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 .
Continued Examination Under 37 CFR 1.114
2. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 23 June 2026 has been entered.
3. Applicant's arguments and amendments to the claims presented in the reply of 23 June 2026 have been fully considered but do not place the application in condition for allowance.
The previous rejection of the claims under 35 U.S.C. 112(b) has been obviated by the amendment to the claims.
Claim Status
4. Claims 1-5, 8-13, 16-17, and 39-48 are pending.
Claims 39-46 are withdrawn from further consideration, there being no allowable generic or linking claim.
Claims 1-5, 8-13, 16, 17 and 47-48 read on the elected invention and have been examined herein. The subject matter in claims 1-5, 8-13, 16, 17 and 47-48 as it relates to the elected species of cg08141395 is allowable over the prior art. The species of cg00438740 has been rejoined with the elected species of cg08141395 and fully examined herein. The claims encompass the non-elected species of CpG sites other than cg08141395 and Cg00438740. Prior to the allowance of the claims, any non-elected subject matter which has not been rejoined with the elected subject matter will be required to be removed from the claims.
In the reply, Applicant states “Since independent claims 1 and 10 should be allowable in view of the amendments and remarks herein, and since claims 39-46 have been amended similarly, Applicant respectfully requests that claims 39-46 be rejoined, the additional species searched, and all of the claims allowed pursuant to MPEP § 821.04.”
However, independent claims 1 and 10 are not allowable for the reasons set forth below. Further, Applicant’s attention is directed to MPEP 803 which states: “If the Markush claim is not allowable, the provisional election will be given effect and examination will be limited to the Markush claim and claims to the elected species, with claims drawn to species patentably distinct from the elected species held withdrawn from further consideration.” Claims 39-46 are not directed to the Markush claim and do not recite the elected species or rejoined species.
Maintained Claim Rejections - 35 USC § 101
5. 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, 8-13, 16, 17 and 47-48 are rejected under 35 U.S.C. 101 because the claimed invention is directed to the judicial exception of a law of nature / natural phenomenon, and/or an abstract idea without significantly more. The judicial exception is not integrated into a practical application and the claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception for the reasons that follow.
Applicant' s attention is directed to MPEP 2106 “Patent Subject Matter Eligibility” which discusses the Alice/Mayo two-part test for evaluating subject matter eligibility.
Regarding Step 1 of the subject matter eligibility test set forth at MPEP 2106III, the claims are directed to the statutory category of a process.
Regarding Step 2A, prong one, the claims recite the judicial exception of a law of nature. The claims recite the correlation between the level of methylation at CpGs in differentially methylated regions (DMRs), and particularly the CpG site cg08141395” and the identity of a cell as being either a buccal cell or a white blood cell. As in Mayo Collaborative Services v. Prometheus, the recited relationship is a natural phenomenon that exists apart from any human action. See also Cleveland Clinic Foundation v. True Health Diagnostic, LLC, 2018-1218 (Fed Cir. 2019) which states that “The re-phrasing of the claims does not make them less directed to a natural law.”
The claims also recite the judicial exception of an abstract idea and particularly mental processes.
MPEP 2106.04(a) states that the enumerated groupings of abstract ideas include:
“1) Mathematical concepts – mathematical relationships, mathematical formulas or equations, mathematical calculations (see MPEP § 2106.04(a)(2), subsection I);…
3) Mental processes – concepts performed in the human mind (including an observation, evaluation, judgment, opinion) (see MPEP § 2106.04(a)(2), subsection III).”
The claims require applying or using a formula based on the methylation status of a CpG site to determine the amount of cellular heterogeneity in a sample. This step can be accomplished through critical thinking processes and thereby is an abstract step. Alternatively, the step can be accomplished using a generic computer or software program. The use of a generic computer or software program to implement an abstract idea does not itself impart patent eligibility.
As stated in MPEP 2106.04(a)(2) III “The courts do not distinguish between mental processes that are performed entirely in the human mind and mental processes that require a human to use a physical aid (e.g., pen and paper or a slide rule) to perform the claim limitation….Nor do the courts distinguish between claims that recite mental processes performed by humans and claims that recite mental processes performed on a computer.”
Herein, the use of a generic computer to apply a formula does not constitute something more than an abstract idea.
