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 .
Claims Status
The amendment filed on 07/23/2026 has been entered. No new matter was added.
Claim 2 was amended in the claim set filed on 07/23/2026.
Applicant’s specie election without traverse of Claim 2 in the reply filed on 03/13/2026 is acknowledged. Claims 1-4 read upon the elected specie.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claims 5-7 were withdrawn, drawn to a non-elected specie.
Claims 1 was canceled in the claim set filed on 07/23/2026.
Claims 2-7 are pending.
Claims 2-4 in the claim set filed on 07/23/2026 are currently under examination.
Response to the Arguments
Objections to the Specification in the previously mailed non-final have been withdrawn.
Applicants arguments with respect to claim 1 are moot, as claim 1 has been canceled in the claim set filed on 07/23/2026.
Applicant’s arguments regarding the determined priority date have been considered but are not persuasive, as documented below in the Priority section in this office action on Pg. 4.
Applicant’s arguments regarding previous rejection(s) of claim(s) 2-4 under 35 U.S.C. 112 have been fully considered and are persuasive. The 35 U.S.C. 112 (b) rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 6.
Applicant’s arguments regarding previous rejection(s) of claim(s) 2-4 under 35 U.S.C. 101 have been fully considered but are not persuasive. Applicant' s argument on Pg. 6, states that “As amended, claim 2 (and amended claim 5) does not merely claim abstract computations or mental comparisons. Instead, the claim integrates these evaluation steps into a concrete, highly specific, and practical clinical application of genetic testing.” The 35 U.S.C. 101 rejections documented in the previously mailed non-final have been maintained and revised in light of applicants claim amendments and arguments on Pg. 6-7, as documented below in the 35 U.S.C. 101 rejection in this office action on Pg. 4-10.
As necessitated by amendment, the 35 U.S.C. 103 rejections of claim(s) 2-4 documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments. However, upon further consideration and search, new grounds of rejection are made as documented below in the 35 U.S.C. 103 rejection in this office action on Pg. 11-22.
The revised and new grounds of rejection for claims 2-4 are documented below in this Final Office Action are necessitated by claim amendments filed on 07/23/2026.
Priority
This application is a Continuation of International Application No. PCT/JP2022/006737, filed February 18, 2022, which claims priority to Japanese Patent Application No. 2021-028913, filed February 25, 2021.
Acknowledgment is made of applicant's claim for foreign priority based on an application filed in Japan on February 25, 2021. The certified copy of JP 2021-028913 has been submitted of the record on 10/25/2023. An English translation of the foreign application JP 2021-028913 is required for the record to be considered for the priority date of February 25, 2021. Accordingly, the priority date of the instant claim set is determined to be February 18, 2022.
Response to Arguments
Applicants’ argument: “Applicant's Representative then confirmed that the foreign priority application was within the scope of the instant application. The Examiner indicated that she would reconsider the foreign priority document once this response was filed.”
Response: Applicant’s arguments have been fully considered and found unpersuasive because while the certified copy was found to be filed on 10/25/203, filed under a different doc code which has since been changed, however an English translation has not been filed of the certified copy of the Patent Application No. JP 2021-028913 application. Thus, the priority date of the claims filed 07/23/2026 remains February 18, 2022.
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 2-4 are rejected under 35 U.S.C. 101 because the claimed invention is directed towards abstract ideas of mathematical concepts-calculating an error rate and mental processes-comparing error rate and estimating false positive and false negative rate and routine and conventional steps of preparing a DNA sample, treating a DNA sample with a bisulfite reagent, and measuring degree of methylation, without significantly more. The claim(s) recite(s) abstract ideas and routine and conventional methods. This judicial exception is not integrated into a practical application because no additional elements integrate the judicial exceptions into a practical application. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because no additional elements are considered significantly more than the judicial exceptions.
