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 .
Applicant’s amendment filed on 09/26/2025 has been entered.
Amended claims 91-94, 98, 101-109 and new claim 110 are pending in the present application, and they are examined on the merits herein.
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.
Amended claims 91-93, 98 and 101-110 are rejected under 35 U.S.C. 103 as being unpatentable over Christoffel (US 2013/0225801; IDS) in view of Gillespie et al (US 5,155,018) and Smith et al (WO 99/54340). This is a modified rejection necessitated by Applicant’s amendment.
Amended claims are directed to a kit for nucleic acid purification comprising: (i) a denaturing solvent comprising a chaotropic salt and at least 20% phenol, wherein the denaturing solvent is added directly to a nucleic acid containing sample generating a first mixture; (ii) a binding agent comprising a chaotropic salt and a lower alcohol, wherein the binding agent is added directly to the first mixture generating a second mixture, wherein the chaotropic salt of the binding agent is guanidinium thiocyanate, guanidinium chloride, sodium iodide, sodium perchlorate, urea or thiourea, and wherein the lower alcohol of the binding agent is methanol, ethanol, isopropanol, butanol, or a combination thereof; (iii) a silica substrate, wherein the silica substrate is contacted with the second mixture, wherein the second mixture comprises phenol, does not comprise chloroform, and does not comprise separate aqueous and organic phases prior to binding the nucleic acid to the silica substrate; and (iv) a wash solution comprising a detergent and an alcohol. It is noted that the instant claims are directed to a kit composition and not to a method; and the examiner interprets the “wherein clause with the contacting or adding step” for each of the reagents (i)-(iii) in the kit to be an intended use in a particular method of nucleic acid purification that requires the generation of a first mixture and a second mixture.
Christoffel already taught at least a kit for isolating RNA from a sample comprising RNA and DNA, the kit comprises: (i) an acidic denaturing composition comprising a chaotropic agent and phenol; (ii) a solution for reducing the amount of DNA in an aqueous RNA-containing phase comprising at least one cationic detergent; (iii) optionally a nucleic acid binding solid phase, and (iv) optionally washing and elution buffers; wherein the chaotropic agent is a chaotropic salt that includes guanidinium thiocyanate, guanidinium isothiocyanate, sodium thiocyanate or others (preferably guanidinium hydrochloride and/or guanidinium thiocyanate) comprised in a concentration of 0.1 to 6M or 0.5 to 3M; the phenol is comprised in a concentration of 10% to 70% or 20% to 60%; and the acidic denaturing composition has a pH value below 6, preferably ≤5, and a buffer in an amount sufficient to maintain at an acidic pH as well as sodium thiocyanate for enhancing RNA extraction; and wherein the nucleic acid binding solid phase includes a variety of materials that are capable of binding nucleic acids under suitable condition, with exemplary solid phases include silica, siliceous solid phases in various forms such as silica particles, glass, diatomaceous earth, silica membrane (see at least the Abstract; particularly paragraphs [0049], [0054]-0067], [0071]-[0077]; and claims 33-36). Christoffel also taught specifically that the term solid phase encompasses materials that are porous or non-porous, membranes, filters, sheets, particles, magnetic particles, beads, gels, powders, fibers (paragraph [0049]); and utilized RNeasy mini columns (Qiagen) comprising silica membranes for RNA isolation (see at least Example 1, particularly paragraphs [0097]-[0099]). Christoffel also taught that a washing solution comprises at least one chaotropic agent (e.g., guanidium thiocyanate, guanidinium isothiocyanate), at least one alcohol (e.g., methanol, ethanol, propanol, isopropanol, butanol), at least one detergent and/or at least one buffering component being used in one or more washing steps that are performed while the RNA bound to the nucleic acid binding solid phase (paragraph [0071]).
Christoffel did not teach specifically a kit comprising a binding agent comprising a chaotropic salt (other than a chaotropic salt in a denaturing solvent and/or a washing solution), a lower alcohol that is selected from methanol, ethanol, isopropanol, butanol, or a combination thereof (other than an alcohol in a washing solution); and/or the silica substrate is a (para)magnetic bead.
