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 .
Claim Status and Formal Matters
This action is in response to papers filed 10/28/2025.
Claims 1-5, 7-12, 17, 21-22, 26-27, 29, 32, 46-47 are pending.
Claims 46-47 have been added by amendment.
Claims 1, 17have been amended.
Claims 1-5, 8-12, 17, 21, 46-47 are being examined.
Applicant's election with traverse of group I, claims 1-5, 8-12, 15-17 in the reply filed on 2/20/2024 is acknowledged. The traversal is on the ground(s) that the response asserts there is no search burden. This is not found persuasive because this argument has been thoroughly reviewed but is not considered persuasive as the instant application is a National Stage Entry of a PCT and thus is examined under unity of invention principles. .The response continues by providing arguments with respect to the nickase of the art not being random. This argument has been thoroughly reviewed but is not considered persuasive as the instant specification fails to provide a clear standard by which to differentiate random from non-random.
Claims 7, 22, 26-27. 29, 32 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 2/20/2024.
The written description rejection has been withdrawn in view of the amendment.
Priority
The instant application was filed 02/16/2021 is a National Stage entry of PCT/US2019/046937 with an International Filing Date: 08/16/2019 and claims Priority from Provisional Application 62765073 , filed 08/17/2018.
Improper Markush Group
Claims 1-5, 8-12, 17, 21, 46-47 are rejected under the judicially approved ‘‘improper Markush grouping’’ doctrine. (See Federal Register, Vol. 76, No. 27, Wednesday, February 9, 2011, page 7166). This rejection is appropriate when the claim contains an improper grouping of alternatively useable species. See In re Harnisch, 631 F.2d 716, 719–20 (CCPA 1980). A Markush claim contains an ‘‘improper Markush grouping’’ if: (1) the species of the Markush group do not share a ‘‘single structural similarity,’’ or (2) the species do not share a common use. Members of a Markush group share a ‘‘single structural similarity’’ when they belong to the same recognized physical or chemical class or to the same art-recognized class. However, when the Markush group occurs in a claim reciting a process or a combination (not a single compound), it is sufficient if the members of the group are disclosed in the specification to possess at least one property in common which is mainly responsible for their function in the claimed relationship, and it is clear from their very nature or from the prior art that all of them possess this property. See MPEP § 803.02.
Here each species is considered to be a nicking nuclease or strand displacing polymerase. .
The recited alternative species in the groups set forth here do not share a single structural similarity, as each nicking enzyme and strand displacement polymerase have different temperature of activity and conditions (table 1 of specification).. Each nicking enzyme and strand displacement polymerase have a different T as evidenced by table 1 of the specification.. While each member of the nicking nuclease and strand displacement polymerase have the function of nicking or strand displacement polymerase, they all have different properties as each nicking enzyme and each strand displacement polymerase have different , thus different properties.
Following this analysis, the claims are rejected as containing an improper Markush grouping.
Response to Arguments
This is a new ground of rejection necessitated by amendment.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-5, 8-12, 17, 21, 46-47 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 has been amended to recite, “forming a reaction mixture comprising: the input dsDNA, a nicking nuclease active at a temperature T, wherein the nicking nuclease is selected from the group consisting of: double-stranded DNA specific nuclease (DSN), Shrimp- based double strand specific nucleases (dsDNase), HL-dsDNAse, and DNAse polymerase, wherein the strand-displacing polymerase is selected from the group consisting of: a Bst DNA polymerase, phi29 polymerase, and Klenow fragment of DNA polymerase I The claims as written requires, “a nicking nuclease active at a temperature T” and “strand-displacing polymerase active at the temperature T,” however the specification in table 1 teaches DSN and BST do not work at the same temperatures as the rest of the nicking enzymes and strand-displacing polymerase provided in the claims.