Claim analysis
The instant claim 2 is directed towards: A method of a genetic test which analyzes a degree of methylation of a gene of a subject, the method comprising the following (i) to (iii):
(i) evaluating a plurality of bisulfite reagents, wherein the evaluating comprises the following (a) to (c) for each bisulfite reagent: (a) preparing a DNA sample 1 in which a CpG site to be measured is not methylated, treating the DNA sample 1 with a bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 1, where the error rate 1 is an average value of the degree of methylation of the CpG site to be measured; (b) preparing a DNA sample 2 which has the same sequence as the DNA sample 1 and in which the CpG site to be measured is methylated, treating the DNA sample 2 with the bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 2, where the error rate 2 is calculated by 100 - (an average value of the degree of methylation of the CpG site to be measured); and (c) comparing the error rate 1 with the error rate 2, wherein when a relationship of the error rate 1 < the error rate 2 is satisfied, the bisulfite reagent is evaluated as having an inappropriate conversion tendency, and wherein when a relationship of the error rate 1 > the error rate 2 is satisfied, the bisulfite reagent is evaluated as having a failed conversion tendency; (ii) selecting a bisulfite reagent from the evaluated plurality of bisulfite reagents; and (iii) treating DNA of the subject with the selected bisulfite reagent, wherein, for the subject who has no history of illness in regard to a target disease, a bisulfite reagent which has been evaluated in (c) as having an inappropriate conversion tendency is selected in (ii) when a disease onset or progression of the target disease correlates with methylation of the gene, and a bisulfite reagent which has been evaluated in as having a failed conversion tendency is selected in (ii) when a disease onset or progression of the target disease correlates with unmethylation of the gene.
Abstract ideas (mental process) related to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work:
The evaluating a plurality of bisulfite reagent is considered to be an abstract idea.
The comparing the error rate 1 with the error rate 2 is considered to be an abstract idea.
The selecting a bisulfite reagent is considered to be an abstract idea.
Abstract ideas (mental process) related Mathematical Relationships / Formulas and Performing Mathematical Calculations:
The calculate an error rate 1 is considered to be an abstract idea.
The error rate 1 is an average value of the degree of methylation is considered to be an abstract idea.
The calculate an error rate 2 is considered to be an abstract idea.
The error rate 2 is calculated by 100 - (an average value of the degree of methylation of the CpG site to be measured) is considered to be an abstract idea.
The comparing the error rate 1 with the error rate 2 is considered to be an abstract idea.
The wherein when a relationship of the error rate 1 ≤ the error rate 2 is satisfied, the bisulfite reagent is evaluated as having an inappropriate conversion tendency is considered to be an abstract idea.
The wherein when a relationship of the error rate 1 > the error rate 2 is satisfied, the bisulfite reagent is evaluated as having a failed conversion tendency is considered to be an abstract idea.
The steps of preparing a DNA sample, treating a DNA sample with a bisulfite reagent, measuring degree of methylation, treating DNA of the subject with the selected bisulfite reagent are considered to be active step. The active steps are routine and conventional as demonstrated by the 35 USC § 103 rejections stated below.
Dependent claims set forth further limitations about estimation of false positive and false negative rate and disease.
The instant claim 3 is directed towards: The method according to claim 2, wherein a false positive rate and a false negative rate in the test are estimated from values of the error rate 1 and the error rate 2 in the evaluation of the bisulfite reagent.
The false positive rate and a false negative rate in the test are estimated from values of the error rate 1 and the error rate 2 in the evaluation of the bisulfite reagent is considered to be an abstract idea.
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? Yes. 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 ideas.
