pH 8.6 buffer and subjecting the silica spin column to a final centrifugation step (p. 8, [0085], lines 1-9). This method satisfies all limitations of steps b), c), and d) of independent claim 1.
Finally, Haesendonckx teaches that, in some instances, binding conditions comprising tetramethylammonium chloride perform better than binding conditions comprising guanidinium salts. For example, Figure 10 demonstrates that at pH 4.8-5.4, higher yields are achieved with TMAC compared with guanidinium thiocyanate and butanol, as evidenced by the lower Ct values (Figure 10 on document p. 11, see right panel of figure; description on pp. 9-10, [0100]). Although Haesendonckx recognizes the effect as unpredictable (p. 9, [0100], lines 1-5), the whole of Haesendonckx’s disclosure demonstrates these superior results under some conditions.
Finally, Haesendonckx teaches binding conditions comprising tetramethylammonium chloride that perform comparably for isolating DNA compared with binding conditions comprising guanidinium salts, as evidenced by comparable Ct values shown in these figures (for example, see document p. 4, Figure 3, conditions with GuSCN/ButOH vs. 1M TMAC pH 5; document p. 5, Figure 4, same conditions as in Figure 3). In addition, in some instances, Haesendonckx teaches binding conditions comprising tetramethylammonium chloride perform better than binding conditions comprising guanidinium salts. For example, Figure 10 demonstrates that at pH 4.8-5.4, higher yields are achieved with TMAC compared with guanidinium thiocyanate and butanol, as evidenced by the lower Ct values (Figure 10 on document p. 11, see right panel of figure; description on pp. 9-10, [0100]). Although Haesendonckx recognizes the effect as unpredictable (p. 9, [0100], lines 1-5), the whole of Haesendonckx’s disclosure demonstrates these superior results under some conditions.
Haesendonckx does not teach a nucleic acid extraction process that includes step a) recited in claim 1, requiring lysing a sample to be extracted with a lysis solution, wherein the lysing releases nucleic acids from the sample.
Montesclaros teaches as described in the above rejection under 35 U.S.C. 103.
It would have been prima facie obvious to modify the method of Haesendonckx with a cellular lysis step to extract intracellular nucleic acid as well as extracellular or cell-free nucleic acid. One of ordinary skill in the art would have been motivated to modify the method of Haesendonckx with a cellular lysis step to extract intracellular nucleic acid as well as extracellular or cell-free nucleic acid because Haesendonckx teaches a method of extraction of DNA using TMAC to facilitate binding to a silica material (spin column), specifically demonstrating that these conditions for binding DNA to silica perform comparably to conditions comprising GuSCN, and because Montesclaros teaches a method of DNA extraction that comprises the same steps as Haesendonckx, including binding to a solid support with a chaotrope, suggesting quaternary ammonium salts as said chaotropes, and further teaches a lysis step prior to binding to the solid support. Therefore, one of ordinary skill in the art would have recognized that the method of Haesendonckx may be reasonably practiced on a sample comprising nucleic acid sample that was previously subjected to a lysis step, such as the lysis step taught by Montesclaros.
In this instance, the rationale “simple substitution of one known element for another to obtain predictable results” would apply. Haesendonckx teaches a method for extraction of nucleic acids from plasma using tetramethylammonium chloride as a salt, including teaching a method of binding, washing, and eluting the nucleic acid as required by steps b) – d) of claim 1, and Montesclaros teaches a related method that includes a proteinase K lysis step and binding with guanidinium thiocyanate. Therefore, one of ordinary skill in the art would have recognized that the method of Haesendonckx, which utilizes tetramethylammonium chloride in the binding solution, may be reasonably combined with the method of Montesclaros that involves first lysing cells prior to isolation of their DNA.
Regarding the limitation of claim 1 that requires “neutralizing the negative charge of the nucleic acids with an agent for neutralizing the negative charge of the nucleic acids and facilitating binding of one or more of the nucleic acids to the adsorbing material, and binding one or more of the nucleic acids to an adsorbing material using a binding solution;” this is believed to be necessarily present when practicing the method of Haesendonckx using tetramethyl ammonium chloride. As described above, Haesendonckx in view of Montesclaros renders obvious a method that involves adding a chaotrope to facilitate binding of the nucleic acid to an adsorbing material, wherein the chaotrope is one of the quaternary ammonium salts recited above.
Because the quaternary ammonium salt carries a formal positive charge and the gDNA carries a formal negative charge, the addition of the quaternary ammonium salt to the binding buffer would necessarily have the effect of neutralizing the charge of the nucleic acid, absent evidence to the contrary.
Moreover, as evidenced by the instant specification, a certain concentration of quaternary ammonium salts can neutralize the negative charge of nucleic acids, such that the nucleic acids can bind to the adsorbing material or be precipitated in the presence of polar solvents (ethanol, isopropanol, etc.) by hydrogen bond, hydrophobic interaction and electrostatic interaction (p. 3, lines 25-29).
In addition, because Haesendonckx teaches that a salt consisting of, for example, a tetramethylammonium (TMN) cation combined with a weak chaotropic chloride or bromide anion creates at acidic pH unique conditions for isolating dsDNA on a silica solid support (p. 2, [0021], lines 9-16), one of ordinary skill in the art would have contemplated the addition of said salts for the purposes of facilitating DNA binding to the adsorbent material.
This is further supported by evidence from the instant specification providing that nucleic acids can reversibly bind to the nucleic acid-adsorbing material, glass particle, glass fiber, magnetic bead, diatomite, silica gel, in solution or precipitate in the presence of polar organic solvent, and that high concentrations of chaotropic salts can facilitate the binding of nucleic acids to these materials (p. 1, lines 33-37).
Therefore the limitations wherein the quaternary ammonium salt neutralizes the negative charge of the nucleic acids and facilitates binding of one or more of the nucleic acids to the adsorbing material are believed to be necessarily present when practicing the method obvious over Haesendonckx in view of Montesclaros using tetramethylammonium chloride for binding to the solid support.
Therefore the invention taken as a whole is prima facie obvious.
Response to Applicant’s arguments: As described in the above rejection, the presently claimed method is obvious over Haesendonckx in view of Montesclaros. In this instance, Haesendonckx teaches all steps of present claim 1, except for a step of lysing a sample to be extracted with a lysis solution.
Haesendonckx disclose conditions that provide approximately the same yield of extracted DNA with trimethylammonium chloride compared with guanidinium thiocyanate, as evidenced by very close Ct values achieved when amplifying DNA isolated using these conditions.
Haesendonckx also demonstrates that under specific conditions (i.e., when a proteinase K step is performed as part of their method), higher yields of extracted DNA may be achieved with trimethylammonium chloride compared with guanidinium thiocyanate, as evidenced by lower Ct values achieved when amplifying DNA isolated using these conditions
The difference between the method of Haesendonckx and the presently claimed method is the addition of a lysis step. Therefore, absent evidence that specific lysis conditions contribute to nucleic acid binding in the present of the compound of Formula I, persuasive evidence of nonobviousness in the form of unexpected results should account for Haesendonckx expressly teaching a step of binding DNA to a solid support in the presence of trimethylammonium chloride.
Demonstrating superior extraction yield with a quaternary ammonium salt compared with a guanidinium salt would not be sufficient evidence to overcome the rejection over Haesendonckx in view of Montesclaros, because Haesendonckx expressly teaches the step of isolating DNA by binding to a solid support in the presence of a quaternary ammonium salt.
Conclusion
No claims are allowed.
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/B.M.B./Examiner, Art Unit 1693
/ANDREA OLSON/Primary Examiner, Art Unit 1693