Claim 46 has been amendment and recites, “wherein the nicking nuclease does not rely on the presence of a DNA recognition sequence to incorporate single-stranded breaks into dsDNA.” However claim 1 requires, “double-stranded DNA specific nuclease (DSN), Shrimp- based double strand specific nucleases (dsDNase), HL-dsDNAse, and DNAse I.” Thus this appears to be an inherent property of the recited nicking enzymes. Thus it is unclear how this further limits the recited nicking enzymes.
Claim 47 has been amendment and recites, “, wherein the strand-displacing polymerase recognizes a single-stranded break in dsDNA, and, in the presence of nucleotide triphosphates, extends the single strand having the break and displaces the ssDNA fragment that is 3' relative to the break.” However claim 1 requires, “wherein the strand- displacing polymerase, wherein the strand-displacing polymerase is selected from the group consisting of: a Bst DNA polymerase, phi29 polymerase, and Klenow fragment of DNA polymerase I .” Thus this appears to be an inherent property of the recited strand-displacing polymerase. Thus it is unclear how this further limits the recited strand-displacing polymerase.
Response to Arguments
This is a new grounds of rejection.
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.
Claim(s) 1-2, 4-5, 9-10, 12, 17, 46-47 is/are rejected under 35 U.S.C. 103 as being unpatentable over Cole (US2005/0136417).
With regards to claim 1, Cole teaches, “[0002] The methods of the invention relate generally to amplification of nucleic acids by generating a nick and extending from the nick with a strand displacing polymerase.” Cole teaches, “[0006] In preferred embodiments the DNA polymerase is a strand displacing enzyme, for example the Klenow fragment or phi29. Nicking and extension may be performed under isothermal conditions,”
Cole teaches, “[[0048] Polymerases useful in this method include those that will initiate 5' to 3' polymerization at a nick site. The polymerase preferably should displace the polymerized strand downstream from the nick, and preferably lacks substantial 5' to 3' exonuclease activity. Enzymes that may be used include, for example, the Klenow fragment of DNA polymerase I, Bst polymerase large fragment, Phi29 and others. DNA Polymerase I Large (Klenow) Fragment consists of a single polypeptide chain (68 kDa) that lacks the 5'->3' exonuclease activity of intact E. coli DNA polymerase I, but retains its 5'->3' polymerase, 3'->5' exonuclease and strand displacement activities. The Klenow fragment has been used for strand displacement amplification (SDA). See, e.g., U.S. Pat. Nos. 6,379,888; 6,054,279; 5,919,630; 5,856,145; 5,846,726; 5,800,989; 5,766,852; 5,744,311;5,736,365; 5,712,124; 5,702,926; 5,648,211;5,641,633; 5,624,825; 5,593,867; 5,561,044; 5,550,025; 5,547,861; 5,536,649; 5,470,723; 5,455,166; 5,422,252; 5,270,184, all incorporated herein by reference. SDA is an isothermal in vitro method for amplification of nucleic acid. SDA initiates synthesis of a copy of a nucleic acid at a free 3' OH that may be provided, for example, by a primer that is hybridized to the template. The DNA polymerase extends from the free 3' OH and in so doing displaces the strand that is hybridized to the template leaving a newly synthesized strand in its place. Repeated nicking and extension with continuous displacement of new DNA strands results in exponential amplification of the original template.. “ Cole teaches, “0051] Any polymerase with strand displacing activity may be used. Examples of other enzymes that may be used include: exo minus Vent (NEB), exo minus Deep Vent (NEB), Bst (BioRad), exo minus Pfu (Stratagene), Pfx (Invitrogen), 9.degree.N.sub.m.TM. (NEB), Bca (Panvera), and other thermostable polymerases. Other characteristics of strand displacing enzymes that may be taken into consideration are described, for example, in U.S. Pat. No. 6,692,918.”