With regard to claim 2, the claim recites “A method of a genetic test which analyzes a degree of methylation of a gene of a subject, the method comprising the following (i) to (iii):
(i) evaluating a plurality of bisulfite reagents, wherein the evaluating comprises the following (a) to (c) for each bisulfite reagent: (a) preparing a DNA sample 1 in which a CpG site to be measured is not methylated, treating the DNA sample 1 with a bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 1, where the error rate 1 is an average value of the degree of methylation of the CpG site to be measured; (b) preparing a DNA sample 2 which has the same sequence as the DNA sample 1 and in which the CpG site to be measured is methylated, treating the DNA sample 2 with the bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 2, where the error rate 2 is calculated by 100 - (an average value of the degree of methylation of the CpG site to be measured); and (c) comparing the error rate 1 with the error rate 2, wherein when a relationship of the error rate 1 < the error rate 2 is satisfied, the bisulfite reagent is evaluated as having an inappropriate conversion tendency, and wherein when a relationship of the error rate 1 > the error rate 2 is satisfied, the bisulfite reagent is evaluated as having a failed conversion tendency; (ii) selecting a bisulfite reagent from the evaluated plurality of bisulfite reagents; and (iii) treating DNA of the subject with the selected bisulfite reagent, wherein, for the subject who has no history of illness in regard to a target disease, a bisulfite reagent which has been evaluated in (c) as having an inappropriate conversion tendency is selected in (ii) when a disease onset or progression of the target disease correlates with methylation of the gene, and a bisulfite reagent which has been evaluated in as having a failed conversion tendency is selected in (ii) when a disease onset or progression of the target disease correlates with unmethylation of the gene.”
Abstract ideas (mental processes) related to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work: The evaluating a plurality of bisulfite reagent; the comparing the error rate 1 with the error rate 2; and the selecting a bisulfite reagent are considered to be an abstract idea (mental processes).
Abstract ideas (mathematical concepts) related Mathematical Relationships / Formulas and Performing Mathematical Calculations: The calculate an error rate 1; The error rate 1 is an average value of the degree of methylation; The calculate an error rate 2; The error rate 2 is calculated by 100 - (an average value of the degree of methylation of the CpG site to be measured); the comparing the error rate 1 with the error rate 2; the wherein when a relationship of the error rate 1 ≤ the error rate 2 is satisfied, the bisulfite reagent is evaluated as having an inappropriate conversion tendency; and the wherein when a relationship of the error rate 1 > the error rate 2 is satisfied, the bisulfite reagent is evaluated as having a failed conversion tendency are considered to be an abstract idea (mathematical concepts).
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? No, there are no additional steps that integrate the claims into a practical application.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? No, there are no additional elements that are significantly more than the judicial exceptions.
Regarding claim 2, the claim requires the routine and conventional active steps of preparing a DNA sample, treating a DNA sample with a bisulfite reagent, measuring degree of methylation, treating DNA of the subject with the selected bisulfite reagent similar to that of Holmes et al. (“Holmes”; (2014). Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PloS one, 9(4), e93933.) in view of Genereux et al. (“Genereux”; (2008). Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies. Nucleic acids research, 36(22), e150.) and Lan et al. (“Lan”; (2019). An Internal Control for Evaluating Bisulfite Conversion in the Analysis of Short Stature Homeobox 2 Methylation in Lung Cancer. Asian Pacific journal of cancer prevention : APJCP, 20(8), 2435–2443.), as discussed below in the 35 U.S.C. 103 section. Thus, the claim does not provide additional steps which are significantly more.
Dependent claims require a method of estimation of false positive and false negative rate which include additional abstract ideas (mathematical concepts) and steps that are routine and conventional based on Holmes et al. (“Holmes”; (2014). Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PloS one, 9(4), e93933.) in view of Genereux et al. (“Genereux”; (2008). Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies. Nucleic acids research, 36(22), e150.) and Lan et al. (“Lan”; (2019). An Internal Control for Evaluating Bisulfite Conversion in the Analysis of Short Stature Homeobox 2 Methylation in Lung Cancer. Asian Pacific journal of cancer prevention : APJCP, 20(8), 2435–2443.
