DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. This Application has been transferred to Examiner Haney in Art Unit 1682.
3. Applicant’s election without traverse of Group I in the reply filed on March 16, 2026 is acknowledged.
Claims 1-2, 6, 9, 11, 14, 16, 21, 24-27, 34-39, and 47-48 are currently pending.
Claims 47-48 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on March 16, 2026.
Claim Rejections - 35 USC § 112
4. 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 14, 24, and 34-39 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 14 recites the limitation “the one or more lytic reagents”. There is insufficient antecedent basis for this limitation in the claim because while the claims previously refer to “one or more lytic agents”, they do not previously refer to “one or more lytic reagents”.
Claim 24 recites the limitation “the enzyme having a hyperthermophile polymerase activity”. There is insufficient antecedent basis for this limitation in the claim because the claims do not previously refer to a “enzyme having a hyperthermophile polymerase activity”.
Claims 34-37 are rejected over the recitation of the phrase “the one or more lytic agents are capable of denaturing” the ribonucleases/deoxyribonucleases. Capability is a latent characteristic and the claims do not set forth the criteria by which to determine capability. That is, it is not clear as to whether the lytic agents do in fact denature the ribonucleases/deoxyribonucleases or if they only have this ability under some under some unspecified conditions.
Claims 34-37 are rejected over the recitation of the phrase “the one or more reducing agents are capable of renaturing” the ribonucleases/deoxyribonucleases. Capability is a latent characteristic and the claims do not set forth the criteria by which to determine capability. That is, it is not clear as to whether the reducing agents do in fact renature the ribonucleases/deoxyribonucleases or if they only have this ability under some under some unspecified conditions.
Claim 36 is rejected over the recitation of the phrase “wherein renatured ribonucleases and/or renatured deoxyribonucleases are enzymatically active”. This phrase lacks antecedent basis and is confusing because claims 1, 2, and 34 do not refer to renatured ribonucleases or deoxyribonucleases.
Claim 38 recites the limitation “the reverse transcriptase”. There is insufficient antecedent basis for this limitation in the claim because the claims do not previously refer to a reverse transcriptase.
Claim 38 is rejected over the recitation of the phrase “about 1.1-fold more sample ribonucleic acids are employed by the reverse transcriptase as template”. “Employed” is not an art recognized term in this context. Because the term “employed” has not been clearly defined in the specification and because there is no art recognized definition for this term as it relates to RNA and reverse transcriptase, one of skill in the art cannot determine the meets and bounds of the claimed subject matter.
Claim 38 is rejected over the recitation of the phrase “about 1.1-fold more sample deoxyribonucleic acids are employed by enzyme having a hyperthermophile polymerase activity as template”. “Employed” is not an art recognized term in this context. Because the term “employed” has not been clearly defined in the specification and because there is no art recognized definition for this term as it relates to DNA and hyperthermophile polymerase activity, one of skill in the art cannot determine the meets and bounds of the claimed subject matter.
Claims 38 and 39 are rejected over the recitation of the phrase “a comparable method”. This phrase in considered indefinite because “a comparable method” is not defined by the specification and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. In other words it is not clear which methods would be considered “comparable” and which ones would not.
Claims 38 and 39 are rejected over the recitation of the phrase “a comparable method wherein the lysis buffer does not comprise one or more reducing agents” in claims 38 and 39. It is unclear if the claim requires comparing two different methods which both use lysis buffers without reducing agents OR if the claim requires comparing two different methods wherein one uses a lysis buffer with a reducing agent and the other uses a lysis buffer without a reducing agent. If it is the later, then the claims are confusing because claims 38 and 39 both depend from claim 1 and claim 1 does not require a lysis buffer that comprises a reducing agent. Clarification is required.
Claim 39 is rejected over the recitation of the phrase “wherein the amplification reaction mixture comprises an at least 1.1-fold lower nuclease activity ten minutes after step (b) and/or step (c)”. This recitation is confusing because claim 39 depends from claim 1 and claim 1 does not require that the amplification reaction mixture comprises a nuclease. Clarification is required.
Claim Rejections - 35 USC § 103
5. 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.
