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 11/3/2025 and 11/10/2025.
It is noted 37 CFR1.11 states, “(2) Supplemental replies.
(i) A reply that is supplemental to a reply that is in compliance with § 1.111(b) will not be entered as a matter of right except as provided in paragraph (a)(2)(ii) of this section. “ However in order to promote compact prosecution and customer service the instant response will be examined however, future supplemental amendments may not be entered and/or examined.
Claims 1, 6, 9 have been amended.
Applicant's election with traverse of Group I, 1) the chemical modification is m6A modification; 2) the DNA polymerase is 7th DNA polymerase; 3) the ligase is SplintR ligase; 4) the threshold cycle of PCR reference is a threshold cycle of first PCR reference; 5) when the threshold cycle of PCR is more than the threshold cycle of first PCR reference, it is determined that the target chemical modification is present in the RNA target site X; 6) the amount of PCR amplification product reference is an amount of first PCR amplification product reference; 7) when the amount of PCR amplification product is less than the amount of first PCR amplification product reference, it is determined that the target chemical modification is present in the RNA target site X;
8) the first reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II comprises a nucleotide sequence sharing a same nucleotide sequence with a nucleotide sequence | in the target RNA segment, wherein: when the site N is located upstream of the site X, the nucleotide sequence | is a nucleotide sequence from a nucleotide that is complementary paired with a nucleotide of 3'- terminal of the up probe Pn1 of the site N to a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Px2 of the site X in the target RNA segment; when the site N is located downstream of the site X, the nucleotide sequence | is a nucleotide sequence from a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X to a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Pnz2 of the site N in the target RNA segment; and no target chemical modification is present in an RNA ) treating the RNA sample with an RNA demodification enzyme; and 10) the RNA sample is total RNA extracted from cells, with traverse. in the reply filed on 2/28/2024 s acknowledged. The traversal is on the ground(s) that The reasons are as follows. Mullis (US4683195) discloses a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof, and does not involve the single-gene single-base resolution ratio detection method for RNA chemical modification of the present application. This is not found persuasive because This argument has been thoroughly reviewed but is not considered persuasive as all the claims require PCR amplification. Claim 14 requires an RNA demodification enzyme, while 16requies gradient dilution. Further, Stewart (Nucleic Acids Research, 1998, Vol. 26, No. 4 961–966) and Khodakov (Advanced Drug Delivery Reviews 105 (2016) 3–19) teach the use of two probes, elongation and ligation with PCR was known. Thus the other sets are different and lack unity of invention.
Claims 14-17 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 2/29/2024.
Claims 1, 6, 9-12, 18-20 are being examined.
The previous objection to the claims has been with drawn in view of the amendment to the claims.
The new matter rejection has been withdrawn in view of the amendment.
Priority
The instant application was filed 03/30/2021 is a National Stage entry of PCT/CN2018/109145 with an international filing date: 09/30/2018.
Specification
The amendment filed 1/31/2025 is objected to under 35 U.S.C. 132(a) because it introduces new matter into the disclosure. 35 U.S.C. 132(a) states that no amendment shall introduce new matter into the disclosure of the invention. The added material which is not supported by the original disclosure is as follows:
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Further the response does not indicate what supports the amendment with respect to PBCV-1 DNA ligase.
Applicant is required to cancel the new matter in the reply to this Office Action.
Response to Arguments
The response traverses the objection in view of Evidence 1, herewith. This argument has been thoroughly reviewed but the instant response does not appear to have anything labeled Evidence 1.
Claim Objections
Claims 1, 6, 9-12, 18-20 are objected to because of the following informalities:
Claim 1 recites, ”the target chemical modification.” The claim does not previously recite this, but recite, “chemical modification.” Claims are clearer and more concise when using the same terminology throughout.
Claim 1 is objected to as it recites “nt” but does not recite the full terminology for the acronym (or abbreviation). Claims are more concise when the first time an acronym (or abbreviation) is presented the full terminology is also presented. Finally an acronym (or abbreviation) may have alternative meanings to an artisan.
. Appropriate correction is required.
Response to Arguments
This is a new ground of objection.