Cole teaches, “[0069] DSN is available, for example, from Evrogen. The enzyme is from the Kamchatka crab. It is specific for dsDNA or DNA in a DNA:RNA hybrid but does not cleave ssDNA. The enzyme has been used to identify SNPs as it can discriminate between perfectly matched short DNA-DNA duplexes (8-12 bp) and duplexes of the same length with a single mismatch. The activity of the DSN enzyme may be controlled, for example, by the amount of enzyme added, the buffer conditions or the temperature conditions. The Ph and temperature optimum for DSN are 7-8 and 55-65.degree. C. with the enzyme being stable at temps below 75.degree. C. In some embodiments the cleavage and extension reactions are performed simultaneously so that multiple rounds of amplification can take place. The polymerase may be chosen so that it functions under similar conditions as the DSN enzyme. For example a mesophilic polymerase such as Bst1 may be used. For additional information about the DSN enzyme see, for example, Shagin et al. Genome Res 12: 1935-1942 (2002).”
While Cole suggests isothermal production of probes by using nicking enzymes including DSN and strand displacement polymerase including BST polymerase, they do not specifically teach the use of BST and DSN.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use Bst polymerase and DSN nicking enzyme to generate probes in an isothermal reaction. The artisan would be motivated as Cole specifically suggests the use of BST and DSN in the method. The artisan would have a reasonable expectation of success as the artisan is merely using known strand displacement polymerase and nicking enzyme known to work at the same temperature.
With regards to claim 2, 5, Cole teaches, “The first strand cDNA synthesis may be primed by addition of an exogenous primer comprising oligo dT, a plurality of locus specific primers or random primers. First strand cDNA may be synthesized by an RNA dependent DNA polymerase or by a DNA dependent DNA polymerase.” Thus Cole teaches amplification of ssDNA.
With regards to claim 4, Cole teaches a Klenow fragment (0048).
With regards to claim 9, Cole teaches a product size of 20 nt or 16 to 40 nt (0071-0072)
With regards to claim 10, Cole teaches, “Using 50-100 ng of ds-cDNA template, at least 6-8 ug of purified amplified DNA product was obtained.” (0073)
With regards to claim 12, Cole teaches inactivating at 80oC.
With regards to claims 16, Cole teaches Klenow fragment and Bst polymerase (0048).
MPEP 2144.05 II A States:
II. ROUTINE OPTIMIZATION
A. Optimization Within Prior Art Conditions or Through Routine Experimentation
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 reAller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to optimize the concentration of the nicking enzyme and the strand displacement polymerase. The artisan would be motivated to provide the most efficient reaction to produce the greatest amount of product with the least amount of enzymes. The artisan would have a reasonable expectation of success as the artisan is merely using known enzymes in a known method.
With regards to claim 46, Cole teaches DSN
With regards to claim 47, Cole teaches BSTN polymerase
Response to Arguments
The response traverses the rejection has been overcome as the amendment has amended the independent claim to recite, “the nicking nuclease is selected from the group consisting of: double-stranded DNA specific nuclease (DSN), Shrimp- based double strand specific nucleases (dsDNase), HL-dsDNAse, and DNAse I.” This argument has been thoroughly reviewed but is not considered persuasive as Cole teaches, “[0069] DSN is available, for example, from Evrogen. The enzyme is from the Kamchatka crab. It is specific for dsDNA or DNA in a DNA:RNA hybrid but does not cleave ssDNA. The enzyme has been used to identify SNPs as it can discriminate between perfectly matched short DNA-DNA duplexes (8-12 bp) and duplexes of the same length with a single mismatch. The activity of the DSN enzyme may be controlled, for example, by the amount of enzyme added, the buffer conditions or the temperature conditions. The Ph and temperature optimum for DSN are 7-8 and 55-65.degree. C. with the enzyme being stable at temps below 75.degree. C. In some embodiments the cleavage and extension reactions are performed simultaneously so that multiple rounds of amplification can take place. The polymerase may be chosen so that it functions under similar conditions as the DSN enzyme. For example a mesophilic polymerase such as Bst1 may be used. For additional information about the DSN enzyme see, for example, Shagin et al. Genome Res 12: 1935-1942 (2002).”