6. Claims 1, 2, 6, 9, 11, 14, 16, 21, 24-27, 38 are rejected under 35 U.S.C. 103 as being unpatentable over Miller (US 9,617,587 Issued 4/11/2017) in view of Nakamura (US 2014/0322761 Pub 10/30/2014).
Regarding Claim 1 Miller teaches a method for isothermal amplification of nucleic acids (abstract). Miller teaches performing cell lysis. Miller teaches that chemical methods generally employ lysing agents to disrupt cells and extract nucleic acids from the cells, followed by treatment with chaotropic salts (col 8 lines 38-47). Miller teaches an approach for real time detection of Chlamydia genomic DNA using a molecular beacon for detection (Example 1). A master mixes comprising a polymerase, dNTPs and a molecular beacon probe was prepared. A primer set targeting a specific sequence within the 7,500 base pair C. trachomatis cryptic plasmid DNA was used, which included a 10 nucleotide forward primer (5′-AGGCTTATGG-3′ (SEQ ID NO:5)) and a 10 nucleotide reverse primer (5′-TTATACCGCT-3′ (SEQ ID NO:6)). The primers (i.e., 750 nM forward primer and 200 nM reverse primer) were combined with either TE as a NTC or 20,000 copies of chlamydia genomic DNA in reaction wells. All components were incubated at 65° C. for 2 minutes, then combined to initiate the isothermal reaction carried out at 65° C. The reaction products were detected at various time points by real-time fluorescence readouts of the molecular beacon, as shown in FIG. 4 (col 32, lines 14-50). Miller further teaches that the amplification process is conducted over a length of time within about 20 minutes or less (col 13, lines 15-32). Thus Miller teaches a method for detecting a target nucleic acid sequence in a sample, comprising: (a) contacting a sample comprising biological entities (cells) with a lysis buffer to generate a treated sample, wherein the lysis buffer comprises one or more lytic agents capable of lysing the biological entities to release sample nucleic acids comprised therein, and wherein the sample nucleic acids are suspected of comprising a target nucleic acid sequence; (b) contacting a reagent composition with the treated sample to generate an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents (polymerase, dNTPs, molecular beacon, primers); (c) amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby generating a nucleic acid amplification product; and (d) detecting the nucleic acid amplification product, wherein the detecting is performed in less than about 20 minutes from the time the reagent composition is contacted with the treated sample.
Regarding Claim 2 Miller teaches that components of an amplification reaction may include, for example, one or more primers, nucleic acid targets, one or more polymerases, nucleotides, and a suitable buffer (e.g., a buffer comprising a detergent, a reducing agent, monovalent ions, and divalent ions) (col 10 lines 43-50). Thus Miller teaches a method wherein the reagent composition comprises a reducing agent.
Regarding Claim 6 Miller teaches that components of an amplification reaction may include, for example, one or more primers, nucleic acid targets, one or more polymerases, nucleotides, and a suitable buffer (e.g., a buffer comprising a detergent, a reducing agent, monovalent ions, and divalent ions). An amplification reaction may further include a reverse transcriptase, in some embodiments. (col 10 lines 43-52). Additionally Miller teaches that the polymerase may be a hyperthermophile DNA polymerase (col 22, lines 36-37). Thus Miller teaches a method wherein the one or more amplification reagents comprise a reverse transcriptase and/or an enzyme having a hyperthermophile polymerase activity.
Regarding Claim 9 Miller teaches that the enzymes (e.g., polymerase(s) and/or reverse transcriptase(s)) may be provided in a separate container from the primers. The components may, for example, be lyophilized, freeze dried, or in a stable buffer. In one example, polymerase(s) and/or reverse transcriptase(s) are in lyophilized form in a single container, and the primers are either lyophilized, freeze dried, or in buffer, in a different container. In some embodiments, polymerase(s) and/or reverse transcriptase(s), and the primers are, in lyophilized form, in a single container (col 30, lines 9-15). Thus Miller teaches a method wherein the reagent composition is lyophilized, heat-dried, and/or comprises one or more additives.
Regarding Claim 14 Miller teaches that cell lysis procedures and reagents are known in the art and may generally be performed by chemical (e.g., detergent, hypotonic solutions, enzymatic procedures, and the like, or combination thereof), physical (e.g., French press, sonication, and the like), or electrolytic lysis methods. Any suitable lysis procedure can be utilized (col 8, lines 38-43). Thus Miller teaches a method wherein the one or more lytic reagents comprise a detergent.