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, 6, 9-12, 18-20 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 1 is indefinite because it lacks a positive active step relating back to the preamble. The preamble recites a method for detecting a chemical modification of an RNA target site X, however the last positive active step is drawn to comparing the threshold cycle of PCR of the target RNA segment to a threshold cycle of PCR reference, or comparing the amount of PCR amplification product to an amount of PCR amplification product reference. Therefore it is unclear as to whether the method is drawn to detecting a chemical modification of an RNA target site X or comparing the threshold cycle of PCR of the target RNA segment to a threshold cycle of PCR reference, or comparing the amount of PCR amplification product to an amount of PCR amplification product reference. Further it is unclear how the comparing provides for determining presence of a chemical modification.
Claim 1 recites, “wherein the second reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II comprises a nucleotide sequence sharing a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein the nucleotide sequence I is a nucleotide sequence in the RNA target segment that starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Px2 of the site X, and the target chemical modification is present in an RNA target site X2 of the second reference sequence corresponding to the RNA target site X of the target RNA segment; or wherein, in step (4), the amount of PCR amplification product reference is an amount of first PCR amplification product reference or an amount of PCR second amplification product reference, wherein when the amount of PCR amplification product is less than the amount of first PCR amplification product reference, it is determined that the target chemical modification is present in the RNA target site X; or when the amount of PCR amplification product is equal to the amount of second PCR amplification product reference, it is determined that the target chemical modification is present in the RNA target site X, wherein: the amount of first PCR amplification product reference is: an amount of PCR amplification product of a first reference sequence determined by a method as same as the method for determining the amount of PCR amplification product of the target RNA segment, wherein the first reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein: the nucleotide sequence I in the RNA target segment starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide at 5'-terminal of the down probe Px2 of the site X, and no target chemical modification is present in an RNA target site X1 of the first reference sequence corresponding to the RNA target site X of the target RNA segment; or wherein, the amount of second PCR amplification product reference is: an amount of PCR amplification product of a second reference sequence determined by a method as same as the method for determining the amount of PCR amplification product of the target RNA segment, wherein the second reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II comprises a nucleotide sequence sharing a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein the nucleotide sequence I is a nucleotide sequence in the RNA target segment that starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Px2 of the site X, and the target chemical modification is present in an RNA target site X2 of the second reference sequence corresponding to the RNA target site X of the target RNA segment..” The recitation is vague, confusing and unclear. It is unclear as the claim says a method as same as RNA target segment . It then provides limitations with respect to a nucleotide sequence II shares a nucleotide sequence and continues further recitations of “a nucleotide sequence.”. The use of the indefinite article suggests the claim does not require the full sequence. Thus the metes and bounds are unclear what is required.
Response to arguments
The response traverses the rejections in view of the amendments. This is noted as rejections not reproduced above have been withdrawn.
It is noted the amendment to add further wherein clauses does not overcome the issue with the last active step of the claim being comparing.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1, 5-6, 8-12, 18-20 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a mental step without significantly more. The claim(s) recite(s) the abstract idea or mental step of comparing. The judicial exception is not integrated into a practical application because there are no steps which depend from or otherwise integrate. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because if there is no deletion or an amplification or unchanged expression or increased expression no treatment step is required.
Claim analysis
The instant claim 1 is directed towards A method for detecting a chemical modification of an RNA target site X, comprising: (1) obtaining an RNA sample, wherein a target RNA segment containing an RNA target site is in the RNA sample, and wherein the RNA sample is total RNA, mRNA, rRNA, or long non-coding RNA (IncRNA) extracted from cells; (2) a single-base elongation- and ligation-based PCR amplification (SELECT) step: elongating a down probe Px2 designed for a downstream sequence of the RNA target site X within the target RNA segment with a DNA polymerase to obtain an elongated down probe Px2, and ligating an up probe Px1 designed for an upstream sequence of the RNA target site X within the target RNA segment and the elongated down probe Px2 with a ligase to obtain a SELECT product; wherein the DNA polymerase isa Bst DNA polymerase; and the ligase is selected from the group consisting of PBCV-1 DNA ligase, T3 