Response to Arguments
Applicant's arguments filed 07/23/2026 have been fully considered but they are not
persuasive. To clarify some instances argued in the response filed 07/23/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “As amended, claim 2 (and amended claim 5) does not merely claim abstract computations or mental comparisons. Instead, the claim integrates these evaluation steps into a concrete, highly specific, and practical clinical application of genetic testing. Specifically, the method requires physically selecting a particular bisulfite reagent from a plurality of pre-evaluated reagents and using that selected reagent to treat the subject's DNA. This selection is governed by a precise clinical context: (1) the patient's clinical background (whether they have a history of illness, which dictates the clinical need for prioritizing sensitivity vs. specificity) and (2) the biological mechanism of the target disease (methylation vs. unmethylation correlation).”(Pg. 6).
Response: Applicant’s arguments have been fully considered and found unpersuasive because applicants amendments do not overcome the lack of patentably matter under U.S.C. 35 101. The previously presented and amended claims recite abstract ideas of mathematical concepts-calculating an error rate and mental processes-comparing error rate and estimating false positive and false negative rate and routine and conventional steps of preparing a DNA sample, treating a DNA sample with a bisulfite reagent, and measuring degree of methylation, without significantly more. The judicial exceptions are not integrated into a practical application because the claim limitations do not appear to improve the current technology or technical field beyond well-understood, routine, conventional activity. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims provide no specific limitations that provide significantly more.
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 2-4 is rejected under 35 U.S.C. 103 as being unpatentable over Holmes et al. (“Holmes”; (2014). Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PloS one, 9(4), e93933.) in view of Genereux et al. (“Genereux”; (2008). Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies. Nucleic acids research, 36(22), e150.) and Lan et al. (“Lan”; (2019). An Internal Control for Evaluating Bisulfite Conversion in the Analysis of Short Stature Homeobox 2 Methylation in Lung Cancer. Asian Pacific journal of cancer prevention : APJCP, 20(8), 2435–2443.
Holmes discloses “DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis.” (Abstract)
Regarding claim 2 step (i), Holmes teaches a method comprising “The aim of this study is to evaluate the performance of the most widely used kits: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen GmbH), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, and EZ DNA Methylation-Lightning Kit (Zymo Research). Furthermore, the innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena) and related kits (innuCONVERT Bisulfite Basic Kit and innuCONVERT Bisulfite All-In-One Kit) were analyzed. Table 1 provides an overview of the analyzed kits and their applicability to different sample types” (Pg. 3, Col. 1, Para. 1) and “The kit performance was compared with regard to conversion efficiency… The suitability for different starting materials … were tested. The performance was tested by means of real-time PCR (yield, purity, stability), clone sequencing (conversion), UV (purity and yield), HPLC (inappropriate conversion), and gel electrophoresis (DNA degradation).” (Pg. 3, Col. 1, Para. 2). Holmes teaches a method comprising “Bisulfite conversion and subsequent purification was performed according to the respective kit protocols” (Pg. 3. Bisulfite Conversion, Para. 1).