Before the effective filing date of the present application (07/8/2012), Gillespie et al already taught a process and kit for isolating and purifying RNA from a biological source, in which process the use of a binding solution comprising concentrated, acidified chaotropic salt (e.g., guanidine thiocyanate, guanidine isothiocyanate) for selective binding of RNA, but not DNA, to siliceous material (e.g., finely divided glass or diatomaceous earth)(see at least Abstract; Summary of the Invention; particularly col. 3, lines 32-40; Example 5; and issued claims 15-17). Gillespie et al also taught that a binding solution may be acidified with any organic or inorganic acid, and the final pH of the binding solution should be between about 2 and 6 (col. 4, lines 50-54); and that the selective RNA binding process is applied to previously disrupted biological cells containing the RNA of interest (col. 5, lines 42-45).
Additionally, Smith et al also taught at least the use of a binding agent selected from the group consisting of: a chaotropic agent, a salt which not a chaotropic agent, a low molecular weight alcohol, or a combination thereof to promote formation of a complex between a target nucleic acid and a second silica matrix for purification of the target nucleic acid in a method for removing endotoxins from nucleic acids (e.g., RNA and DNA) (see at least Abstract; Brief Summary of the Invention; particularly pages 16, line 6 continues to line 10 on page 19). Smith et al stated specifically that “The proportions of each binding agent used depend upon how much of each other agent is present in the resulting binding solution….Smaller concentrations of non-chaotropic salt and other binding agents can be used where more than one binding agent is present in the binding solution” (page 16, lines 17-22). Smith et al disclosed that when a chaotropic agent (guanidinium salts, guanidine hydrochloride) is the only binding agent in a binding solution, the concentration of chaotropic agent therein must be sufficiently high to cause the nucleic acid to form a complex with the second silica matrix, but not so high as to substantially denature, degrade, or cause the target nucleic acid to precipitate out the binding solution (e.g., RNA and small DNA molecules remain undegraded and in solution at chaotropic agent concentrations between 2 and 5 molar) (page 16, line 23 continues to line 14 at page 17). Smith et al also taught that when a low molecular weight alcohol is the only binding agent present in the binding solution, the minimum amount of alcohol necessary to promote formation of the complex between the second silica matrix and the nucleic acid depends on the nature of the alcohol and the type of nucleic acid to form the complex with the second silica matrix; specifically RNA tends to form a complex with the second silica matrix more readily than DNA, in the presence of binding solution concentration of polar low molecular weight alcohols (e.g., isopropanol) of up to 30% by volume; and it is preferably to use a less polar alcohol (e.g., ethanol) at concentration of at least 80% (page 17, line 15 continues to line 4 on page 18). Smith et al further taught that the silica matrix is in a non-magnetic form such as silica gel particles, ground glass, controlled pore glass particles, diatomaceous earth, a silica filter or a filter embedded with silica particles; or in magnetic silica matrices such as paramagnetic particles from Promega, or magnetic silica particles (page 9, lines 1-18).
Accordingly, it would have been obvious for an ordinary skilled artisan to modify the kit of Christoffel by also including at least a binding solution comprising an acidified chaotropic salt (e.g., guanidine thiocyanate, guanidine isothiocyanate) at pH about 2-6 and/or a low molecular weight alcohol (e.g., ethanol and isopropanol), as well as utilizing silica (para)magnetic beads as a nucleic acid binding solid phase for isolating RNA from a sample comprising RNA and DNA; in light of the teachings of Gillespie et al and Smith et al as set forth above.