Regarding Claim 16 Miller teaches isothermal amplification of Chlamydia genomic DNA with 9 Degrees North polymerase. Miller teaches a primer set targeting a specific sequence within the 7,500 base pair C. trachomatis cryptic plasmid DNA was used, which included a 10 nucleotide forward primer (5′-AGGCTTATGG-3′ (SEQ ID NO:5)) and a 10 nucleotide reverse primer (5′-TTATACCGCT-3′ (SEQ ID NO:6)). The assay was designed to generate a 25 base DNA product, which included a 5 base spacer. The spacer includes 5 nucleotides between the 3′ ends of each primer, and these 5 nucleotides are not present in either of the primer sequences (col 32 lines 14-43). Thus Miller teaches a method comprising amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other in an isothermal amplification condition, wherein the amplifying comprises contacting a nucleic acid comprising the target nucleic acid sequence with: i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the second primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, wherein the nucleic acid amplification product comprises: (1) the sequence of the first primer, and the reverse complement thereof, (2) the sequence of the second primer, and the reverse complement thereof, and (3) a spacer sequence flanked by (1) the sequence of the first primer and the reverse complement thereof and (2) the sequence of the second primer and the reverse complement thereof, wherein the spacer sequence is 1 to 10 bases long.
Regarding Claim 21 Miller further teaches a method wherein the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA may be synthesized prior to or during the amplification step by reverse transcription (col 10, lines 39-42). Thus Miller teaches a method comprising (c1) contacting sample ribonucleic acids with a reverse transcriptase and/or a reverse transcription primer to generate a cDNA; (c2) contacting the cDNA with an enzyme having a hyperthermophile polymerase activity to generate a double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, and wherein the target nucleic acid sequence comprises a first strand and a second strand complementary to each other; (c3) amplifying the target nucleic acid sequence under an isothermal amplification condition, wherein the amplifying comprises contacting the dsDNA with: (i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the second primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and (ii) the enzyme having a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, wherein the nucleic acid amplification product comprises: (1) the sequence of the first primer, and the reverse complement thereof, (2) the sequence of the second primer, and the reverse complement thereof, and (3) a spacer sequence flanked by (1) the sequence of the first primer and the reverse complement thereof and (2) the sequence of the second primer and the reverse complement thereof, wherein the spacer sequence is 1 to 10 bases long.
Regarding Claim 24 Miller teaches a method wherein the amplification reaction components comprise a hyperthermophile DNA polymerase comprising an amino acid sequence of SEQ ID NO: 8 or a functional fragment of SEQ ID NO:8 (col 23 lines 20-22). It is noted for the record that SEQ ID NO: 8 is 100% identical to SEQ ID NO: 1 of the instant application. Thus Miller teaches a method wherein the enzyme having a hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof.
Regarding Claim 25 Miller teaches an all components were incubated at 65° C. for 2 minutes, then combined to initiate the isothermal reaction carried out at 65° C (col 32, lines 14-50). Thus Miller teaches a method wherein amplifying the target nucleic acid sequence is performed at a constant temperature of about 55 °C to about 75 °C, or at a constant temperature of about 65 °C.
Regarding Claim 26 Miller teaches isothermal amplification of Chlamydia genomic DNA with 9 Degrees North polymerase. Miller teaches a primer set targeting a specific sequence within the 7,500 base pair C. trachomatis cryptic plasmid DNA was used, which included a 10 nucleotide forward primer (5′-AGGCTTATGG-3′ (SEQ ID NO:5)) and a 10 nucleotide reverse primer (5′-TTATACCGCT-3′ (SEQ ID NO:6)). The assay was designed to generate a 25 base DNA product, which included a 5 base spacer. The spacer includes 5 nucleotides between the 3′ ends of each primer, and these 5 nucleotides are not present in either of the primer sequences (col 32 lines 14-43). Thus Miller teaches a method wherein the first and second primer are about 8 to 17 bases long; the nucleic acid amplification product is about 20 to 40 bases long; and/or the spacer sequence comprises a portion of the target nucleic acid sequence.