DNA ligase, T4 RNA ligase 2, and T4 DNA ligase; wherein, the up probe Px1 is complementary paired with the upstream sequence of the RNA target site X, and the first nucleotide of 5'-terminal of the up probe Px1 is complementary paired with a nucleotide located at a site with a distance of 1 nt from the RNA target site X at the upstream sequence of the RNA target site X; the down probe Px2 is complementary paired with the downstream sequence of the RNA target site X, and the first nucleotide of 3'-terminal of the down probe Px2 is complementary paired with a nucleotide located at a site with a distance of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nt from the RNA target site X at the downstream sequence of the RNA target site X; (3) PCR amplification step: performing PCR amplification of the SELECT product obtained in step (2), determining a threshold cycle of PCR of the target RNA segment or an amount of PCR amplification product, wherein said PCR is qPCR; and (4) comparing the threshold cycle of PCR of the target RNA segment to a threshold cycle of PCR reference, or comparing the amount of PCR amplification product to an amount of PCR amplification product reference, to determine if the target chemical modification is present at the RNA target site Xi wherein the chemical modification is N6-methyladenosine modification; and wherein, in step (4), the threshold cycle of PCR reference is a threshold cycle of first PCR reference or a threshold cycle of second PCR reference, wherein when the threshold cycle of PCR of the target RNA segment is more than the threshold cycle of first PCR reference, it is determined that the target chemical modification is present in the RNA target site X; or when the threshold cycle of PCR of the target RNA segment is equal to the threshold cycle of second PCR reference, it is determined that the target chemical modification is present in the RNA target site X, wherein: the threshold cycle of first PCR reference is: a threshold cycle of PCR of a first reference sequence determined by a method as same as the method for determining the threshold cycle of PCR of the target RNA segment, wherein the first reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein: the nucleotide sequence I in the RNA target segment starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Px2 of the site X, and no target chemical modification is present in an RNA target site X1 of the first reference sequence corresponding to the RNA target site X of the target RNA segment; or the threshold cycle of second PCR reference is: a threshold cycle of PCR of a second reference sequence determined by a method as same as the method for determining the threshold cycle of PCR of the target RNA segment, wherein the second reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein the nucleotide sequence Ireference, it is determined that the target chemical modification is present in the RNA target site X, wherein: the amount of first PCR amplification product reference is:an amount of PCR amplification product of a first reference sequence determined by a method as same as the method for determining the amount of PCR amplification product of the target RNA segment, wherein the first reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein: the nucleotide sequence I in the RNA target segment starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide at 5'-terminal of the down probe Px2 of the site X, and no target chemical modification is present in an RNA target site X1 of the first reference sequence corresponding to the RNA target site X of the target RNA segment; or wherein, the amount of second PCR amplification product reference is: an amount of PCR amplification product of a second reference sequence determined by a method as same as the method for determining the amount of PCR amplification product of the target RNA segment, wherein the second reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein the nucleotide sequence I in the RNA target segment starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Px2 of the site X, and the target chemical modification is present in an RNA target site X2 of the second reference sequence corresponding to the RNA target site X of the target RNA segment... The selecting and comparing steps are a mental step or abstract idea.
The obtaining an RNA sample, elongating and ligating and PCR amplification are considered positive active steps.
Dependent claims set forth further limitations to about the reference level, chemical modifications, ligase and polymerase.
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case the Step 1 requirement is satisfied as the claims are directed towards a process.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract idea.
With regards to claim 1, the claim recites, “comparing the threshold cycle of PCR of the target RNA segment to a threshold cycle of PCR reference, or comparing the amount of PCR amplification product to an amount of PCR amplification product reference, to determine if the target chemical modification is present at the RNA target site Xi wherein the chemical modification is N6-methyladenosine modification; and wherein, in step (4), the threshold cycle of PCR reference is a threshold cycle of first PCR reference or a threshold cycle of second PCR reference, wherein when the threshold cycle of PCR of the target RNA segment is more than the threshold cycle of first PCR reference, it is determined that the target chemical modification is present in the RNA target site X; or when the threshold cycle of PCR of the target RNA segment is equal to the threshold cycle of second PCR reference, it is determined that the target chemical modification is present in the RNA target site X, wherein: the threshold cycle of