The claims also recite the judicial exception of a mathematical concept of the formula that is used to correct for cellular heterogeneity, including the particular formula recited in claims 8 and 16 See MPEP 2106.04(a)(2):
“A claim that recites a mathematical calculation, when the claim is given its broadest reasonable interpretation in light of the specification, will be considered as falling within the “mathematical concepts” grouping. A mathematical calculation is a mathematical operation (such as multiplication) or an act of calculating using mathematical methods to determine a variable or number, e.g., performing an arithmetic operation such as exponentiation. There is no particular word or set of words that indicates a claim recites a mathematical calculation. That is, a claim does not have to recite the word “calculating” in order to be considered a mathematical calculation. For example, a step of “determining” a variable or number using mathematical methods or “performing” a mathematical operation may also be considered mathematical calculations when the broadest reasonable interpretation of the claim in light of the specification encompasses a mathematical calculation.”
Regarding Step 2A, prong two, having determined that the claims recite a judicial exception, it is then determined whether the claims recite additional elements that integrate the judicial exception into a practical application.
Herein, the claims do not recite additional steps or elements that integrate the recited judicial exceptions into a practical application of the exception(s). The additionally recited steps of providing a biological sample and determining methylation of a CpG site (and a target nucleic acid sequence in claims 47 and 48) are part of the data gathering process necessary to observe the judicial exception. These steps do not practically apply the judicial exception.
Regarding Step 2B, the next question is whether the remaining elements/steps – i.e., the non-patent-ineligible elements/steps - either in isolation or combination, amount to significantly more than the judicial exception.
Herein, the claims as a whole are not considered to recite any additional steps or elements that amount to significantly more than routine and conventional activity and do not add something “significantly more” so as to render the claims patent-eligible. The additionally recited steps of providing a biological sample, and determining methylation status of a CpG site (and a target nucleic acid in claims 47 and 48), including determining methylation by contacting nucleic acids in the biological sample with bisulfite under alkaline conditions and performing methylation-sensitive PCR on the bisulfite treated nucleic acids were well-known, routine and conventional in the prior art. This finding is evidenced by the teachings in the specification. For example, the specification (para [0059]; paragraph numbering herein is with respect to the published application) states:
“Methods of determining the methylation status of a target nucleic acid sequence and/or a DMR loci (e.g., one or more CpG dinucleotides or of a CpG island within a target sequence or a DMR loci) are known in the art. It would be appreciated that the most common method for evaluating the methylation status of DNA begins with a bisulfite-based reaction on the DNA (see, for example, Frommer et al., 1992, PNAS USA, 89(5):1827-31). Commercial kits are available for bisulfite-modifying DNA. See, for example, EpiTect Bisulfite or EpiTect Plus Bisulfite Kits (Qiagen).”
See also para [0061] of the specification.
Further, see:
Guetg, D. (U.S. 20040248120, para [0049]) and Chen, B. (U.S. Patent No. 6180344; col. 11, lines 1-15) which each teach methods of methylation-sensitive PCR of DNA that has been modified by treatment with bisulfite (e.g., para [0049]);
Herman et al (EP0954608; published 2006) which teaches methods of methylation-specific PCR of DNA that has been modified by treatment with bisulfite under alkaline conditions (e.g., para [0011] and [0021]);
Eipel (Aging. May 2016. 8(5): 1034; cited in the IDS) which teaches methods for determining the relative level of buccal cells and leukocytes / white blood cells in a buccal swab sample by assaying for the methylation status of two cell-type specific CpGs - i.e., cg07380416 and cg20837735 (p. 1038, col. 2); and
McCord et al (U.S. 10053740) also teaches methods comprising obtaining a saliva (oropharyngeal sample) that contains buccal and white blood cells and determining the methylation status of CpG sites, including the CpG site cg00438740, wherein differential methylation at the CpG site is indicative of the composition of the sample as containing buccal cells or white blood cells (e.g.., Table 1; Figure 4; col. 3, lines 48-56; col. 4, lines 54-67).
Additionally MPEP 2106.05(d) II states:
The courts have recognized the following laboratory techniques as well-understood, routine, conventional activity in the life science arts when they are claimed in a merely generic manner (e.g., at a high level of generality) or as insignificant extra-solution activity.
Determining the level of a biomarker in blood by any means, Mayo, 566 U.S. at 79, 101 USPQ2d at 1968; Cleveland Clinic Foundation v. True Health Diagnostics, LLC, 859 F.3d 1352, 1362, 123 USPQ2d 1081, 1088 (Fed. Cir. 2017);
Using polymerase chain reaction to amplify and detect DNA, Genetic Techs. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016); Ariosa Diagnostics, Inc. v. Sequenom, Inc., 788 F.3d 1371, 1377, 115 USPQ2d 1152, 1157 (Fed. Cir. 2015);
Detecting DNA or enzymes in a sample, Sequenom, 788 F.3d at 1377-78, 115 USPQ2d at 1157); Cleveland Clinic Foundation 859 F.3d at 1362, 123 USPQ2d at 1088 (Fed. Cir. 2017); …
Amplifying and sequencing nucleic acid sequences, University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 764, 113 USPQ2d 1241, 1247 (Fed. Cir. 2014); and
viii. Hybridizing a gene probe, Ambry Genetics, 774 F.3d at 764, 113 USPQ2d at 1247.