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.
Claim(s)1-2, 4-5, 8-10, 12, 17, 21, 46-47 is/are rejected under 35 U.S.C. 103 as being unpatentable over Van Ness (WO2003/080645), Zhao (Chem. Rev. 2015, 115, 12491−12545) and Gerasimova ( Chem. Soc. Rev., 2014, 43, 6405-6438).
Van Ness discloses a method of generating probes from a sample of input double-stranded DNA (dsDNA) (The present invention relates to compounds, kits and methods for ... preparing single-stranded nucleic acid probes, Abstract; In another aspect, the present invention provides a method that includes: (a) Forming a mixture of (i) a partially or fully double-stranded template nucleic acid, Pg. 55, Lns. 29-30), the method comprising: (a) forming a reaction mixture comprising: the input dsDNA, a nicking nuclease active at a temperature T, a strand-displacing polymerase active at the temperature T, wherein the strand-displacing polymerase recognizes a single-stranded break in dsDNA, and, in the presence of nucleotide triphosphates, extends the single strand having the break and displaces the ssDNA fragment that is 3' relative to the break, and deoxynucleotide triphosphates (dNTPs) (In another aspect, the present invention provides a method that includes: (a) Forming a mixture of (i) a partially or fully double-stranded template nucleic acid molecule that comprises a nicking agent recognition sequence (NARS), (ii) a nicking agent (NA) that recognizes the NARS, (iii) a DNA polymerase, and (iii) one or more deoxyribonucleoside triphosphates, Pg. 55, Lns. 29-33; The amplification is performed by repetitively nicking the template and then extending from the nicking site to reproduce the template. Thus, the template nucleic acid is first nicked by a NA that recognizes the NARS(s) of the template to thereby produce a 3' terminus at the NS. A DNA polymerase that has a strand displacement ability, but lacks a 5'->3' exonuclease activity, is then used to extend the nicked template from the 3' terminus at the NS, thus displacing the downstream single stranded nucleic acid, Pg. 97, Lns. 5-11; Both the nicking and the extension reaction will work at the same temperature or within the same narrow temperature range, Pg. 103, Lns. 26-28); and (b) subjecting the reaction mixture to the temperature T under which both the nicking nuclease and the strand-displacing polymerase are active, thereby forming the probes ((b) Maintaining the mixture of step (a) under conditions that allows for the amplification of a single-stranded nucleic acid fragment, where under the conditions the single-stranded nucleic acid fragment is capable of spontaneously dissociating from the template nucleic acid in the absence of any strand displacement activity of the DNA polymerase or a strand displacement facilitator, Pg. 55, Ln. 33 - Pg. 56, Ln. 1; Figure 28 is a schematic diagram of major steps in an exemplary method of the present invention for preparing single-stranded nucleic acid probe. Immobilized poly(dT) probe is used to isolate mRNA and to function as a primer for synthesizing cDNA. The synthesized cDNA is then ligated with an exemplary adaptor of the present invention, which comprises a nicking endonuclease recognition sequence (NERS) and a type lls restriction endonuclease recognition sequence (TRERS). The ligated nucleic acid fragment is then digested with a type lls restriction endonuclease and used as the template for synthesizing short single-stranded nucleic acid probes in the presence of a DNA polymerase and a nicking endonuclease that recognizes the NERS in the nucleic acid adaptor, Pg. 66, Lns. 23-32). Van Ness teaches Klenow fragment of DNA polymerase, phi29 polymerase (page 102, top) VAness teaches Bst poymeerase (104, top)
Van Ness fails to explicitly disclose wherein the nicking nuclease incorporates random single-stranded breaks into dsDNA, and wherein the dNTPs comprise one or more the following dNTPs: deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP); deoxycytosine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP).