first PCR reference is: a threshold cycle of PCR of a first reference sequence determined by a method as same as the method for determining the threshold cycle of PCR of the target RNA segment, wherein the first reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein: the nucleotide sequence I in the RNA target segment starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Px2 of the site X, and no target chemical modification is present in an RNA target site X1 of the first reference sequence corresponding to the RNA target site X of the target RNA segment; or the threshold cycle of second PCR reference is: a threshold cycle of PCR of a second reference sequence determined by a method as same as the method for determining the threshold cycle of PCR of the target RNA segment, wherein the second reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein the nucleotide sequence IRNA segment; or wherein, in step (4), the amount of PCR amplification product reference is an amount of first PCR amplification product reference or an amount of PCR second amplification product reference, wherein when the amount of PCR amplification product is less than the amount of first PCR amplification product reference, it is determined that the target chemical modification is present in the RNA target site X; or when the amount of PCR amplification product is equal to the amount of second PCR amplification product reference, it is determined that the target chemical modification is present in the RNA target site X, wherein:the amount of first PCR amplification product reference is:an amount of PCR amplification product of a first reference sequence determined by a method as same as the method for determining the amount of PCR amplification product of the target RNA segment, wherein the first reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein: the nucleotide sequence I in the RNA target segment starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide at 5'-terminal of the down probe Px2 of the site X, and no target chemical modification is present in an RNA target site X1 of the first reference sequence corresponding to the RNA target site X of the target RNA segment; or wherein, the amount of second PCR amplification product reference is: an amount of PCR amplification product of a second reference sequence determined by a method as same as the method for determining the amount of PCR amplification product of the target RNA segment, wherein the second reference sequence comprises at least a nucleotide sequence II, and the nucleotide sequence II shares a same nucleotide sequence with a nucleotide sequence I in the target RNA segment, wherein the nucleotide sequence I in the RNA target segment starts with a nucleotide that is complementary paired with a nucleotide of 3'-terminal of the up probe Px1 of the site X and ends with a nucleotide that is complementary paired with a nucleotide of 5'-terminal of the down probe Px2 of the site X, and the target chemical modification is present in an RNA target site X2 of the second reference sequence corresponding to the RNA target site X of the target RNA segment... The selecting and comparing steps are a mental step or abstract idea.” This is an abstract idea or mental step. (UNIVERSITY OF UTAH RESEARCH v. AMBRY GENETICS CORPORATION). The broadest reasonable interpretation of the wherein clauses is they are mental steps.
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? The answer is no as there are no steps which depend from or otherwise integrate the judicial exception.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? No, the claim appears to merely provide routine conventional steps of obtaining RNA, elongating, ligating and PCR amplifying.
According to the manufacturer's instructions, total RNA was extracted with TRIzol reagent (purchased from ThermoFisher Scientific) (page 20, top)
The SELECT product obtained in step 3 was subjected to a real-time quantitative PCR (qPCR) reaction in Applied Biosystems ViiAm7 real-time PCR system (Applied Biosystems, USA) (page 20).
Stewart (Nucleic Acids Research, 1998, Vol. 26, No. 4 961–966), Shi ( J. Am. Chem. Soc. 2015, 137, 13804−13806), Wang ( Chem. Sci., 2016, 7, 1440-1446), Tocigl (US 2016/0319354), Ma (BioMed Research International Volume 2016, Article ID 2906484, 8 ), Tanner (Biotechniques (2012)) 53:2, 81-89) demonstrate that use of upstream and downstream probes which are elongated and ligated are routine and conventional.
Response to Arguments’
The response traverses the rejection asserting step (4) is a method step. This argument has been thoroughly reviewed but is not considered persuasive as step (4) recites, “comparing.” This is a mental step and/or abstract idea (UNIVERSITY OF UTAH RESEARCH v. AMBRY GENETICS CORPORATION).
The response asserts the comparing integrates the judicial exception. This argument has been thoroughly reviewed but is not considered persuasive as the comparing is the judicial exception which provides for interpretation of the data of the proceeding steps.
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, 5-6, 8-12, 18-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Stewart (Nucleic Acids Research, 1998, Vol. 26, No. 4 961–966), Shi ( J. Am. Chem. Soc. 2015, 137, 13804−13806), Wang ( Chem. Sci., 2016, 7, 1440-1446), Tocigl (US 2016/0319354), Ma (BioMed Research International Volume 2016, Article ID 2906484, 8 ), Tanner (Biotechniques (2012)) 53:2, 81-89)
With regards to claim 1, Stewart teaches:
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Stewart teaches design of probes, extension, ligation, PCR and comparison in figure 2.
Stewart while teaching DNA polymerase and DNA ligase does not specifically teach the use of BST 2.0 polymerase or SplintR Ligase or m6 adenine.