Regarding claim 2 step (i), Holmes teaches a method comprising “The CFP assay was used to quantify the specific conversion of cytosine to uracil. Altogether 421 clones comprising 18900 conversion sites were analyzed. Conversion rates above 98.7% were obtained with all kits (Figure 8)” (Pg. 8, Conversion Efficiency and Specificity, Para. 1), “inappropriate conversion was studied using an 11-mere DNA oligonucleotide containing a centered methylated cytosine (T5 MeCT5)” (Pg. 9, Col. 1, Para. 1) and “Performance differences between the compared bisulfite kits were examined by t-test and ANOVA. Correlation between conversion efficiency and inappropriate conversion was tested using Pearson correlation” (Pg. 6, Data Evaluation and Statistical Analysis, Para. 1). Holmes teaches that “The EZ DNA Methylation-Direct Kit showed the highest conversion rate of 99.9%” (Pg. 8, Conversion Efficiency and Specificity, Para. 1). Holmes also teaches that “The kits led to an inappropriate conversion between 0.9% (innuCONVERT kit family) and 2.7% (EZ DNA Methylation-Gold Kit) (Figure 9 B). A significant correlation between conversion efficiency and inappropriate conversion (Pearson’s correlation coefficient 0.83, p = 0.04) of the used protocol was found. This is expected since a bisulfite treatment under harsh conditions leads to a more complete conversion on the one hand but to an increased inappropriate conversion on the other hand” (Pg. 9, Col. 1, Para. 1). Thus, Holmes suggests a method comprising step (i) evaluating a plurality of bisulfite reagents, wherein the evaluating comprises the following (a) to (c) for each bisulfite reagent: (a) preparing a DNA sample 1 in which a CpG site to be measured is not methylated, treating the DNA sample 1 with a bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 1, where the error rate 1 is an average value of the degree of methylation of the CpG site to be measured; (b) preparing a DNA sample 2 which has the same sequence as the DNA sample 1 and in which the CpG site to be measured is methylated, treating the DNA sample 2 with the bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 2, where the error rate 2 is calculated by 100 - (an average value of the degree of methylation of the CpG site to be measured); and (c) comparing the error rate 1 with the error rate 2, wherein when a relationship of the error rate 1 < the error rate 2 is satisfied, the bisulfite reagent is evaluated as having an inappropriate conversion tendency, and wherein when a relationship of the error rate 1 > the error rate 2 is satisfied, the bisulfite reagent is evaluated as having a failed conversion tendency.
Regarding claim 2 (ii-iii), Holmes teaches that “The choice of a suitable kit for a specific application is not trivial and should be based on the specific performance requirements with regard to the respective sample material and biological/clinical question” (Pg. 11, Discussion, Para. 1) and “The methods for measuring bisulfite conversion reaction rates as described herein might be used in the future to identify the optimal reaction conditions allowing for sufficient conversion of cytosines but leading to only limited inappropriate conversion of methylated cytosines” (Pg. 12, Col. 2, Para. 1). Holmes also teaches that “Genereux et al. provide a comprehensive analysis and discussion of the two types of bisulfite-conversion errors: failure of conversion of unmethylated cytosines to uracils and inappropriate conversion of methylated cytosines to thymines. These errors will lead to false positive and false negative methylation signals, respectively, in downstream analyses” (Pg. 12, Discussion, Para. 4 ). Furthermore, Holmes teaches that “There is an emerging need for protocols and kits that allow for the preparation of bisulfite DNA from low abundance biomarkers in… fluidic samples of clinical relevance, … Several studies describe the sensitive analysis of DNA methylation biomarkers in plasma or serum as promising tests for early detection of various tumors. The low abundance of tumor DNA in blood, especially in blood from patients with early stage tumors where screening is particularly valuable, necessitates the usage of high volumes of plasma or serum in order to increase the likelihood of presence of methylated tumor DNA and therefore reach sufficient sensitivity of the test. To date the only commercially available kit enabling the preparation of bisulfite DNA from high volume (up to 3 ml) body fluid is the innuCONVERT Bisulfite Body Fluids Kit” (Pg. 14, Col. 2, Para. 3). Thus, Holmes suggests a method comprising steps (ii) selecting a bisulfite reagent from the evaluated plurality of bisulfite reagents; and step (iii) treating DNA of the subject with the selected bisulfite reagent, wherein, for the subject who has no history of illness in regard to a target disease, a bisulfite reagent which has been evaluated in (c) as having an inappropriate conversion tendency is selected in (ii) when a disease onset or progression of the target disease correlates with methylation of the gene, and a bisulfite reagent which has been evaluated in as having a failed conversion tendency is selected in (ii) when a disease onset or progression of the target disease correlates with unmethylation of the gene.
However, Holmes does not explicitly teach the limitations of step (i) (a-c) and step (iii).