Regarding Claim 27 Miller teaches that another approach for real-time detection of chlamydia genomic DNA is to use molecular beacons for detection. Miller discloses a molecular beacon (Fam-ccgcgagccttATACCGCTTAACTCg*c*g*g-IBFQ (SEQ ID NO:7)) which contained a 14-base sequence complementary to a portion of the forward product (14-base sequence is shown in upper case lettering) (col 32, lines 16-32). Thus Miller teaches a method
further comprising contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the amplification product, wherein the signal- generating oligonucleotide comprises a fluorophore, a quencher, or both.
Miller does not teach a method wherein the reagent composition further comprises a protectant (clm 1). Miller does not teach a method wherein the one or more protectants comprises a cyclodextrin compound of formula (I) (clm 11).
However Nakamura teaches a method of preparing a sample for nucleic acid amplification reaction, including a procedure of dissolving a solid phase reagent at least containing a DNA polymerase, cyclodextrin, and a binder, in a liquid containing a nucleic acid (abstract). Nakamura teaches that the cyclodextrin contained in the solid phase reagent is a component for suppressing deterioration of activity of an enzyme such as the DNA polymerase contained in the solid phase reagent (refer to Example 1). The solid phase reagent is prepared such that a reagent is made into a predetermined component and the predetermined component is subsequently dried or freeze-dried. There is a concern that an enzyme contained in the solid phase reagent is deactivated depending on such a preparation process or a dried state after the preparation process. In the solid phase reagent according to an embodiment of the present technology, it is possible to suppress the deterioration of the activity of the enzyme using the reagent containing the cyclodextrin (refer to Example 1) (para 0034). Nakamura further teaches that cyclodextrin also has an effect of suppressing inhibition of nucleic acid amplification reaction of an ionic surfactant contained in a liquid containing a nucleic acid to be described (refer to Example 2) (para 0035). Additionally Nakamura discloses numerous cyclodextrins and teaches that it is preferable that the cyclodextrin have the cyclodextrin group, for example, hydroxyprophyl-β-cyclodextrin (HPβCD). Since HPβCD has higher water solubility compared to the β-cyclodextrin, it is easy to add a sufficient amount of cyclodextrin to the solid phase reagent in order to obtain an effect with respect to the ionic surfactant to be described.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Miller by adding a protectant, such as hydroxyprophyl-β-cyclodextrin (HPβCD) to the reagent composition as suggested by Nakamura. One of skill in the art would have been motivated to add HPβCD to a reagent composition comprising a polymerase particularly since Nakamura teaches that cyclodextrin suppresses deterioration of DNA polymerase activity and suppresses inhibition of nucleic acid amplification reactions in the presence of ionic surfactants.
Regarding Claim 38 the combined references do not teach a method wherein: about 1.1-fold more sample ribonucleic acids are employed by the reverse transcriptase as template to generate cDNA as compared to a comparable method wherein the lysis buffer does not comprise one or more reducing agents; and/or about 1.1-fold more sample deoxyribonucleic acids are employed by enzyme having a hyperthermophile polymerase activity as template to generate a nucleic acid amplification product to a comparable method wherein the lysis buffer does not comprise one or more reducing agents (clm 38). However the claims are included in this rejection because claim scope is not limited by claim language (such as wherein clauses) that suggests or makes optional but does not require steps to be performed. In the instant case the recited “wherein” clause does not modify any of the active process steps that are recited in claim 1 and does not limit the claims.
7. Claim 34 is rejected under 35 U.S.C. 103 as being unpatentable over Miller (US 9,617,587 Issued 4/11/2017) in view of Nakamura (US 2014/0322761 Pub 10/30/2014) as applied to claims 1 and 2 above and in further view of Gerdes (US 2016/0289735 Pub 10/6/2016). The rejection is further evidenced by Hollis (US 2005/0277204 Pub 12/15/2005) and Pasloske (US 6,777,210 Issued 8/17/2004).
The teachings of Miller and Nakamura are presented above.
The combined references do not teach a method wherein the sample comprises a plurality of ribonucleases, and wherein: the one or more lytic agents are capable of denaturing the ribonucleases to generate denatured ribonucleases, wherein the one or more reducing agents are capable of reducing the ribonucleases to generate reduced ribonucleases, and wherein the one or more reducing agents and the one or more lytic agents are capable of generating reduced- denatured ribonucleases.