Note that while the claims recite detecting the methylation status at a particular CpG site - i.e., cg08141395 - the identity of the CpG is part of the judicial exception and not something in addition to the recited judicial exceptions.
Further, to the extent that the claims encompass the use of a computer to apply the formula and calculate a correction for cellular heterogeneity, MPEP 2106.05(a) states that ”Limitations that the courts have found not to be enough to qualify as “significantly more” when recited in a claim with a judicial exception include: i. Adding the words “apply it” (or an equivalent) with the judicial exception, or mere instructions to implement an abstract idea on a computer, e.g., a limitation indicating that a particular function such as creating and maintaining electronic records is performed by a computer, as discussed in Alice Corp., 134 S. Ct. at 2360, 110 USPQ2d at 1984 (see MPEP § 2106.05(f))”
In Mayo v. Prometheus, the Supreme Court stated: "[t]o put the matter more succinctly, the claims inform a relevant audience about certain laws of nature; any additional steps consist of well understood, routine, conventional activity already engaged in by the scientific community; and those steps, when viewed as a whole, add nothing significant beyond the sum of their parts taken separately."
This is similar to the present situation wherein the additional steps and elements are recited at a high degree of generality and are all routine, well understood and conventional in the prior art. The recited steps and elements do not provide the inventive concept necessary to render the claims patent eligible. See also Genetic Technologies Ltd. v. Merial L.L.C. 818 F.3d at 1377, 1379 (Fed. Cir. 2016).
For the reasons set forth above, when the claims are considered as a whole, the claims are not considered to recite something significantly more than a judicial exception and thereby are not directed to patent eligible subject matter.Response to Remarks:
The response argues that he claimed methods are an improvement and provide “significantly” more because they solve a problem that is not solved by Eipel. It is argued that the claimed methods “provide a simple way to accurately evaluate the methylation status of virtually any loci using a buccal sample that was not previously available prior to the present disclosure.” Applicant asserts that the claimed methods “provide a simple way to accurately evaluate the methylation status of virtually any loci using a buccal sample that was not previously available prior to the present disclosure.”
Applicants’ arguments have been fully considered but are not persuasive. A claim is not considered to be patent eligibility under 35 U.S.C. 101 based on a general
finding that an invention is an improvement. There is no showing that the claimed
method results in the improvement of the function of a computer or an improvement to
another / different technology or technical field (see MPEP 2106.04(d)(1) and
2106.05(a).
In particular, see MPEP 2016.04(d)(I) which states:
“It should be noted that while this consideration is often referred to in an abbreviated manner as the "improvements consideration," the word "improvements" in
the context of this consideration is limited to improvements to the functioning of a computer or any other technology/technical field, whether in Step 2A Prong Two or in Step 2B.” (Emphasis added).
MPEP 2106.04(d)(I) provides examples of what the courts have found to be
indicative of an additional element (or combination of elements) that integrate the
exception into a practical application. The present claims do not recite such elements. Rather, the asserted improvements are improvements to the judicial exception itself of the correlation between methylation levels and the identity of a cell as being a buccal cell or white blood cell. The response does not point to a particular technology or technical field that is improved upon by the claimed methods.
The response argues that “the relative contribution of buccal and white blood cell DNA to saliva DNA can be directly assessed and used to correct other methylation assessments of health condition-related loci to impute health status
more accurately.”
However, MPEP 2016.04 II states that when evaluating whether the claim as a whole integrates the exception into a practical application of that exception, “if the additional claim elements merely recite another judicial exception, that is insufficient to integrate the judicial exception into a practical application.” Herein, the additional element of applying a formula to obtain an absolute value is the judicial exception of an abstract idea and the judicial exception of a mathematical formula. The step of determining the amount of cellular heterogeneity in the sample (based on the results of using the formula) is the judicial exception of an abstract idea. Thus, these steps are not steps in addition to the judicial exception since they themselves are a judicial exception. Further, the identity of the particular CpG sites within a DMR locus which are assayed to determine the methylation status and provide the asserted accurate results is part of the judicial exception itself - i.e., the natural law of the correlation between the methylation status at the CpG sites and the occurrence of a buccal cell versus a white blood cell in a oropharyngeal biological sample. Therefore, these limitations in the claims are not an application of the law of nature, but rather are part of the law of nature. Thus, these elements are also not something in addition to the recited judicial exceptions.