Genereux discloses “Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcytosine to thymine, and failure to convert unmethylated cytosine to uracil. Conventional bisulfite treatment requires hours of exposure to low-molarity, low-temperature bisulfite (‘LowMT’) and, sometimes, thermal denaturation. An alternate, high-molarity, high-temperature (‘HighMT’) protocol has been reported to accelerate conversion and to reduce inappropriate conversion. We used molecular encoding to obtain validated, individual-molecule data on failed- and inappropriate-conversion frequencies for LowMT and HighMT treatments of both single-stranded and hairpin-linked oligonucleotides. After accounting for bisulfite-independent error, we found that: (i) inappropriate-conversion events accrue predominantly on molecules exposed to bisulfite after they have attained complete or near-complete conversion; (ii) the HighMT treatment is preferable because it yields greater homogeneity among sites and among molecules in conversion rates, and thus yields more reliable data; (iii) different durations of bisulfite treatment will yield data appropriate to address different experimental questions; and (iv) conversion errors can be used to assess the validity of methylation data collected without the benefit of molecular encoding.” (Abstract).
Regarding claim 2 Step (i) (a-b), Genereux teaches a method comprising “a DNA substrate with which to measure conversion-error frequencies, we designed and ordered from GeneLink, Hawthorne, NY four single-stranded synthetic oligonucleotides. Two of these, methylated top strand (‘TM’) and methylated bottom strand (‘BM’), contained 5-methylcytosine at all 10 of their CpG sites” (Pg. 3, Methods- Design and assembly of molecularly encoded oligonucleotides) and “The other two oligonucleotides, (‘TU’), unmethylated top strand, and (‘BU’), unmethylated bottom strand, were identical in sequence to TM and BM, except that they contained cytosines at the sites that were occupied by 5-methylcytosine in TM and BM.” (Pg. 3, Methods- Design and assembly of molecularly encoded oligonucleotides). Genereux teaches a method comprising “In preparation for HighMT treatment, molecularly encoded oligonucleotides (20 μl) were first denatured in 0.3 N NaOH at 42°C for 20 min, added to the HighMT bisulfite solution (180 μl), and held at 70°C for durations ranging from 5 to 200 min.” (Pg. 4, Methods- Bisulfite treatment of molecularly encoded oligonucleotides). Genereux teaches a method comprising “PCR products were cycle-sequenced using either the T7 primer or the M13R primer, and Big-Dye Terminator v3.1 (ABI, Foster City, CA), then sequenced on an ABI3100, using a 36 cm POP6 array (Comparative Genomics Center, University of Washington, Seattle, WA).” (Pg. 4, Methods- Post-bisulfite cleanup, PCR amplification and sequencing) and “DNA sequencing is a standard component of protocols designed to collect DNA methylation patterns. To investigate how often errors were introduced during the sequencing and base-calling processes, we performed repeat sequencing on a subset of our clones”. Genereux teaches a method comprising “we calculated point estimates as the number of events divided by the number of opportunities.” (Pg. 4, Methods- Data analysis). Genereux teaches a method comprising “we examined both population-mean and single-molecule error frequencies for single-stranded … molecules treated under the LowMT and HighMT conditions” (Pg. 8, Investigating bisulfite-conversion error by analysis of individual molecules; Fig. 4 ;Table 3). Genereux teaches a method comprising the proportions of error frequencies: failed conversions (%) and inappropriate conversions (%) due to changes in conditions such as the molar concentrations of components in a bisulfite reagent, the reaction time, the reaction temperature, the substrate structure, etc. were confirmed. Thus, Holmes and Genereux suggest a method comprising step (i) (a) preparing a DNA sample 1 in which a CpG site to be measured is not methylated, treating the DNA sample 1 with a bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 1, where the error rate 1 is an average value of the degree of methylation of the CpG site to be measured and (b) preparing a DNA sample 2 which has the same sequence as the DNA sample 1 and in which the CpG site to be measured is methylated, treating the DNA sample 2 with the bisulfite reagent to be evaluated, and subsequently measuring a degree of methylation of the CpG site to be measured to calculate an error rate 2, where the error rate 2 is calculated by 100 - (an average value of the degree of methylation of the CpG site to be measured)
