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 December 17, 2021, is a national stage application of PCT/EP2020/068080, filed June 26, 2020, and claims priority to foreign priority application EP19183297.1, filed June 28, 2019.
Status of the Application
Applicant’s amendment, received September 3, 2025, wherein claims 1, 2, 4, 6-8, 10, 11, 13, 14, 16, 18, 19, 22, 25, and 26 are amended, is acknowledged.
Claims 1-14, 16, 18-19, 22, and 24-26 are pending and examined on the merits herein.
Withdrawn Rejections
Applicant’s amendment, received September 3, 2025, with respect to the rejection of claims 4, 7, 11, 16, 18, 19, and 22 under 35 USC § 112(b) over the scope of the claim due to the phrase “such as” has been fully considered and found to be persuasive to remove the rejection because these claims are amended to replace the phrase “such as” with “optionally,” which makes clear that any limitations following the term “optionally” are not required by the claim. Therefore the rejection is withdrawn.
Applicant’s amendment, received September 3, 2025, with respect to the rejection of claims 6, 7, 8, 10, 11, 13, 14, 16, 18, 22, 25, and 26 under 35 USC § 112(b) over the scope of the claim due to the term “preferably” has been fully considered and found to be persuasive to remove the rejection because these claims are amended to remove limitations following the term “preferably” or replace the term “preferably” with “optionally,” which makes clear that any limitations following the term “optionally” are not required by the claim. Therefore the rejection is withdrawn.
Applicant’s amendment, received September 3, 2025, with respect to the rejection of claims 7, 10, and 11 under 35 USC § 112(b) for reciting both broad and narrow limitations together in the same claim has been fully considered and found to be persuasive to remove the rejection because the narrower limitations recited in these claims are now optional, and thus the scope of the claim is now definite. Therefore the rejection is withdrawn.
The following rejections are maintained from the previous office action, mailed April 3, 2025, and updated here to specifically cite the data presented by Wisniewski discussed in the previous office action.
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 1-14, 16, 18, 19, 22, 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Hawkins (Publication no. WO 1996009379 A1; cited in IDS received December 17, 2021) in view Wisniewski (Publication no. WO 2010115016 A2; cited in previous office action).
Claim 1 recites a poly(alkylene oxide) polymer based size selective method for enriching nucleic acid molecules having a length below a cut-off value from a nucleic acid containing sample comprising the steps (a) – (e) recited in claim 1, wherein the nucleic acid containing sample is a cell-free or cell-depleted body fluid sample and wherein the eluted extracellular nucleic acid molecules. Claim 2 requires the eluted nucleic acid molecules are extracellular DNA, claims 3 and 4 specify specific cutoff values, claim 5 requires eluting the nucleic acids from step (f), claim 6 specifies requirements about the solid phase, claim 7 specifies requirements about the poly(alkylene oxide) polymer, claim 8 specifies requirements about the salt, claim 9 requires in step (a) adding a binding reagent to the nucleic acid containing sample to prepare the binding mixture, claim 10 specifies requirements about the binding reagent or conditions, claim 11 specifies requirements about the elution composition, claim 12 requires providing the elution composition of step ( c) by diluting the binding reagent with a dilution solution, claim 13 specifies requirements about the nucleic acid containing sample.
Claim 14 depends from claim 1 and requires the features listed therein. claim 16 requires providing the elution composition of step ( c) by diluting the binding reagent with a dilution solution and wherein the solid phase with the bound nucleic acid is in step (c) only contacted with a single elution composition (c) but not with further reagents, and claim 18 requires the binding reagent has a pH value that lies in a range of 4.5 to 9.5 and wherein the solid phase is provided by carboxylated magnetic particles.
Claims 19 and 22 depend from claim 1 and require the method with additional limitations listed therein. Claim 24 depends from claim 1 and requires the eluted nucleic acids comprise extracellular nucleic acid molecules, and the method comprises analyzing the eluted extracellular nucleic acid molecules to identify, detect, screen for, monitor or exclude a disease, an infection and/or at least one fetal characteristic.
Claims 25 and 26 recite a kit for the size selective enrichment of nucleic acid molecules, having a length below a cut-off value from a nucleic acid containing sample, comprising the reagents listed therein.
Hawkins teaches a method of separating polynucleotides in solution by reversibly and non-specifically binding the polynucleotides to a solid surface (cover page, Abstract, lines 1-2). Hawkins teaches that when binding DNA to a solid surface, sufficient salt and polyethylene glycol are added to a solution containing magnetic microparticle-bound DNA to result in a final concentration of from about 0.5 M to about 5.0 M salt and from about 7% to about 13% polyethylene glycol, and as a result, DNA is bound non-specifically to the surfaces of the magnetic microparticles (p. 4, lines 1-7) (emphasis added).