However, Tocigil teaches, “[0304] Other enzymes that are thermostable and deprived of 5′ to 3′ exonuclease activity and of 3′ to 5′ exonuclease activity include AmpliTaq Gold. Other DNA polymerases, which are at least substantially equivalent, may be used like other N-terminally truncated Thermus aquaticus (Taq) DNA polymerase I. The polymerase named KlenTaq I and KlenTaq LA are quite suitable for that purpose. Of course, any other polymerase having these characteristics can also be. [0305] Other polymerases include those that have strand displacement activity. In one embodiment the enzymes are Bst polymerase and the warm-start Bst 2.0 polymerase from NEB or Lucigen's polymerase called OmniAmp.”
Tocigil teaches, “[0139] Samples may be ligated using any DNA ligase able to ligate the oligonucleotides. For example, PBCV-1 DNA ligase (also known as Chlorella virus ligase or commercially as SplintR ligase; New England Biolabs Inc.), T4 DNA ligase, E. coli DNA ligase, etc. may be used. In some particular examples, either PBCV-1 DNA ligase (also known as Chlorella virus ligase or commercially as SplintR ligase) or T4 DNA ligase may be used. T4 DNA ligase may be used, such as around 0.5 U/l (or less than 1 U/ul, less than 5 U/ul, etc.).”
Shi teaches, “To verify if Bst DNA polymerase, large fragment (Bst LF) and Vent (exo−) DNA polymerase (Vent exo−) had innate reverse transcriptase activities, 50 nt hepatitis C virus (HCV) RNA was synthesized as a template (sequence listed in Supplementary Table S1). The classical AMV reverse transcriptase (AMV) was used as a positive control here. HCV RNA was reverse transcribed by Vent exo−, AMV and Bst LF, respectively, then the reaction products were further digested by RNase H, followed by denaturing polyacrylamide gel electrophoresis (PAGE). If RNA was successfully reverse transcribed, hybrid of RNA and cDNA would generate. With addition of RNase H, RNA was digested, and only cDNA bands appeared on gel. As shown in Figure 1, there was only primer fragment (Klenow LF) and Klenow exo−, Bst LF, Bst 2.0 DNA polymerase (Bst 2.0) and Bst 2.0 WarmStart DNA polymerase (WS Bst 2.0) were used to reverse transcribe, respectively, all of their corresponding fluorescence intensities of PCR significantly increased. This result indicated that cDNA templates for real-time PCR were produced, showing the five types of DNA polymerases like AMV possessing reverse transcriptase activities. However, no change of fluorescence intensity was observed, when DNA polymerase I (E. coli) (DNA poly I), Vent exo− or Taq DNA polymerase (Taq) was added, which implied that these three types of DNA polymerase had no reverse transcriptase activities of their own. Surprisingly, the fluorescence signals of RT-PCR by Bst LF, Bst 2.0 and WS Bst 2.0 appeared earlier than that of AMV. Thus, the reverse transcriptase activities of a series of Bst DNA polymerase were superior to that of AMV at the tested length of 50 nt.” (13804 2nd column-13805, 1st colum top).
Wang teaches
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Wang teaches m6A in template slows the rate of incorporation of nucleotides by BST DNA polymerase relative to controls without m6A.
Ma (BioMed Research International Volume 2016, Article ID 2906484, 8 ) is presented in the response of 3/28/2025. Ma teaches, “Bst DNA polymerase is only available, popular in some international companies as NewEngland Biolabs at high price. Therefore, it is urgent to design, produce, and purify a new and efficient Bst DNA polymerase to satisfy growing demand of less developed area. In this study, we cloned the gene of Bst DNA polymerase I from a new strain (Geobacillus stearothermophilus GIM1.543) and make various site-directed substitutions of four conserved residues in the polymerase active site which is important in binding the DNA primer terminus and dNTP to catalyze the polymerase reaction [15, 16]. Three mutants showed higher polymerization activity than the commercial Bst DNA polymerase and, consequently, would lay the roots for promoting its wide application..” Ma concludes with, “the modified Bst DNA polymerase employed in IMSA technology, on the one hand, breaks application restrictions of the patent in commercial kit in China; on the other hand, it improved the understanding of the three-dimensional structure of Bst DNA polymerase and the mechanism of DNA amplification. This study has established a fast, simple, accurate, and sensitive diagnostic method for rapidly detecting HFMD associated with EV71which provides an efficient way for quick identification, especially for the primary diagnostics setting in rural and under developed areas.” Thus MA discloses a polymerase different than BST DNA polymerase or BST 2.0 DNA polymerase which have trademarks.