However Gerdes teaches that the addition of DTT to a lysis buffer containing SDS showed rapid lysis of sperm cells in less than 5 min (para 0068). It is noted for the record that DTT is a reducing agent and SDS is a denaturing agent. As evidenced by the prior art of Pasloske, DTT inactivates RNases (col 6, lines 62-65). As evidenced by the prior art of Hollis SDS is a detergent that denatures proteins, including Rnases (para 0475).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Miller and Nakamura by using a lysis reagent comprising DTT and SDS as suggested by Gerdes. One of skill in the art would have been motivated to use the lysis reagent of Gerdes since the reference teaches that the addition of DTT to a lysis buffer containing SDS showed rapid lysis of sperm cells in less than 5 min (para 0068). As discussed above such a lysis buffer would be expected to result in the generation of reduced-denatured ribonucleases.
8. Claims 35-37 are rejected under 35 U.S.C. 103 as being unpatentable over Miller (US 9,617,587 Issued 4/11/2017) in view of Nakamura (US 2014/0322761 Pub 10/30/2014) and Gerdes (US 2016/0289735 Pub 10/6/2016) as applied to claims 1, 2, and 34 above and in further view of Huang (US 2005/0048486 Pub 3/3/2005). The rejection of claim 34 was further evidenced by Hollis (US 2005/0277204 Pub 12/15/2005) and Pasloske (US 6,777,210 Issued 8/17/2004). The rejection of claim 36 is further evidenced by Gibbons (US 2006/0002936 Pub 1/5/2006).
The teachings of Miller, Naikamura, and Gerdes are presented above.
The combined references do not teach a method wherein the denatured ribonucleases are capable of renaturing upon passage of time and/or upon reduced physical interaction with the one or more lytic agents, thereby generating renatured ribonucleases (claim 35). The combined references do not teach a method wherein the denatured ribonucleases are enzymatically inactive, and wherein renatured ribonucleases are enzymatically active (clm 36). The combined references do not teach a method wherein reduced-denatured ribonucleases renature at least about 1.1-fold more slowly than denatured ribonucleases (clm 37).
However Huang teaches that some Rnases renature upon cooling (a phenomenon called reversible thermal denaturation) (para 0004). Thus Huang teaches a method wherein denatured ribonucleases can renature upon passage of time (cooling). As evidenced by the prior art of Gibbons it is a property of denatured ribonucleases that they are enzymatically inactive and then when renatured become enzymatically active again (para 0080).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Miller, Naikamura, and Gerdes by renaturing the denatured ribonuclease as suggested by Huang. One of skill in the art would have been motivated to renature a nuclease for the benefit of recovering its enzymatic activity. Further based on the prior art of record there was a reasonable expectation that denatured ribonucleases would renature faster than those that are denatured and reduced since they only have one modification (denatured) opposed to having two modifications (reduced and denatured).
9. Claim 39 is rejected under 35 U.S.C. 103 as being unpatentable over Miller (US 9,617,587 Issued 4/11/2017) in view of Nakamura (US 2014/0322761 Pub 10/30/2014) as applied to claim 1 above and in further view of Pasloske (US 6,777,210 Issued 8/17/2004).
The teachings of Miller and Nakamura are presented above.
The combined references do not teach a method wherein the amplification reaction mixture comprises an at least 1.1-fold lower nuclease activity ten minutes after step (b) and/or step (c) as compared to a comparable method wherein the lysis buffer does not comprise one or more reducing agents.
However Pasloske discloses a method for irreversibly inactivating ribonucleases. The methods comprise the steps of obtaining a sample, obtaining a reducing agent, admixing the reducing reagent and sample, and heating (col 3, lines 59-64). Pasloske teaches that to effectively inactivate Rnases, the preferred method is to add DTT to a final concentration of 20 mM to the sample and then incubate at 60ºC for 8 min (col 6, lines 62-65).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Miller and Nakamura by including a reducing reagent in an amplification reaction. One of skill in the art would have been motivated to include a reducing reagent in an amplification reaction for the benefit of being able to inactivate any RNase as taught by Pasloske.
10. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Shen can be reached at 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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