Hawkins teaches that small polynucleotides require higher salt concentrations for strong binding to the microparticles, and thus salt concentrations can be selectively manipulated to release polynucleotides bound to magnetic microparticles on the basis of size (p. 3, lines 20-24).
In addition, Hawkins teaches that an embodiment of their invention refers to a method of separating a mixture of polynucleotide fragments based on size (p. 12, lines 31-33) (emphasis added). Hawkins teaches this method involves combining a solution of DNA fragments of different sizes with magnetic microparticles having a carboxyl group-coated surface under conditions appropriate for non-specific binding of DNA, then separating the magnetic microparticles from supernatant (p. 12, line 33 to p. 13, line 4). Hawkins teaches that the magnetic microparticles are then washed with an elution buffer of appropriate ionic strength to elute the smaller size polynucleotide fragments, while leaving the larger size polynucleotide fragments bound to the magnetic microparticles (p. 13, lines 8-13) (emphasis added). Hawkins teaches that the smaller polynucleotide fragments in the elution buffer can then be isolated in the usual manner or processed further, (e.g., subjected to further biochemical reactions) (p. 13, lines 13-16).
In addition, Hawkins teaches that the separation of polynucleotide fragments based on size can also be accomplished by the method of the present invention by adjusting the PEG concentration, the molecular weight of the PEG used or both (p. 13, lines 30-33), and that an elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles (p. 10, lines 28-32) (emphasis added). This is interpreted as teaching the method of Hawkins is intended to also be used to size select target nucleic acid fragments that are smaller than the non-target nucleic acid fragments, and this selective elution of smaller nucleic acids may be accomplished by adjusting the ionic strength and/or PEG present in the elution buffer.
Hawkins teaches an example wherein DNA PCR products are bound to the solid-phase using the method described (p. 22, lines 26-27; Example 6). Hawkins teaches PCR amplification of DNA and mixing with carboxyl coated magnetic particles, and subsequent addition of an equal volume of binding buffer comprising 20% PEG 8000 and 2.5 M NaCl (p. 23, lines 3-12), before washing with a wash buffer and elution in water (p. 23, lines 13-20). This example is interpreted as teaching conditions wherein nucleic acids may be bound non-specifically to a solid-phase that comprises carboxyl coated magnetic particles.
Finally, Hawkins teaches kits comprising magnetic microparticles and a binding buffer which contains a suitable salt and polyalkylene glycol at concentrations suitable for reversibly binding polynucleotide onto solid surfaces, such as to the surfaces of magnetic microparticles. Hawkins teaches the kit may additionally comprise a suitable wash buffer, elution buffer, reagents for preparing such buffers (p. 2, lines 16-23).
Hawkins does not expressly teach step (c) of claim 1, requiring contacting the solid phase with the bound nucleic acid molecules at least once with an elution composition comprising a poly(alkylene oxide) polymer and a salt to selectively elute nucleic acid molecules having a length below the cut-off value from the solid phase while larger nucleic acid molecules having a length above the cut-off value remain bound to the solid phase, wherein the concentration of the poly(alkylene oxide) polymer in the elution composition is lower than the concentration of the poly(alkylene oxide) polymer in the binding mixture of (a), as required by claim 1. Furthermore, Hawkins does not teach the nucleic acid containing sample is a cell-free or cell-depleted body fluid sample and wherein the eluted nucleic acid molecules are extracellular nucleic acid molecules the nucleic acids as extracellular nucleic acid molecules, as required by claim 1.
In addition, Hawkins does not teach the eluted extracellular nucleic acids as extracellular DNA, the specific size cutoffs for the method, establishing a cutoff value by adjusting the concentration of poly(alkylene oxide) polymer, further purification of bound nucleic acids, or, specific limitations regarding the elution composition, the requirements of the nucleic acid sample, or further analyzing the eluted nucleic acid to monitor a disease infection, or fetal characteristic, as required by claims 2-5, 11, 13-14, 9, 22, and 24. Hawkins does not teach the requirement that the elution composition is obtained by diluting the binding reagent, as required by claim 12 and 16. Finally, Hawkins does not teach the specific kit requirements of claims 25 and 26.
Wisniewski teaches methods and compositions to extract and enrich relatively short nucleic acids from a nucleic acid composition containing a high background of longer nucleic acids (cover page, Abstract, lines 1-2) (emphasis added).