An ordinary skilled artisan would have been motivated to carry out the above modification because Gillespie et al already taught the use of a binding solution comprising concentrated, acidified chaotropic salt (e.g., guanidine thiocyanate, guanidine isothiocyanate) at pH about 2-6 for selective binding of RNA, but not DNA, to siliceous material; while Smith et al also taught the use of a binding agent such as a chaotropic agent (e.g., guanidinium salts, guanidine hydrochloride) and/or a low molecular weight alcohol (e.g., ethanol and isopropanol) to promote formation of a complex between a target nucleic acid and a second silica matrix (e.g., silica (para)magnetic particles/beads) for purification of the target nucleic acid with reduced endotoxins. The further inclusion of a binding solution comprising a chaotropic salt and an alcohol in a kit enables further purification of a desired RNA under selective conditions from DNA and other contaminants such as endotoxins.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Christoffel, Gillespie et al and Smith et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified kit resulting from the combined teachings of Christoffel, Gillespie et al and Smith et al as set forth above has the same components and it is indistinguishable from the kit of the present application; and each of the components (i)-(iii) in the modified kit can be used for its intended use in a nucleic acid purification method. With respect to new claim 91 reciting the new limitation “wherein the kit does not comprise chloroform”, it is noted that at least the kit of the primary Christoffel reference does not contain chloroform, and that the kit also optionally contains a nucleic acid binding solid phase, and optionally contains washing and elution buffers (see at least claims 33-36 in the Christoffel reference).
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Arguments
Applicant’s arguments related to the above modified 103 rejection in the Amendment filed on 09/26/2025 (pages 4-6) have been fully considered, but they are respectfully not found persuasive for the reason discussed below.
Once again, Applicant argued that the cited references do not provide a motivation to combine Christoffel’s kit, the only one with phenol and does not use siliceous material, with Gillespie’s or Smith’s kits. This is because Gillespie’s and Smith’s kits use siliceous material for RNA isolation and do not contain phenol; and Gillespie teaches that “contacting a cell or tissue homogenate with phenol or a mixture of phenol and chloroform, thereby denaturing proteins and precipitating them while leaving nucleic acids in solution….while still widely used, is hazardous, laborious, and of limited utility for isolation of RNA” (col. 1, lines 34-46); while Smith only mentions the use of phenol/chloroform extraction in a step prior to the silica-based purification described. Additionally, independent claim 91 has been amended such that the silica substrate is contacted with the second mixture, which comprises phenol, and does not comprise separate aqueous and organic phases prior to binding to the silica substrate; and the cited prior art does not teach contacting silica with a mixture comprising phenol or phases that are unseparated. Moreover, Applicant argued that the kits as claimed provide “surprising and unexpected” results as evidenced by the Forman Declaration (IDS dated July 22, 2021 as reference C16) showing highly reproducible purification of intact RNA from cells, non-biased recovery of miRNA, and efficiently recovery of total RNA, including small RNAs, and without requiring phase separation or phenol removal that is highly suitable for automation and a significant improvement over phenol-based purification methods in the art.
First, the instant claims are directed to a kit for nucleic acid purification comprising: (i) a denaturing solvent comprising a chaotropic salt and at least 20% phenol, wherein the denaturing solvent is added directly to a nucleic acid containing sample generating a first mixture; (ii) a binding agent comprising a chaotropic salt and a lower alcohol, wherein the binding agent is added directly to the first mixture generating a second mixture, wherein the chaotropic salt of the binding agent is guanidinium thiocyanate, guanidinium chloride, sodium iodide, sodium perchlorate, urea or thiourea, and wherein the lower alcohol of the binding agent is methanol, ethanol, isopropanol, butanol, or a combination thereof; (iii) a silica substrate, wherein the silica substrate is contacted with the second mixture, wherein the second mixture comprises phenol, does not comprise chloroform, and does not comprise separate aqueous and organic phases prior to binding the nucleic acid to the silica substrate; and (iv) a wash solution comprising a detergent and an alcohol. It is noted that the instant claims are directed to a kit composition and not to a method; and the examiner interprets the “wherein clause with the contacting or adding step” for each of the reagents (i)-(iii) in the kit to be an intended use in a particular method of nucleic acid purification that requires the generation of a first mixture and a second mixture.