However, Gerasimova teaches, “The main disadvantage of NEases and REases as enzymes for EATR approaches is their sequence-specificity: both types of enzymes require a specific nucleotide sequence to be present in the probe and its complementary target. It imposes the limitation on the target sequence. To overcome this limitation, the use of sequence-independent nucleases has been suggested.
Examples of such enzymes include exonuclease III, l exonuclease, RNases HI and HII, apurinic/apyrimidinic (AP) endonuclease, duplex-specific nuclease (DSN), DNase I, and T7 exonuclease (Table 2). These enzymes catalyze the cleavage of phosphodiester
bonds in either DNA or RNA. Although the abovementioned nucleases do not require a specific recognition sequence in their substrate, they still display some substrate preference. For example, l exonuclease needs a cleavable DNA strand to have a phosphate group on its 5’-end. AP endonuclease makes a cut at the 5’ of an AP site. Most of the above mentioned nucleases use a double-stranded DNA or DNA–RNA hybrid as a substrate, while DNase I, for example, is active on both single- and double-stranded DNA, as well as on DNA in DNA–RNA hybrids. Among the sequence-independent nucleases, exonuclease III seems to be more commonly used for EATR.” (6418, 1st column-2nd column).
Zhao teaches, “Nucleases can cleave the phosphodiester bonds of nucleic acids and include deoxyribonuclease (DNase), which cleaves DNA, and ribonuclease (RNase), which cleaves RNA, and can be further classified into Exos and Endos. Generally, nuclease assisted strategies involve recycling cleavage of nucleic acids catalyzed by different nucleases, such as flap endonuclease (FEN),88 NEase,89−98 duplex-specific nuclease (DSN),99−104 REases,105,106 Exos,107−117 DNase I,118−121 and RNase H.122 NEase-assisted methods and Exo-assisted ones are widely used, and FEN-based methods (invader or invasive assay) have been commercialized for SNP genotyping and virus detection by Third Wave Technologies.”
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use random or non-specific nickases such as DSN in the enzyme assisted amplification method of Van Ness with bst polymerase. The artisan would be motivated as Gerasimova teaches, “The main disadvantage of NEases and REases as enzymes for EATR approaches is their sequence-specificity: both types of enzymes require a specific nucleotide sequence to be present in the probe and its complementary target. It imposes the limitation on the target sequence. To overcome this limitation, the use of sequence-independent nucleases has been suggested. Examples of such enzymes include exonuclease III, l exonuclease, RNases HI and HII, apurinic/apyrimidinic (AP) endonuclease, duplex-specific nuclease (DSN), DNase I, and T7 exonuclease (Table 2)..” (6418, 1st column-2nd column). The artisan would have a reasonable expectation of as the artisan is merely substituting one nickase for another nickase.
Van Ness discloses further comprising, prior to forming the reaction mixture, forming the input dsDNA from input ssDNA (In some embodiments where the target nucleic acid is a RNA molecule (e.g., mRNA), a DNA polymerase using a RNA molecule (e.g., a reverse transcriptase) as a template may be required. The single-stranded DNA molecule synthesized by the DNA polymerase may be further used as a template to synthesize and/or amplified double-stranded DNA molecules, Pg. 94, Lns. 13-18).
With regards to claim 4, Van Ness teaches, “The single-stranded cDΝA template can then be used for oligonucleotide-directed second strand cDΝA synthesis, PCR, random-primed probe production or gene-expression studies using labeled oligonucleotides.” Van Ness teaches use of Klenow fragment.