Wisniewski teaches and claims a method for enriching relatively short nucleic acid from a nucleic acid composition, which comprises:
(a) contacting nucleic acid of a nucleic acid composition with a solid phase under association conditions, wherein:
(i) the nucleic acid of the nucleic acid composition comprises relatively
short nucleic acid and relatively long nucleic acid,
(ii) the relatively short nucleic acid is about 300 base pairs or less, and
(iii) the relatively long nucleic acid is larger than about 300 base pairs;
whereby the relatively short nucleic acid and the relatively long nucleic
acid are associated with the solid phase;
(b) introducing the solid phase after (a) to dissociation conditions that comprise a volume exclusion agent and a salt, wherein:
(i) the salt is not a chaotropic salt, and
(ii) the relatively short nucleic acid preferentially dissociates from the solid phase under the dissociation conditions as compared to the relatively long nucleic acid, thereby yielding dissociated nucleic acid; and
(c) separating the dissociated nucleic acid from the solid phase, whereby the
relatively short nucleic acid is enriched in the dissociated nucleic acid relative to in the
nucleic acid composition (p. 94, claim 1).
Wisniewski claims wherein the nucleic acid composition is a biological composition (p. 94, claim 2), wherein the biological composition is a substantially cell-free biological composition (p. 94, claim 3), wherein the nucleic acid is cell-free nucleic acid (p. 94, claim 4), wherein the substantially cell-free biological composition is blood serum (p. 95, claim 7), blood plasma (p. 95, claim 8), or urine (p. 95, claim 9).
Wisniewski further claims wherein the association conditions comprise a volume exclusion agent (p. 97, claim 33), and wherein the volume exclusion agent comprises a polyalkyl alcohol (p. 98, claim 47) which may be polyethylene glycol, or more specifically, PEG 8000 (p. 98, claims 48 and 49).
Wisniewski further claims the salt and the volume exclusion agent are present in the dissociation conditions at concentrations according to Table 1 (p. 98, claim 51). Wisniewski teaches the percent recovery of specific size fractions of nucleic acid in Table 1 under different, specific dissociation conditions (p. 2, see table and caption). Specifically, Wisniewski teaches that for recovery of small fragments eluted during step (b) of their method (see description of step p. 80, Step 5, lines 7-13), the concentration of PEG 8000 and/or NaCl may be adjusted to adjust the size cutoff of nucleic acids eluted during this method (see different dissociation conditions recited in Table 1 on p. 82). These conditions recited in Table 1 include a concentration of NaCl from 0.25M to 0.5M and a concentration of PEG 8000 of 10% or 18%. These dissociation conditions disclosed by Wisniewski, wherein the volume exclusion agent is PEG and the salt is NaCl, satisfy the requirement of claim 1 step (c) wherein the elution compositions comprise a poly(alkylene oxide) polymer and a salt.
These data shown in Table 1 demonstrate that a greater proportion of nucleic acids are retained on the solid-phase when eluting with a higher concentration of PEG compared to eluting with a lower concentration of PEG. For example, as the concentration of PEG decreases from 18% to 10% under the same salt concentrations, the nucleic acid cutoff shifts from eluting smaller nucleic acids to eluting larger nucleic acids (see for example, the small fragments isolated with 0.5M NaCl and 10% PEG8000 or 18% PEG8000). Therefore, in view of these data in Table 1, one of ordinary skill in the art would have recognized that a composition that includes a lower concentration of PEG than the binding conditions would elute more nucleic acids. In addition, based on the data in Table 1, lower concentrations of PEG also elute longer nucleic acids in addition to the shorter nucleic acids eluted at higher concentrations of PEG.
Accordingly, one of ordinary skill in the art would have recognized that, at equal concentrations of NaCl, higher concentrations of PEG result in a lower nucleic acid size cutoff for target nucleic acids (i.e., elution of nucleic acids below a lower size), whereas lower concentration of PEG result in a larger nucleic acid size cutoff for target nucleic acids. Accordingly, one of ordinary skill in the art would have recognized that the concentration of PEG 8000, NaCl, or both may be optimized to adjust the size cut off for the desired size of nucleic acid.
Wisniewski further teaches that the solid phase in their method may be magnetic beads, such as magnetically responsive silica dioxide beads (p. 22, line 9).
Wisniewski teaches that the eluted smaller fragments may be additionally bound to beads and further purified (p. 80, Steps 6 and 7, lines 14-27) (emphasis added), and that both the smaller nucleic acids and longer nucleic acids may be eluted and prepared for further analysis (p. 80, Steps 7 and 8, lines 24-35) (emphasis added).