The supplemental response further cites Tanner (Biotechniques (2012)) 53:2, 81-89) Tanner teaches, “using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases.” Tanner teaches, “The reaction was found to proceed faster when using either the standard or WarmStart versions of Bst 2.0 DNA polymerase as compared with wild-type Bst DNA polymerase (Figure 2 and Supplementary Figure S1). Therefore, Bst 2.0 DNA polymerase was used in all subsequent experimentation.”
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 substitute BST 2.0 polymerase ( or its derivatives) and SplintR Ligase taught by for the ligase and polymerase taught by Stewart. The artisan would be motivated as Tocigil suggests the ligase and polymerase taught by Tocigil and art accepted alternatives to the those of Stewart. The artisan would further be motivated as to use BST or BST2 DNA polymerase as Shi teaches it has reverse transcriptase activity, while Wang demonstrates BST DNA polymerase allows for differentiation of m6A based. Further the teachings of Shi suggest BST 2.0 DNA polymerase is an obvious variant. The artisan would have a reasonable expectation of success as the artisan is merely substituting one polymerase and ligase for another.
With regards to claim 5, it would have been prima facie obvious to one of ordinary skill in the art at the filing date of the claims, the slowed incorporation of BST (or BST2.0) DNA polymerase with m6A in the target sequence taught by Wang would result in less product and greater amplification to provide a greater threshold cycle relative to unmodified probes. The artisan would be motivated as Wang suggest the use of BST DNA polymerase to differentiate m6A modified nucleotides from non-m6A modified nucleotides. The artisan would have a reasonable expectation of success as the artisan is merely using known reagents and conditions.
With regards to claim 8, it would have been prima facie obvious to one of ordinary skill in the art at the filing date of the claims, the slowed incorporation of BST (or BST2.0) DNA polymerase with m6A in the target sequence taught by Wang would result in less product and greater amplification to provide a greater threshold cycle relative to unmodified probes. The artisan would be motivated as Wang suggest the use of BST DNA polymerase to differentiate m6A modified nucleotides from non-m6A modified nucleotides. The artisan would have a reasonable expectation of success as the artisan is merely using known reagents and conditions.
With regards to claim 10, Stewart teaches an RNA sample, dNTP, DNA polymerase and ligase.
With regards to claim 11, Stewart teaches, “Cycling conditions included incubation at 50C for extension and ligation.” (page 962, 2nd column, bottom).
With regards to claim 18, Stewart teaches, “WTLig-1, 54-GGA TGG GCG TTG TGT GGT TAT C-34; WTLig-2, 54-GTG TAT TCT GTA TTG GGC TCC GCA G-34.”(page 963, 1st column, middle.)
With regards to claim 19, Stewart teaches, “Reaction products were precipitated with ethanol and resolved in a 10% denaturing polyacrylamide gel.” (page 962, 2nd column, bottom)
With regards to claim 20, Tocigil teaches, “[0304] Other enzymes that are thermostable and deprived of 5′ to 3′ exonuclease activity and of 3′ to 5′ exonuclease activity include AmpliTaq Gold. Other DNA polymerases, which are at least substantially equivalent, may be used like other N-terminally truncated Thermus aquaticus (Taq) DNA polymerase I. The polymerase named KlenTaq I and KlenTaq LA are quite suitable for that purpose. Of course, any other polymerase having these characteristics can also be. [0305] Other polymerases include those that have strand displacement activity. In one embodiment the enzymes are Bst polymerase and the warm-start Bst 2.0 polymerase from NEB or Lucigen's polymerase called OmniAmp.”
Tocigil teaches, “[0139] Samples may be ligated using any DNA ligase able to ligate the oligonucleotides. For example, PBCV-1 DNA ligase (also known as Chlorella virus ligase or commercially as SplintR ligase; New England Biolabs Inc.), T4 DNA ligase, E. coli DNA ligase, etc. may be used. In some particular examples, either PBCV-1 DNA ligase (also known as Chlorella virus ligase or commercially as SplintR ligase) or T4 DNA ligase may be used. T4 DNA ligase may be used, such as around 0.5 U/l (or less than 1 U/ul, less than 5 U/ul, etc.).”