With regards to claim 5, Van Ness discloses the method of claim 2. Keck Graduate Institute further discloses wherein forming the input dsDNA comprises: forming a reaction mixture comprising the input ssDNA, Cine or more oligonucleotides that are complementary to part of the input ssDNA; and subjecting the reaction mixture to a temperature and a polymerase under which the oligonucleotides extend to form dsDNA (In some embodiments where the target nucleic acid is a RNA molecule (e.g., mRNA), a DNA polymerase using a RNA molecule (e.g., a reverse transcriptase) as a template may be required. The single-stranded DNA'molecule synthesized by the DNA polymerase may be further used as a template to synthesize and/or amplified double-stranded DNA molecules, Pg. 94, Lns. 13-18; The single-stranded cDNA template can then be used for oligonucleotide- directed second strand cDNA synthesis, PCR, random-primed probe production or gene-expression studies using labeled oligonucleotides, Pg. 209, Lns. 33-35).
With regards to claim 7, Van Ness discloses a method of generating probes from a sample of input single-stranded DNA (ssDNA) (The present invention relates to compounds, kits and methods for ... preparing single-stranded nucleic acid probes, Abstract; In some embodiments where the target nucleic acid is a RNA molecule (e.g., mRNA), a DNA polymerase using a RNA molecule (e.g., a reverse transcriptase) as a template may be required. The single-stranded DNA molecule synthesized by the DNA polymerase may be further used as a template to synthesize and/or amplified double-stranded DNA molecules, Pg. 94, Lns. 13-18), the method comprising: (a) forming a reaction mixture comprising: the input ssDNA, one or more oligonucleotides that are complementary to at least a part of the input ssDNA, wherein the oligonucleotides are capable of extending in the presence of a strand-displacing polymerase to form dsDNA, a nicking nuclease active at a temperature T, wherein the nicking nuclease incorporates random single-stranded breaks into dsDNA, a strand-displacing polymerase active at the temperature T, wherein the stranddisplacing polymerase recognizes a single-stranded break in dsDNA, and, in the presence of nucleotide triphosphates, extends the input ssDNA and/or single strand of dsDNA having the break and displaces the ssDNA fragment that is 3' relative to the break, and deoxynucleotide.triphosphates (dNTPs) (In some embodiments where the target nucleic acid is a RNA molecule (e.g., mRNA), a DNA polymerase using a RNA molecule (e.g., a reverse transcriptase) as a template may be required. The single-stranded DNA molecule synthesized by the DNA polymerase may be further used as a template to synthesize and/or amplified double-stranded DNA molecules, Pg. 94, Lns. 13-18; The single-stranded cDNA template can then be used for oligonucleotide- directed second strand cDNA synthesis, PCR, random-primed probe production or gene-expression studies using labeled oligonucleotides, Pg. 209, Lns. 33-35;; Both the nicking and the extension reaction will work at the same temperature or within the same narrow temperature range, Pg. 103, Lns. 26-28); and (b) subjecting the reaction mixture to the temperature T under which both the nicking nuclease and the strand-displacing polymerase are active, thereby forming the probes ((b) Maintaining the mixture of step (a) under conditions that allows for the amplification of a single-stranded nucleic acid fragment, where under the conditions the single-stranded nucleic acid fragment is capable of spontaneously dissociating from the template nucleic acid in the absence of any strand displacement activity of the DNA polymerase or a strand displacement facilitator, Pg. 55, Ln. 33 - Pg. 56, Ln. 1; Figure 28 is a schematic diagram of major steps in an exemplary method of the present invention for preparing single-stranded nucleic acid probe. Immobilized poly(dT) probe is used to isolate mRNA and to function as a primer for synthesizing cDNA. The synthesized cDNA is then ligated with an exemplary adaptor of the present invention, which comprises a nicking endonuclease recognition sequence (NERS) and a type lls restriction endonuclease recognition sequence (TRERS). The ligated nucleic acid fragment is then digested with a type lls restriction endonuclease and.used as the template for synthesizing short single-stranded nucleic acid probes in the presence of a DNA polymerase and a nicking endonuclease that recognizes the NERS in the nucleic acid adaptor, Pg. 66, Lns. 23-32). Keck Graduate Institute fails to explicitly disclose wherein the nicking nuclease incorporates random single-stranded breaks into dsDNA, and wherein the dNTPs comprise one or more the following dNTPs: deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP); deoxycytosine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP).