Wisniewski teaches that the target nucleic acid may be extracellular nucleic acid, and may be derived from a source having substantially no cells, including blood plasma, blood serum, or urine (p. 16, lines 19-23). Wisniewski teaches that the method comprises detecting the presence or absence of fetal nucleic acid, and in some embodiments comprises detecting the presence or absence of a fetal specific nucleotide sequence, such as Y-chromosome sequence (p. 5, lines 25-29). This is interpreted as monitoring for at least one fetal characteristic, as recited in claim 25. In addition, a Y chromosome sequence that is an extracellular nucleic acid would be a sample of extracellular DNA, as required by claim 2.
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 Hawkins with the elution conditions disclosed by Wisniewski. One of ordinary skill in the art would have been motivated to modify the method of Hawkins with the elution conditions disclosed by Wisniewski because Hawkins teaches methods of size selecting nucleic acids that involves a first step of non-specific binding of a nucleic acid to a solid support in the presence of PEG and salt, teaches that the sample may be washed with an elution buffer of appropriate ionic strength to elute the smaller size polynucleotide fragments while leaving the larger size polynucleotide fragments bound to the magnetic microparticles, and teaches that these smaller size fragments may be further used, and because Wisniewski teaches a method of size selecting smaller fragments of nucleic acids by selectively eluting smaller nucleic acid fragments from a solid surface by adjusting the salt and/or PEG concentration. Therefore, one of ordinary skill in the art would have recognized that the elution/dissociation conditions disclosed by Wisniewski may be substituted in place of the elution conditions taught by Hawkins, with an expectation that this substitution will still enable size selection of the smaller target nucleic acids, as suggested by both Hawkins and Wisniewski. In addition, because Hawkins and Wisniewski both teach that PEG and salt concentrations may be adjusted to elute nucleic acids from a solid support, and because Wisniewski provides that data teaching both PEG and salt as variables that may be optimized to adjust the size of the target nucleic acid isolated, one of ordinary skill in the art would have recognized the elution conditions taught by Wisniewski as relevant to the method of size selection of DNA taught by Hawkins.
In this instance, the rationale “simple substitution of one known element for another to obtain predictable results.” Because Hawkins teaches the method of claim 1 but does not disclose specific conditions for elution of nucleic acids from the solid support, but teaches generally that the salt concentration and PEG concentration are below the ranges required for binding of DNA and that PEG concentration may be adjusted to selectively elute specific size DNA, one of ordinary skill in the art would have considered prior art conditions that similarly teach size-selective elution of nucleic acids from a solid phase. Because Wisniewski teaches size-selective elution of smaller nucleic acids from a solid phase, and further teaches that both PEG and salt concentration may be adjusted to alter the size of nucleic acid isolated from the solid support, one of ordinary skill in the art would have reasonably considered modifying the method of Hawkins and using the elution conditions taught by Wisniewski, because each are selectively eluting a specific size of nucleic acid from a solid support.
Regarding the requirement that the concentration of the concentration of the poly(alkylene oxide) polymer in the elution composition is lower than the concentration of the poly(alkylene oxide) polymer in the binding mixture, in view of Hawkins teaching that the elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles and Wisniewski teaching the relationship between PEG concentration and the size of nucleic acid eluted from the solid support, one of ordinary skill in the art would have recognized that the concentration of PEG should, assuming comparable salt conditions, be lower in the elution composition than in the binding mixture.
Regarding the specific binding cutoffs recited in claims 3, 4, 14, and 19, in view of Wisniewski and Hawkins teaching that the concentration of PEG may be used to precipitate or elute different sizes of nucleic acids, one of ordinary skill in the art would have recognized the concentration of PEG as a result-effective variable.
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).” In this instance, because the concentration of PEG is taught by Wisniewski as a variable that affects the size of nucleic acids eluted from the solid phase, one of ordinary skill in the art would have contemplated adjusting the concentration of PEG in order to modify the size cutoff of nucleic acids isolated using the method obvious over Hawkins in view of Wisniewski. In addition, the specific limitations regarding salt concentration and the molecular weight of PEG are satisfied by the examples disclosed by Hawkins and/or Wisniewski.
Regarding the dilutions recited in claims 12 and 16, because Hawkins teaches dilution of the binding reagent when adding to the mixture comprising nucleic acid sample and solid support to achieve the final, desired concentration of PEG and NaCl (see p. 23, lines 5-10), and because Hawkins teaches the elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles, one ordinary skill in the art would have reasonably contemplated diluting the binding buffer to arrive at the buffer used to elute the nucleic acids from the solid support, because they would have recognized that the elution buffer must have salt and polyalkylene concentrations below what was required for binding to the solid support.