In the present claims, there is no improvement that is made to the technologies of providing a biological sample from a subject and determining the methylation status of CpG site, which are the only non-patent-ineligible elements (non-judicial exceptions) recited in the claims. The claimed methods rely on well-known, routine and conventional methods of treating DNA with bisulfite and sequencing the DNA or performing PCR on the DNA to determine the methylation status of CpGs. The Applicant has identified a new natural phenomena - the relationship between the differential methylation status of the CpGs in buccal and white blood cells and the ability to distinguish between buccal and white blood cells based on the differential methylation status at the CpGs - and applied routine and conventional technologies to perform data gathering steps required for application of this judicial exception. See e.g. MPEP 2106.05(g) relating to insignificant extra-solution activity that is performed to observe a judicial exception.
In summary, it is maintained that Applicant has not established that the claimed method provides an improvement to the functioning of a computer or an improvement to another technology or technical field so as to establish that the claims recite additional elements or steps that integrate the recited judicial exception into a practical application of that exception.
Note that the following is to clarify the record. The response does not accurately characterize the complete teachings of Eipel (Aging. May 2016. 8(5): 1034; cited in the IDS). The response states “Eipel compared the use of three CpG sites ("the 3-CpG-model") with the use of five CpG sites ("the 5-CpG-model") to estimate the epigenetic age of a person. Significantly, the method described by Eipel is relevant only to estimating the epigenetic age of a person.”
However, while Eipel does teach a panel of three CpG sites (in the PDE4C, ASPA and ITGA2B genes) that can be used to predict epigenetic age, Eipel also teaches methods for determining the relative level of buccal cells and leukocytes / white blood cells in a buccal swab sample by assaying for the methylation status of two cell-type specific CpGs in the CD6 and SERPINB5 genes - i.e., cg07380416 and cg20837735 (p. 1038, col. 2 and abstract). Eipel (p. 1038) states “We reasoned that epigenetic characteristics of buccal epithelial cells and leukocytes might be utilized to determine the cellular composition in buccal swabs.” Eipel teaches that the methylation status of the individual CpGs of cg07380416 and cg20837735 can be used to determine the amount of buccal and white blood cells in a sample (see abstract and “Analysis of the composition of buccal epithelial cells” at p. 1037 -1038).
Priority
6. The present claims are entitled to the filing date of International Application PCT/US2020/29266, filed 04/22/2020. It is noted that a claim as a whole is assigned an effective filing date rather than the subject matter within a claim being assigned individual effective filing dates. U.S. Application No. 62/836,890, filed 04/22/2019 does not provide support for each of the embodiments in the claims. The ‘890 application is limited to methods in which the DMR is DMR16 having SEQ ID NO: 1 therein and the CpG in the DMR is cg05575921. In particular, the ‘890 application teaches that in methods that correct for cellular heterogeneity, the biological sample from a subject is a saliva sample. The ‘890 application does not teach “DMR11” or cg08141395 or the other CpG sites listed in claims1 and 10. The ‘890 application also does not teach methods in which the sample is sputum as recited in present claim 2. The ‘890 application does not teach that the absolute difference between methylation levels recited in claims 3-9 and 11-13, or the formulas listed in claims 8 and 16. The ‘890 application does not disclose that determining the methylation status comprises sequencing, as recited in present claims 9 and 17. If Applicant asserts that the present claims are entitled to priority to the provisional applications, Applicant should point to specific teachings (e.g., by page and line number) in the priority applications to establish priority for each of the recitations set forth in the claims.
New Claim Rejections - 35 USC § 103
7. 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, 9, and 47 is/are rejected under 35 U.S.C. 103 as being unpatentable over McCord et al (U.S. 10053740) in view of Eipel (Aging. May 2016. 8(5): 1034-1044 and Supplemental Data p. 1045-1048; previously cited).
McCord (col. 3, lines 43-47) states that “(s)ome loci on the genome called ‘tissue-specific differentially methylated regions’ can therefore be used for cell identification because they present different levels of DNA methylation depending on the cell studied.” McCord teaches using DNA methylation levels at tissue-specific CpG sites to identify the cells present in a body fluid sample (e.g., col. 3, line 48 -col. 4 line 67).