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Regarding claim 2 Step (i) (c), Genereux teaches a method wherein the error frequencies of failed conversions and inappropriate conversions in a bisulfite reaction were compared (Tables 3-5.). Table 3 illustrates the relationship of error rate 1 ≤ the error rate 2 in the error frequencies of table rows indicated by HighMT 80 min and HighMT 200 min (Table 3), where the bisulfite treatment is shown to have primarily inappropriate conversion errors. Genereux teaches a method comprising “inappropriate-conversion events detected in the 200 min sample occurred primarily during the last 120 minutes of bisulfite treatment on molecules that had already achieved complete conversion” (Pg. 8, Single-stranded DNA treated under the HighMT protocol). Table 3 also illustrates the relationship of error rate 1 > the error rate 2 in error frequencies of table rows indicated by HighMT 5 min, HighMT 15 min and HighMT 30 min (Table 3), where the bisulfite treatment is shown to have primarily failed conversion errors. Thus, Holmes and Genereux suggest a method comprising step (c) comparing the error rate 1 with the error rate 2, wherein when a relationship of the error rate 1 ≤ the error rate 2 is satisfied, the bisulfite reagent is evaluated as having an inappropriate conversion tendency, and wherein when a relationship of the error rate 1 > the error rate 2 is satisfied, the bisulfite reagent is evaluated as having a failed conversion tendency.
Regarding claim 2, Lan teaches a method wherein “Lymphocytes were collected from healthy volunteers; formalin-fixed, paraffin-embedded (FFPE) tissue specimens were collected from 97 lung cancer patients… Genomic DNAs were extracted from lymphocytes of healthy volunteers or FFPE lung tissues… DNAs quantified with the NanoDrop2000 (Invitrogen) was submitted to bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen)” (Pg. 2437, Sample collection, genomic DNA isolation and bisulfite conversion). Thus, Holmes, Genereux and Lan suggest a method comprising treating DNA of the subject with a bisulfite reagent.
Lan discloses “The methylation status is considered as powerful diagnostic, prognostic, and predictive biomarkers. However, the limited DNA amount and conversion efficiency after bisulfite treatment are considerable hindrances in quantitative methylation analysis. In this study, we designed an artificial internal control (IC) system that contained the cytosine-free fragment (CFF) following CpG sequences of the SHOX2 promoter whose methylation status has been described as a valuable biomarker of lung cancer. Its performance in quantifying DNA recovery and bisulfite conversion efficiency as well as in detecting false-positive SHOX2 methylation was determined on samples from lung cancer patients.” (Abstract-objective)
Regarding claim 2, Lan teaches a method wherein “SHOX2 gene promoter whose methylation status has been described as a valuable biomarker for lung cancer detection” (Pg. 2436 , Introduction). Lan teaches a method wherein “different commercial bisulfite conversion kits, applied to various DNA sources with high and low molecular weight (HMW/LMW), have been comprehensively evaluated for their DNA recovery and bisulfite conversion efficiencies” (Pg. 2436, Introduction). Lan also teaches “Subsequently, based on the bisulfite conversion efficiency assessed on the IC, false positive SHOX2 methylation level and successful conversion of the SHOX2 sequence was accurately detected in patients with lung cancer patients” (Pg. 2436, Introduction) Genereux teaches a method wherein “For experiments where even a very small number of methylated cytosines would provide important biological information, it will be desirable to minimize the misclassification of unmethylated cytosines as methylated... For experiments where even a very small number of unmethylated cytosines would provide important biological information, it will generally be desirable to minimize the number of inappropriate-conversion events… To assess whether complete or near-complete methylation really does occur in some biological cases, or whether it is instead an artifact of conversion error, it is necessary to choose conditions that yield low inappropriate-conversion frequencies. For this goal, too, HighMT conditions seem preferable. Specifically, our data indicate that for our single-stranded oligonucleotide treated under HighMT conditions, durations of ∼50–80 min would yield successful conversion frequencies between 98% and 99.9%, with few or no inappropriate-conversion events.” (Pg. 15, Modulating bisulfite-conversion error frequencies to address a specific biological question). Thus, Holmes, Genereux and Lan suggest a method wherein when a disease onset or progression of which correlates with methylation of the gene is the target disease, the bisulfite reagent is a bisulfite reagent which has been evaluated by the method according to claim 2 as having an inappropriate conversion tendency, and wherein when a disease onset or progression of which correlates with unmethylation of the gene is the target disease, the bisulfite reagent is a bisulfite reagent which has been evaluated by the method according to claim 2 as having a failed conversion tendency.