Regarding claim 16 and the requirement that the solid phase with the bound nucleic acid is in step (c) only contacted with a single elution composition (c), but not with further reagents, such as further solutions, such a requirement would have been obvious in view of Wisniewski teaching that in step (b) of their method, the only solution that contact the nucleic acid-bound solid phase is the solution comprising volume exclusion agent and salt. In this instance, one of ordinary skill in the art would have recognized that the solid phase may be contacted with the single elution composition, and need not be contacted with further reagents.
Regarding claim 18, because Hawkins teaches washing and resuspending the magnetic particles in 0.5 M EDTA pH 7.2 immediately before adding an equal volume of binding buffer, the actual solution in which the nucleic acids bind to the magnetic beads is interpreted as having a pH between 4 and 9, absent evidence to the contrary.
Regarding the kits recited in claims 25 and 26, in view of Hawkins teaching kits comprising the reagents required to perform their method, and Wisniewski teaching that the smaller target nucleic acids may be selectively eluted using a combination of PEG and NaCl, one of ordinary skill in the art would have contemplated a kit of Hawkins with the elution buffer comprising PEG and NaCl included, to enable a user to perform the method obvious over Hawkins in view of Wisniewski rapidly and with reduced likelihood of error when preparing the required reagents.
Therefore the invention taken as a whole is prima facie obvious.
Response to Applicant’s arguments: Regarding the above rejection of claims 1-14, 16, 18, 19, 22, 24-26 under 35 U.S.C. 103 as unpatentable over Hawkins in view Wisniewski Applicant provides the following arguments:
1. Applicant argues that Hawkins fails to disclose, in the context of a method as defined in claim 1, the following combination of features as defined in step ( c) of claim 1:
(i) contacting the solid phase(. .. ) with an elution composition comprising a poly(alkylene
oxide) polymer and a salt to selectively elute nucleic acid molecules( ... ),
(ii) wherein the concentration (w/v) of the poly(alkylene oxide) polymer in the elution
composition is lower than the concentration (w/v) of the poly(alkylene oxide) polymer
in the binding mixture of (a). Specifically, as noted above (see section 1.), Hawkins discloses washing with elution buffers for size selection wherein said buffers either have an "appropriate ionic strength" or ("alternatively") have an adjusted PEG concentration and/or molecular weight (seep. 13, lines 8-13 and 30-33, p. 15, lines 6-12). Applicant argues that the examples of Hawkins are silent on an elution buffer comprising a poly(alkylene oxide) polymer and a salt, and that the wash buffers used for elution in the examples do not comprise a poly(alkylene oxide) polymer, and this also applies to Example 6 which is specifically dedicated to the selective removal of DNA from the solid phase based on the size of the DNA fragments. (Applicant’s remarks, pp. 20-21).
2. Applicant argues that Hawkins also fails to teach a size selection method wherein the concentration (w/v) of the poly(alkylene oxide) polymer in the elution composition is lower than the concentration (w/v) of the poly(alkylene oxide) polymer in the binding mixture of (a), let alone wherein the elution composition comprises a poly(alkylene oxide) polymer and a salt, as is required in step (c) of claim 1.
Applicant argues that Hawkins teaches manipulating the salt concentration / ionic strength for size selective elution, and does not disclose or suggest a size selective method as claimed wherein both poly(alkylene oxide) polymer and salt are used for size selective elution, and moreover the concentration of poly(alkylene oxide) polymer for size selective elution is lower than the one used for binding. Applicant argues that the passages of Hawkins at best would have distracted the skilled person away from the claimed invention by focusing on the relevance of salt concentrations for size selection. (Applicant’s remarks, pp. 21-22).
3. Applicant argues that Hawkins on p. 13, lines 30-33 merely teaches that the
separation of polynucleotide fragments based on size can be accomplished by "adjusting" or
"varying" the poly(alkylene oxide) polymer concentration, its molecular weight or both, but lacks a direct and unambiguous disclosure of "adjusting" or "varying" the concentration of
poly(alkylene oxide) polymer for size selective elution to a concentration that is lower than the
one used for binding. Applicant argues that size selective elution with a poly(alkylene oxide) polymer containing buffer is also not exemplified in Hawkins (the wash buffers used for elution in Hawkins as explained do not even comprise a poly(alkylene oxide)), and Hawkins also in the general description fails to provide any clear guidance how to "adjust" or "vary" the poly(alkylene oxide) polymer concentration, its molecular weight or both for size selective elution.