With regards to claim 8, Zhao teaches up to 109 amplification.(table 1)
With regards to claim 9, Van Ness teaches , “Z In various embodiments of the invention, this single-stranded nucleic acid fragment has no more than 50, or 45, or 40, or 35, or 30, or 25, or 24, or 23, or 22, or 30 21, or 20, or 19, or 18, or] 7, or 16, or 15, or 14, or 13, or 12, or 11, or 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 nucleotides.” (page 3).
With regards to claim 12, Van Ness teaches characterizing the probes by mass spectroscopy, liquid chromatography or electrophoresis. These methods separate and thus inhibit the nickase and polymerase from the probes and slate probe.
With regards to claim 16, Van Ness teaches Klenow, Phi29
Zhoa and Gerasimova teach the use of DSN and BST DNA polymerase.
They do not specifically teach the concentrations of the claims.
However, MPEP 2144.05 II.A states:
A.Optimization Within Prior Art Conditions or Through Routine Experimentation
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 reAller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to optimize the concentration of the polymerase, nicking enzyme and temperature. The artisan would be motivated to determine the best reaction conditions for the synthesis of probes. The artisan would have a reasonable expectation of success as the artisan is using known reagents with known conditions.
With regards to claim 21, Van Ness teaches ligation of adapters to sequences , followed by washing (page 211). Van Ness teaches combining the probes to a biological sample (claim 12). Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use probes attached to a solid support and hybridizing to target nucleic acids to validate the sequences. The artisan would have a reasonable expectation of success as the artisan is using known method to examine known sequences.
With regards to claim 46,. Zhoa and Gerasimova teach the use of DSN.
With regards to claim 47, Zhoa and Gerasimova teach the use BST DNA polymerase
Response to Arguments
The response traverses the rejection in view of the amendment.
The response continues by citing MPEP 2141(III). The response asserts, “Applicant submits that the skilled person would not have sought to solve the problem of comprehensive isothermal amplification of dsDNA by using a nicking nuclease selected from the group consisting of: double-stranded DNA specific nuclease (DSN), Shrimp-based double strand specific nucleases (dsDNase), HL-dsDNAse, and DNAse; and a strand-displacing polymerase selected from the group consisting of: a Bst DNA polymerase, phi29 polymerase, and Klenow fragment of DNA polymerase over Van Ness in view of the cited documents. Therefore,independent claim 1 is not obvious over Van Ness in view of the additionally cited documents at least in view of the amendments presented herein.” This argument has been thoroughly reviewed but is not considered persuasive in view of the rejection as amended.
Claim(s) 3 is/are rejected under 35 U.S.C. 103 as being unpatentable over Van Ness (WO2003/080645), Zhao (Chem. Rev. 2015, 115, 12491−12545) and Gerasimova ( Chem. Soc. Rev., 2014, 43, 6405-6438) as applied to claims 1-2, 4-5, 8-10, 12, 17, 21, 46-47 above, and further in view of Getts (US20080160581) and Seitz (WU2009/0311754)
Van Ness, Zhoa and Gerasimova fail to explicitly disclose wherein the forming of input dsDNA comprises: forming a reaction mixture comprising; the input ssDNA, deoxynucleotidyl transferase (TdT), wherein the TdT has the ability to add a polyA tail to the 3' end of ssDNA, and poly-dT-primers, wherein the poly-dT primers consist of equal amounts of a polydT-primer with an extra G nucleotide at the 3' end, a poly-dT-primer with an extra C nucleotide at the 3' end, and a poly-dT-primer with an extra A nucleotide at the 3' end; forming a polyA tail on the 3' end of the ssDNA; subjecting the reaction mixture to a temperature under which the poly-dT-primers anneal to the polyA tails on the ssDNA; and subjecting the reaction mixture to a polymerase and to a temperature under which the poly-dT-primers extend to form dsDNA.