Accordingly, Holmes, Genereux and Lan suggest the claim limitations of claim 2.
Holmes, Genereux and Lan are considered to be analogous to the claimed invention because they are in the same field of evaluating bisulfite conversion. Holmes teaches that “There is an emerging need for protocols and kits that allow for the preparation of bisulfite DNA from low abundance biomarkers” (Pg. 14, Col. 2, Para. 3) and “protocols and kits could be further improved”(Pg. 12, Col. 1, Para. 1), hence one of ordinary skill in the art would be motivated to modify the method of Holmes to ideally analyze a degree of methylation of a gene. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of evaluating a bisulfite reagent, selecting a bisulfite reagent, treating DNA of the subject with the selected bisulfite reagent as suggested by Holmes to incorporate the method preparing a sample as suggested by Genereux and to incorporate the method of treating DNA of a subject with a bisulfite reagent and wherein the methylation status of gene correlates with disease as suggested by Lan and provide a method for analyzing the degree of methylation of a gene in a subject. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 2. Doing so would allow for more reliable data based on the needs to answer the clinical question.
The teachings of Holmes, Genereux and Lan are documented above in the rejection of claim 2 under 35 U.S.C. 103. Claims 3 and 4 depend on claim 2.
Regarding claim 3, Genereux teaches that “When inappropriate conversion occurs and is ignored in data analysis, it will lead to underestimates of genomic methylation densities. In contrast, when inappropriate conversion occurs and its frequency is known, it can be included as a parameter in the data analysis. Information on failed- and inappropriate-conversion frequencies is therefore essential for inference from detailed DNA methylation patterns.” (Pg. 2, Introduction). Thus, Holmes, Genereux and Lan suggest a method wherein a false positive rate and a false negative rate in the test are estimated from values of the error rate 1 and the error rate 2 in the evaluation of the bisulfite reagent.
Regarding claim 4, Lan teaches a method wherein “lung cancer” (Pg. 2437, Sample collection, genomic DNA isolation and bisulfite conversion). Thus, Holmes, Genereux and Lan teach a method wherein “biomarker for lung cancer detection” (Pg. 2436, Introduction). Thus, Genereux and Lan suggest a method wherein the disease is cancer.
Response to Arguments
Applicant's arguments filed 07/23/2026 (Pg. 7-11) with respect to claims 2-4 have been fully considered but do not apply to the new grounds of rejection.
Conclusion of Response to Arguments
In view of the amendments, new grounds of rejections and above responses to arguments, no claims are in condition for allowance.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's
disclosure: Patent App. No. WO 2008103761 A2, Aug. 28, 2008 (Pg. 3, methylation of gene and cancer diagnosis; Pg. 89-91 and 99, Bisulfite treatment, Pg. 104 methylation conversion error) Claims 2-4 (in-part).
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 KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