Applicant argues that Hawkins, neither in the above cited passages nor elsewhere, discloses an elution buffer comprising both, salt and poly(alkylene oxide) polymer for size selective elution. Accordingly, Hawkins also fails to disclose or suggest the selective elution of smaller nucleic acids may be accomplished by adjusting the ionic strength and PEG present in the elution buffer (let alone what such "adjustment" should look like).
Applicant further argues that the passage on p. 10, lines 28-31 of Hawkins must be read in its own proper context. It does not relate to a size selective elution method, but instead the passage is part of a section describing an embodiment wherein total DNA is isolated from a solution containing polynucleotide by non-specific binding to magnetic particles. Once separated from the supernatant, the DNA (and hence the total DNA bound) can be removed from the particles with a suitable elution buffer. However, the only concrete exemplary eluents / elution buffers disclosed in said passage (i.e., 20% sucrose, 100% formamide, and (preferred) water), comprise neither salt nor poly(alkylene oxide) polymer. (Applicant’s remarks, pp. 22-23).
4. Applicant argues that Wisniewski - like Hawkins - fails to disclose a size selective method for enriching nucleic acids wherein a poly(alkylene oxide) polymer and a salt are present during both, binding and elution, and the poly(alkylene oxide) polymer is used at a lower concentration for elution than for binding. Applicant argues that Wisniewski simply fails to teach using poly(alkylene oxide) polymer at a lower concentration for elution than for binding to selectively elute nucleic acids below a cut-off value at all. Wisniewski therefore - like Hawkins - regarding present claim 1 fails to disclose the mandatory feature of step (c) that the concentration of the poly(alkylene oxide) polymer in the elution composition is lower than in the binding mixture of step (a). Hence, Wisniewski does not fill in the gap between Hawkins and claim 1, the claimed method is nonobvious over Hawkins in view of Wisniewski.
Applicant further argues that the data of Wisniewski comparing PEG and NaCl concentrations does not support the conclusions drawn by the office. Applicant states that the experimental conditions and protocols according to which the composition comprising 18% PEG was used are not stated in Wisniewski, as the written description of the experiments conducted in Wisniewski only refers to the composition comprising 10% PEG. It is therefore also not known whether the data for the compositions comprising 10% or 18% PEG were prepared under otherwise identical conditions and from the same samples, or whether the data relating to the composition comprising 18% PEG were prepared, e.g., independently and from different samples. Applicant argues it is therefore is unclear whether data pertaining to the composition comprising 10% PEG are at all directly comparable with data pertaining to the composition comprising 18% PEG. In any case, a comparative discussion of the data pertaining to the compositions comprising 10% PEG vs. 18% PEG is also not provided by Wisniewski, and Wisniewski neither states nor suggests that the data could or should be compared with one another. Clearly, Wisniewski also does not state or suggest that the PEG concentration was decreased from 18% to 10% in an experiment. Instead, Wisniewski simply teaches to vary the salt concentration for size specific elution and in doing so is presenting data for two solutions comprising a constant concentration of PEG (either 18% or 10%) and a varied concentration of NaCl. Applicant further presents examples wherein Wisniewski teaches varying salt concentration to selectively elute nucleic acids of specific sizes.
Applicant concludes by stating that the Office’s analysis of Wisniewski draws from hindsight knowledge, and argues that Wisniewski’s data may reasonably lead to the conclusions recited on pp. 27-28. (Applicant’s remarks, pp. 25-29).
5. Regarding the motivation to combine Hawkins and Wisniewski, Applicant argues that the claimed invention therefore concerns a specific and difficult field, the size selective isolation of extracellular nucleic acid molecules from a cell-free or cell-depleted body fluid sample. Applicant argues that the invention provides a technology for enriching extracellular nucleic acid molecules, such as ccfDNA, having a length below a cut-off value from cell-free or cell-depleted body fluid samples, while simultaneously depleting high molecular weight (HMW) nucleic acid species that are also encountered in cell-free or cell-depleted body fluid samples. Applicant argues that the skilled person would not have been motivated to use the binding conditions of Hawkins, because Hawkins is silent on the isolation of extracellular nucleic acids from cell-free or cell-depleted body fluids, while Wisniewski focusses on the isolation of extracellular nucleic acids and teaches how cell-free or cell-depleted body fluids samples should be processed for size selective nucleic acid isolation, along with concrete exemplification (see Example 2 of Wisniewski). (Applicant’s remarks, pp. 29-31).