Second, Christoffel’s kit for isolating at least RNA from a sample comprises an acidic denaturing composition comprising a chaotropic agent and phenol, and a nucleic acid binding solid phase that include silica, siliceous solid phases in various forms such as silica particles, glass, diatomaceous earth, silica membrane. As already indicated in the above 103 rejection, it would have been obvious for an ordinary skilled artisan to modify the kit of Christoffel by also including at least a binding solution comprising an acidified chaotropic salt (e.g., guanidine thiocyanate, guanidine isothiocyanate) at pH about 2-6 and/or a low molecular weight alcohol (e.g., ethanol and isopropanol), as well as utilizing silica (para)magnetic beads as a nucleic acid binding solid phase for isolating RNA from a sample comprising RNA and DNA because: (i) Gillespie et al already taught the use of a binding solution comprising concentrated, acidified chaotropic salt (e.g., guanidine thiocyanate, guanidine isothiocyanate) at pH about 2-6 for selective binding of RNA, but not DNA, to siliceous material; and (ii) Smith et al also taught the use of a binding agent such as a chaotropic agent (e.g., guanidinium salts, guanidine hydrochloride) and/or a low molecular weight alcohol (e.g., ethanol and isopropanol) to promote formation of a complex between a target nucleic acid and a second silica matrix (e.g., silica (para)magnetic particles/beads) for purification of the target nucleic acid with reduced endotoxins. The further inclusion of a binding solution comprising a chaotropic salt or an alcohol in a kit enables further purification of a desired RNA under selective conditions from DNA and other contaminants such as endotoxins.
Third, there is no teaching away whatsoever by Gillespie in combining with Christoffel and/or Smith. Gillespie stated “One commonly used method for isolating nucleic acids from cells and tissues was the “Sevag” procedure. This method comprises contacting a cell or tissue homogenate with phenol or a mixture of phenol and chloroform, thereby denaturing proteins and precipitating them while leaving nucleic acids in solution. This method, while still widely used, is hazardous, laborious and of limited utility for isolation of RNA from biological sources containing high amounts of ribonuclease (RNAse), an extremely stable enzyme that degrades RNA” (col. 1, lines 36-46). However, these statements are related specifically to the “Sevag” procedure and biological sources containing high amounts of ribonuclease; and Gillespie does not explicitly exclude the use of phenol in any other RNA isolation procedures/methods or isolating RNA from other biological sources that do not contain high amount of ribonuclease. Instead, Gillespie et al also stated “According to another aspect of the present invention, the selective binding process is applied to previously disrupted biological cells containing the RNA of interest. The cells are disrupted by exposing them to a lytic agent, which comprises as its main component, concentrated, chaotropic salt. The lytic agent may also comprise other agents capable of disrupting cell membranes and inactivating intracellular enzymes” (col. 5, lines 42-49). Please note that Christoffel’s kit for isolating at least RNA from a sample comprises an acidic denaturing composition comprising a chaotropic agent and phenol.
Fourth, Smith stated “The first solution can be any solution containing endotoxins and any target nucleic acid. The first solution is preferably obtained by disrupting a cell, such as a gram-negative bacteria cell such as an E coli cell, which contains or has endotoxins associated therewith. Any conventional method can be used to isolated the target nucleic acid from other material in the cell...For example, a solution containing at least 1M concentration of a chaotropic agent can be used to disrupt cells to isolate RNA” (page 13, first full paragraph); and “For example, plasmid DNA can be isolated using isolation means such as: banding by ultracentrifugation in a cesium chloride/ethidium bromide solution; phenol, chloroform or phenol/chloroform extraction; adherence to a silica matrix and elution therefrom after separation of the silica matrix from the solution in which the plasmid DNA was adhered to the matrix” (page 14, last paragraph). Once again, Christoffel’s kit for isolating at least RNA from a sample comprises an acidic denaturing composition comprising a chaotropic agent and phenol.
Fifth, once again the instant claims are directed to a kit composition and not to a method; and the examiner interprets the “wherein clause with the contacting or adding step” for each of the reagents (i)-(iii) in the kit to be an intended use in a particular method of nucleic acid purification that requires the generation of a first mixture and a second mixture. The modified kit resulting from the combined teachings of Christoffel, Gillespie et al and Smith et al as set forth above has the same components and it is indistinguishable from the kit of the present application; and each of the components (i)-(iii) in the modified kit can be used for its intended use in a nucleic acid purification method. With respect to new claim 91 reciting the new limitation “wherein the kit does not comprise chloroform”, it is noted that at least the kit of the primary Christoffel reference does not contain chloroform, and that the kit also optionally contains a nucleic acid binding solid phase, and optionally contains washing and elution buffers (see at least claims 33-36 in the Christoffel reference).