However, Getts teaches ligating a polyA tail to a ssDNA using TdT and amplifying ssDNA using ollgo-dT primers (The oligodeoxynucleotide tail can be incorporated by any means that attaches deoxynucleotides to single stranded DNA. Preferably, the oligodeoxynucleotide tail is attached to the single stranded cDNA using terminal deoxynucleotidyl transferase, or other suitable enzyme, in the presence of
appropriate deoxynucleotides. Preferably, the oligodeoxynucleotide tail is a homopolymeric tail (i.e., polydA, ... ), Para. [0020]; Primers for first strand cDNA synthesis include single strand oligodeoxynucleotides comprising an oligodT tail at their 3' ends, Para. (0018]).
It would have been prima facie obvious to one of ordinary skill in the art at the effective filing date of the claims to modify Van Ness, Zhoa and Gerasimova with the teaching of Getts. The artisan would be motivated to ligating a known sequence to the end of a template of unknown sequence using TdT for recognition and amplification by primers complementary to the ligated sequence to create a dsDNA template. The artisan would have a reasonable expectation of success as the artisan is merely combining known molecular biology methods.
Seitz teaches amplifying a single stranded nucleic acid using a poly-dT-primer comprising ex1ra A, C, or G nucleotides on the 3' end (The discrimination of the cDNA into different pools can also be accomplished by using more than one amplification (PCR) step. Each amplification (PCR) step would be more specific to the pool to be amplified. For instance in a first amplification (PCR) a primer such as dTx could be used for hybridizing to cDNA on the side that is complementary to the poly A tail side of the RNA. This primer would be not discriminating. In a next step, 3 reactions could be used with one being primed with dTxG, one with dTxA and one with dTxC. Thus creating 3 pools, Para. (0086]).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to modify Van Ness, Zhoa and Gerasimova with the teaching of Seitz. The artisan would be motivated to generate dsDNA using primers non-specific to the target sequence by hybridizing to the poly A tail attached to the target sequence. The artisan would have a reasonable expectation of success as the artisan is merely combining known molecular biology methods.
Response to Arguments
The response traverses the rejection for the reasons set forth with respect to the independent claim. These arguments are not persuasive for the reasons of record.
Claim(s) 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Van Ness (WO2003/080645), Zhao (Chem. Rev. 2015, 115, 12491−12545) and Gerasimova ( Chem. Soc. Rev., 2014, 43, 6405-6438) as applied to claims 1-2, 4-5, 8-10, 12, 17, 21, 46-47 above, and further in view of MOk (Nature Scientific reports ((2016) 6:37837 | DOI: 10.1038/srep37837)
Van Ness, Zhoa and Gerasimova fail to explicitly disclose subjecting a mixture at a temperature for 4-5 minutes.
However, Mok teaches a method of isothermal exponential amplification with provides for maximal amplification for sample with target in less than 5 minutes (figure 2c).
However, MPEP 2144.05 II.A states:
A. Optimization Within Prior Art Conditions or Through Routine Experimentation
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 reAller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to subject the reaction mixture to 4-5 at a temperature. The artisan would be motivated as Mok suggests at most concentrations of target sequence 4-5 minutes provide maximum signal. The artisan would have a reasonable expectation of success as the artisan is merely using known reactions times for a known reaction.
Response to Arguments
The response traverses the rejection for the reasons set forth with respect to the independent claim. These arguments are not persuasive for the reasons of record.
Summary
No claims are allowed.
Conclusion
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 STEVEN C POHNERT PhD whose telephone number is (571)272-3803. The examiner can normally be reached Monday- Friday about 6:00 AM-5:00 PM, every second Friday off.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Anne Gussow can be reached at (571)272-6047. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Steven Pohnert/ Primary Examiner, Art Unit 1683
Prosecution Insights