6. Applicant argues that Wisniewski, like Hawkins (without hindsight knowledge of the present invention) teaches to vary the salt concentration to elute nucleic acids of a desired size, and that there is no basis for the skilled person to assume that a size selection method wherein instead a lower concentration of the poly(alkylene oxide) polymer in the elution composition is used than in the binding mixture would be superior over the method of Hawkins. (Applicant’s remarks, pp. 31-32).
Applicant’s remarks have been fully considered but they are not found persuasive.
Regarding arguments 1, 2, and 3 above, Hawkins teaches that an elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles (p. 10, lines 28-32). Although Hawkins may not exemplify this elution buffer with a specific example an elution buffer that includes both PEG and a salt, Hawkins clearly teaches that an elution buffer may include both salt and polyalkylene.
Hawkins further teaches that the separation of polynucleotide fragments based on size can also be accomplished by the method of the present invention by adjusting the PEG concentration, the molecular weight of the PEG used or both (p. 13, lines 30-33). In this instance, Hawkins teaches the fundamental principle of the present method – selective elution of smaller molecular weight nucleic acids from a solid support by varying the conditions of the elution buffer to promote elution of only the smaller molecular weight nucleic acids.
Regarding Applicant’s argument that the Hawkins does not teach the elution buffer having a lower polyalkylene concentration than the binding buffer, as stated, Hawkins teaches that an elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles (p. 10, lines 28-32) (emphasis added). Therefore, based on Hawkins, the PEG concentration may be adjusted to separate polynucleotide fragments based on size, and when adjusting the PEG concentration to separate polynucleotides based on size, the PEG must be below the range required for binding of DNA onto magnetic microparticles. The Office maintains that in view of Hawkins, one of ordinary skill in the art would have contemplated adjusting either or both the salt concentration and PEG concentration to separate polynucleotide fragments based on size, and would have recognized a lower PEG concentration was necessary to elute the nucleic acids from the solid phase.
Regarding Applicant’s argument that the passage on p. 10, lines 28-31 of Hawkins must be read in its own proper context, the passage states that an elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles. Although a specific elution buffer may not be exemplified by Hawkins, the elution buffers considered by Hawkins are not solely those exemplary buffers provided by Applicant. Moreover, regarding context, Hawkins teaches that size selection may be accomplished by varying the concentration of PEG, and that the salt concentration and PEG concentration for elution bust be below the ranges required for binding of DNA onto magnetic microparticles. Therefore, one of ordinary skill in the art would have 1) considered varying the PEG concentration to separate nucleic acids based on size, and 2) would have recognized that, in order to elute the nucleic acid, the PEG concentration must be below that required to bind DNA to the solid support.
Regarding argument 4 above, the present Office action cites to Table 1, which is generated under different dissociation conditions. The examiner agrees that Wisniewski at step 5 does not expressly state testing 18% PEG concentration. However, because Table 1 states that these data are using specific dissociation conditions, and because these data are presented on the same table, the examiner submits that one of ordinary skill in the art would make use of the full data presented and reasonably interpret that both PEG and salt conditions present during dissociation may be compared with one another. Specifically, if one were to compare the 18% PEG concentration with the 10% PEG concentration at equivalent salt concentrations, one would recognize the recovered DNA includes a higher fraction of short fragments at 18% PEG compared with 10% PEG, and in view of these data, would conclude that the PEG concentration may be varied to affect the size of nucleic acid recovered during elution.
However, even if the PEG concentration data of Wisniewski were not directly compared, the teachings from Hawkins that the concentration of PEG may be used to separate nucleic acids based on size, and that one may use an elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles, is a sufficient teaching that a reduction in PEG concentration would have been necessary for the size separation of nucleic acids.
Therefore, even if, as Applicant argues, one of ordinary skill in the art would not have recognized from the teachings of Wisniewski that the concentration of PEG may be selected to adjust the size cut off of the desired nucleic acid, because this same effect is taught by Hawkins, it cannot be considered improper hindsight.
Regarding argument 5 above, although Hawkins is silent on the on the isolation of extracellular nucleic acids from cell-free or cell-depleted body fluids, as stated, Wisniewski teaches their method is intended to size select for the isolation of extracellular nucleic acids from cell-free or cell-depleted body fluids. In addition, although Wisniewski teaches and exemplifies alternative conditions for binding DNA to a solid phase, the prior art, including Hawkins, provides that nonspecific binding of nucleic acid to a solid phase may be accomplished under different conditions. Moreover, Wisniewski claims that binding conditions for binding of the nucleic acid to a solid support may include a volume exclusion agent, which may be PEG. Therefore, one of ordinary skill in the art would have considered substituting the volume exclusion agent exemplified by Wisniewski for an alternative suggested by Wisniewski with a reasonable expectation that such a substitution would reasonably enable binding of a nucleic acid sample to a solid phase.