Sixth, with respect to the “surprising and unexpected” results disclosed in the Forman Declaration it is noted that such results were obtained via a particular method of nucleic acid purification with a specific series of steps and particular solutions/mixtures having particular components at specific concentrations and/or pH; and not simply via the components recited in the kit of the present application.
Claim 94 is rejected under 35 U.S.C. 103 as being unpatentable over Christoffel (US 2013/0225801; IDS) in view of Gillespie et al (US 5,155,018) and Smith et al (WO 99/54340) as applied to claims 91-93, 98 and 101-110 above, and further in view of Jia (US 2010/0222560).
The combined teachings of Christoffel, Gillespie et al and Smith et al were presented above. However, none of the cited references teach specifically a kit further comprising an affinity purification column comprising an array of a plurality of columns.
Before the effective filing date of the present application, Jia disclosed a universal column comprising a binding matrix that includes siliceous matrix (e.g., glass fiber or silica beads) or affinity matrices for separating a compound (e.g., nucleic acids such as DNA or RNA molecules) from impurities under conditions (e.g., the column is spun in a centrifuge; a negative pressure is applied to the bottom of the column via a vacuum manifold) wherein the compound remains bound to the column matrix; and a kit comprising a plurality of the columns (e.g., 10, 15, 20, 25 or more) (Abstract; paragraphs [0018]-[0025]; [0061]-[0063], [0080]-[0092], [0143]; and Fig. 3B). Fig. 3B shows a universal column attached to a vacuum manifold having an array of attachment.
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Accordingly, it would have been obvious for an ordinary skilled artisan to further modify the kit of Christoffel, Gillespie et al and Smith et al by also further including an array of a plurality of universal columns comprising a binding matrix that includes siliceous matrix (e.g., glass fiber or silica beads) or affinity matrices for attachment to a vacuum manifold to isolate RNA from a sample comprising RNA and DNA, in light of the teachings of Jia as set forth above.
An ordinary skilled artisan would have been motivated to carry out the above modification because Jia already successfully taught a kit comprising a plurality of universal columns comprising a binding matrix that includes siliceous matrix (e.g., glass fiber or silica beads) or affinity matrices for separating a compound (e.g., nucleic acids such as DNA or RNA molecules) from impurities under conditions such as a negative pressure being applied to the bottom of each of the columns via a vacuum manifold shown in Fig. 3B in an improved purification method.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Christoffel, Gillespie et al, Smith et al and Jia; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified kit resulting from the combined teachings of Christoffel, Gillespie et al, Smith et al and Jia as set forth above is indistinguishable from the kit of the present application.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Arguments
Applicant’s argument related to the above 103 rejection in the Amendment filed on 09/26/2025 (page 7) has been fully considered, but it is respectfully not found persuasive for the reason discussed below.
Applicant argued basically that Jia does rectify the deficiencies of Christoffel, Gillespie, and Smith as discussed in the rejection of claims 91-93, 98 and 101-110 above. Accordingly, claim 94 is not obvious over Christoffel in view of Gillespie and Smith, and further in view of Jia.
With respect to the deficiencies of Christoffel, Gillespie and Smith, please refer to the examiner’s same responses to Applicant’s arguments for the rejection of claims 91-93, 98 and 101-110 above. Jia is cited primarily to supplement the combined teachings of Christoffel, Gillespie and Smith for the limitation “wherein the purification column comprises an array of a plurality of columns” recited in dependent claim 94.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Quang Nguyen, Ph.D., at (571) 272-0776.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s SPE, James Douglas (Doug) Schultz, Ph.D., may be reached at (571) 272-0763.
To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Group Art Unit 1631; Central Fax No. (571) 273-8300.
Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to (571) 272-0547.
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/QUANG NGUYEN/Primary Examiner, Art Unit 1631