Response to Arguments
The response begins traversing the rejection by asserting, “mRNA or IncRNA in the RNA sample are low-abundance RNA or ultralow-abundance RNA, respectively. According to BACKGROUND OF THE INVENTION in the specification, to date, the RNase H-based SCARLET method is the only one that can quantitatively detect m⁶A status of single mRNA or IncRNA locus but its time-consuming nature and the need for radioactive labeling have limited its wider application. That is, the detection of m⁶A modification of mRNA or IncRNA per se is very hard and highly unpredictable in the art.” This argument has been thoroughly reviewed but is not considered persuasive as the claims are not limited to lCRNA or mRNA, but also encompass total RNA and rrna.
The response continues by reviewing the representatives interpretation and the teachings of the specification. This is noted.
The response continues by asserting, “Stewart at least fails to disclose or teach (i) the method is used for detecting a chemical modification of an RNA target site X, and the chemical modification is N⁶- methyladenosine modification (m⁶A modification); (ii) SELECT step: elongating a down probe Px2 with a DNA polymerase to obtain an elongated down probe Px2, and ligating an up probe Px1 and the elongated down probe Px2 with a ligase to obtain a SELECT product, wherein the DNA polymerase is a Bst DNA polymerase, wherein, the up probe Px1 is complementary paired with the upstream sequence of the RNA target site X, and the down probe Px2 is complementary paired with the downstream sequence of the RNA target site X; (iii) PCR amplification step: performing PCR amplification of the SELECT product obtained in step (2). “ This argument has been thoroughly reviewed but is not considered persuasive as Wang teaches m6A in template slows the rate of incorporation of nucleotides by BST DNA polymerase relative to controls without m6A.
The response continues by asserting Stewart teaches away from the claimed invention. The response further asserting, “That is, Stewart teaches the use of a non-strand displacing thermostable DNA polymerase, e.g. Taq DNA polymerase, in combination with in the gap ligation reaction.” MPEP 2123 states:
“[t]he prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed….” In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004).
Depuy Spine, Inc. v. Medtronic Sofamor Danek, Inc., 567 F.3d 1314 (Fed. Cir. 2009). The courts indicated a reference that does "not criticize, discredit, or otherwise discourage investigation into the invention claimed," does not teach away.”
In the instant case Stewart does not specifically criticize, discredit, or otherwise discourage the use of a strand displacement polymerase. Further the cited prior art demonstrate BST DNA polymerase was known and an obvious variant.
The response continues by asserting Stewart requires a non-strand displacement polymerase. This argument has been thoroughly reviewed but is not considered persuasive as the response fails to disclose where Stewart teaches a non-strand displacement polymerase is required. Thus this is arguments of counsel that is not substantiated by evidence.
The response concedes Tocigl and Tanner teach BST DNA polymerase.
The response continues by providing the representatives interpretation of Shi. This is noted. The response asserts, “This is an experiment of isothermal RNA direct detection, in which Bst LF is only used as inverse transcriptase.” This argument has been thoroughly reviewed but is not considered persuasive as Shi teaches, “We have demonstrated that Bst and Klenow DNA polymerases could be successfully used to reverse transcribe RNA within 125-nt length by real time RT-PCR.”
The response continues by providing the representative interpretation of the art and asserts the claims do not require RT-PCR. This argument has been thoroughly reviewed but is not considered persuasive as whole the claims do not recite RT-PCR, the claims require reverse transcription of the BST DNA polymerase to perform the PCR required of the claim as the claim require RNA as a substrate.
The response continues by providing arguments with respect to working temperatures. This argument has been thoroughly reviewed but is not considered persuasive as the claims provide no limitation with respect to temperature, let alone working temperatures. Thus the response is arguing limitations not in the claims. Further the prior art teaches Bst DNA polymerase and PBCV-1 ligase, if this combination does not work at the same temperature the response is arguing the instant claims have 112(a) issues.
The response continues by providing arguments with respect to Wang individually. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
The response concludes by asserting the prior art does not specifdication teach detection of M6A in mRNA or lncRNA. This argument has been thoroughly reviewed but is not considered persuasive as the claims are not limited ot mRNA and lncRNA, but encompass total RNA or rRNA.
Thus the rejection is maintained.
Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Stewart (Nucleic Acids Research, 1998, Vol. 26, No. 4 961–966), Shi ( J. Am. Chem. Soc. 2015, 137, 13804−13806), Wang ( Chem. Sci., 2016, 7, 1440-1446), Tocigl (US 2016/0319354), Ma (BioMed Research International Volume 2016, Article ID 2906484, 8 ), Tanner (Biotechniques (2012)) 53:2, 81-89) , as applied to claims 1, 5-6, 8, 10-11, 17-20 above, and further in view of Huang (Nucleic Acids Research, 2015, Vol. 43, No. 1 373–384)
The teachings of Stewart, Shi, Wang, Tocigl, Ma and Tanner are set forth above.
While Stewart, Shi, Wang, Tocigl, Ma and Tanner suggest the BST (or BST2.0) DNA polymerases can be used to detect m6A modification of RNA in a PCR reaction. They do not specifically teach the use of FTO or ALKBH5.
However, Huang teaches , “The methylation of internal adenosines at the N6 position (m6A) in messenger RNA (mRNA) was first observed several decades ago (1,2). A grasp of its regulatory function was missing, however, until the discovery of the fat mass and obesity-associated (FTO) enzyme in 2011. An m6A demethylase, FTO, was shown to regulate cellular levels of endogenous m6A residues, which strongly suggested that the m6A modification in mRNA is reversible and might be subject to dynamic regulation (3). The existence of the m6A demethylase FTO and the demonstration of the dynamic regulation of the levels of m6A have stimulated explorations of m6A modifications and related enzymes. Researchers have uncovered a wealth of unequivocal evidence for m6A function. The identification and characterization of the m6A methyltransferase tricomplex (METTL3–METTL14–WTAP) further highlights the biological significance of m6A methylation (4,5). The physiological relevance of this modification remains unclear, however. Two recent RNA ‘methylome’ studies have provided a map of m6A-modified mRNAs (6,7). Both found that the ubiquitous m6A modification plays a fundamental regulatory role in gene expression. In addition, binding proteins selectively recognize the dynamic m6A modification, research shows, in order to regulate translation status and the lifetime of mRNA (8). High-resolution mapping has revealed that methylation of adenosine to m6A in mRNA is particularly important for yeast meiosis (9). These studies have provided new insights into the distribution and functional role of m6A in mRNA (10,11). FTO belongs to the family of Fe2+ and 2-oxoglutarate (2OG) dependent AlkB dioxygenases (12) and contributes to non-syndromic human obesity (13). Besides the internal m6A substrates in mRNA, FTO also oxidatively demethylates N3-methylthymine (dm3T) in single-stranded (ss) DNA and N3-methyluridine (m3U) in ssRNA in vitro (14), but has relatively lower activities compared to other AlkB family enzymes that catalyze a wide range of oxidative reactions (15–19). The FTO gene was initially shown to influence human obesity and energy utilization (20,21). In addition, reports indicate the involvement of the FTO protein itself in various diseases (22–26). Such discoveries make FTO an increasingly interesting target with respect to its functional links to human diseases. Another member of the AlkB family, ALKBH5, was also identified as an m6A demethylase of mRNA using Fe2+ and cofactor 2OG, which together function to oxidatively remove the methyl group in m6A-containing substrates (27). Both FTO and ALKBH5 are localized to nuclei and colocalize with nuclear speckles, indicating the effects of methylation on splicing. Indeed, knockdown of the ALKBH5 gene was shown to affect splicing in tissue culture cells, and displayed a sterility phenotype in male mice. The discovery of these two m6A demethylases within the scientific community highlights the importance of m6A modification in basic biology and human disease. Studies that focus on the inhibition of m6A demethylation will likely (i) shed light on the science of ‘RNA epigenetics’ in chemical biology and (ii) hold promise for future therapeutic developments.”
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 an RNA sample treated with FTO or ALKBH5. The artisan would be motivated to use a control RNA generated from the same sample as it would have the same contaminants that would influences the enzymatic reactions. The artisan would have a reasonable expectation of success as the artisan is merely using known reagents to produce controls with the same contaminants.
Response to Arguments
There are no specific arguments to the instant rejection. Thus the rejection is maintained.
Summary
No claims are allowed.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Lebedev (US 20100003724 A1
Lebedev (20140038181 A1.
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/Steven Pohnert/ Primary Examiner, Art Unit 1683
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