Regarding argument 6, although Hawkins and Wisniewski both teach varying the salt concentration as one method to selectively elute smaller nucleic acids, because Hawkins teaches that the concentration of PEG may be used to separate nucleic acids based on size, and that one may use an elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles, one of ordinary skill in the art would have recognized that either or both the salt concentration and PEG concentration to separate polynucleotide fragments based on size, and would have had a reasonable expectation of success in utilizing an elution buffer with lower PEG concentration to selectively elute smaller molecular weight nucleic acids.
Therefore, for the reasons described above, the present rejection of claims 1-14, 16, 18, 19, 22, and 24 under 35 U.S.C. 103 as unpatentable over Hawkins in view Wisniewski is maintained. For the same reasons, the rejection of the kit recited in claims 25 and 26 is also maintained.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-14, 16, 18-19, 22, and 24-26 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 9-14 and 17 of U.S. patent 8,624,020 (reference application, herein referred to as ‘020) in view of Hawkins (Publication no. WO 1996009379 A1; cited in IDS received December 17, 2021) and Wisniewski (Publication no. WO 2010115016 A2; cited in previous office action).
Both the present application and ‘020 are assigned to Qiagen GmbH.
The present claim 1 recites a poly(alkylene oxide) polymer based size selective method for enriching nucleic acid molecules having a length below a cut-off value from a nucleic acid containing sample comprising the steps (a) – (e) recited in claim 1, wherein the nucleic acid containing sample is a cell-free or cell-depleted body fluid sample and wherein the eluted nucleic acid molecules are extracellular nucleic acid molecules. Claim 2 requires the eluted nucleic acid molecules are extracellular DNA, claims 3 and 4 specify specific cutoff values, claim 5 requires eluting the nucleic acids from step (f), claim 6 specifies requirements about the solid phase, claim 7 specifies requirements about the poly(alkylene oxide) polymer, claim 8 specifies requirements about the salt, claim 9 requires in step (a) adding a binding reagent to the nucleic acid containing sample to prepare the binding mixture, claim 10 specifies requirements about the binding reagent or conditions, claim 11 specifies requirements about the elution composition, claim 12 requires providing the elution composition of step ( c) by diluting the binding reagent with a dilution solution, claim 13 specifies requirements about the nucleic acid containing sample.
The present claim 14 depends from claim 1 and requires the features listed therein. Claim 16 requires providing the elution composition of step ( c) by diluting the binding reagent with a dilution solution and wherein the solid phase with the bound nucleic acid is in step (c) only contacted with a single elution composition (c) but not with further reagents, and claim 18 requires the binding reagent has a pH value that lies in a range of 4.5 to 9.5 and wherein the solid phase is provided by carboxylated magnetic particles.
The present claims 19 and 22 depend from claim 1 and require the method with additional limitations listed therein. Claim 24 depends from claim 1 and requires the eluted nucleic acids comprise extracellular nucleic acid molecules, and the method comprises analyzing the eluted extracellular nucleic acid molecules to identify, detect, screen for, monitor or exclude a disease, an infection and/or at least one fetal characteristic.
The present claims 25 and 26 recite a kit for the size selective enrichment of nucleic acid molecules, having a length below a cut-off value from a nucleic acid containing sample, comprising the reagents listed therein.
Claim 9 of ‘252 claims a method for the extraction of nucleic acids from a solution, comprising the steps a) – e) recited in the claims. Claims 10 limits the surface material used for binding nucleic acids in claim 9 to materials including silica and a carboxylated surface, claim 11 limits the surface to materials including a silica-based magnetic particle, and claim 12 claims the binding mediator is selected from the group that includes polyethylene glycol 200. Claims 13 and 14 claim a reagent kit for washing nucleic acids immobilized on one or more surfaces comprising a wash buffer, a binding mediator, and an eluant, and claim 17 claims the kit for the extraction of nucleic acids from biological materials.
The claims of ‘020 do not claim the size selective method of claim 1 for enriching nucleic acid molecules having a length below a cut-off value or the specific steps and conditions required by said method.
However, as described in the above rejection under 35 U.S.C. 103, the presently claimed method, including the size selective method for enriching nucleic acid molecules having a length below a cut-off value by eluting nucleic acid from a solid phase with a buffer comprising lower concentration of PEG than is used to bind the nucleic acid to the solid phase, as well as the steps and conditions recited in the present claims, would have been obvious over Hawkins in view of Wisniewski.
It would therefore have been pr