McCord teaches a method comprising: providing a saliva sample (i.e., a oropharyngeal biological sample) that contains buccal cells and white blood cells; determining the methylation status of a locus that includes the CpG site cg00438740; and determining that the saliva sample contains blood cells or buccal cells based on the determined methylation status (e.g.., Table 1; Figure 4; col. 3, lines 48-56; col. 4, lines 54-67). In the method of McCord, the CpG site cg00438740 is present in the INPP50 gene, and the locus comprising this site is SEQ ID NO: 11 therein (Table 1 and col. 4, lines 54-67).
In particular, McCord (col. 4, lines 54-67) states:
“The blood cell specific locus having the sequence of SEQ ID NO: 11 is also referred to as INPP5D200 TM60 in this disclosure. INPP5D200 TM60 is a locus of 156 base pairs on chromosome 2 and occupies the chromosomal location from 233060162 to 233060317. The genome coordinates for INPP5D200 TM60 are chr2: 233060162-233060317. INPD200_TM60 locus contains CpG sites that are hypomethylated in the genomes of blood cells, particularly, white blood cells, compared to other cells, particularly, vaginal epithelial cells, semen, or buccal epithelial cells. Particularly, cytosine residues located at positions 24, 26, 33, 59, and 130 are hypomethylated in the genomes of vaginal epithelial cells compared to other cells, particularly, blood cells, semen, or buccal epithelial cells.”
McCord does not specifically recite the limitation of “applying a formula based on the methylation status of the at least one locus in the white blood cells and in the buccal cells of the biological sample to determine the relative amount of white blood cells and buccal cells in the biological sample.”
However, McCord does teach calculating the number of cells in a sample, including a saliva sample, that are white blood cells based on the methylation status. For instance, McCord teaches determining methylation levels at the cg00438740 site (figure 4 and Table 1) and states (col. 21, lines 16-21): “(t)he level of methylation or methylation status refers to the percentage of cells in a sample that do or do not have a methylation group on such cytosines. For example, if 50 cells in a pool of 100 cells contain methylated cytosines at a CpG site, the level of methylation or methylation status of the CpG site is 50%.”
Further, Eipel teaches methods for determining the cellular composition of saliva and buccal swabs. Eipel (p. 1033 col. 1 final para to col. 2 first para) teaches:
“(s)aliva as well as mouth swabs are very heterogeneous in their composition of
buccal epithelial cells and leukocytes.” Eipel (p. 1038, col. 1) further states “(w)e reasoned that epigenetic characteristics of buccal epithelial cells and leukocytes might be utilized to determine the cellular composition in buccal swabs.” Eipel teaches applying a formula to correct for the cellular heterogeneity of a sample by taking into consideration the quantity of buccal epithelial cells and white blood cells in a sample when determining the methylation status of a target CpG (p. 1038, col. 1 and p. 1042, col. 2 to p. 1043, col. 1). See also Figure 2 of Eipel “Prediction of the cellular composition in mouth swab samples.….The percentage of buccal epithelial cells versus leukocytes was determined by cell counting in 11 stained mouth swab smears. DNAm levels at the two cell type‐specific CpGs were determined by pyrosequencing and
correlated with cell counts.”
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 McCord so as to have used the formulas of Eipel to calculate the relative amounts of buccal and white blood cells in the saliva samples in the method of McCord based on the level of methylation at the CpG site cg00438740. One would have been motivated to have done so because Eipel teaches that saliva samples can contain both buccal epithelial cells and white blood cells / leukocytes and teaches that the formulas and assays therein provide an effective mean for identifying and determining the cellular composition of a sample.
Such a modification of the method of McCord necessarily determines the amount of cellular heterogeneity in the biological sample since the modified method determines the relative amounts of the white blood cells and buccal cells.
Regarding claims 3-5, since the CpG site of McCord is that same as that in the present claim, in the absence of evidence to the contrary, the absolute difference between the methylation status of the CpG site at cg00438740 of McCord in whole blood as compared to buccal cells is at least 0.5, at least 0.8, and at least 0.9.
Regarding claim 9, both McCord (e.g., col. 11, line 64 to col. 65, line 12; and col. 16, lines 7-21) and Eipel (e.g. p. 1042, col. 1 and Figure S5) teach determining the methylation status at the CpG site by performing a sequencing assay.
Regarding claim 47, McCord teaches that the method further comprises determining the methylation status of another CpG site in the sample (e.g., col. 5, lines 33-63).
8. Claim(s) 10-13, 17 and 48 is/are rejected under 35 U.S.C. 103 as being unpatentable over McCord et al (U.S. 10053740) in view of Eipel (Aging. May 2016. 8(5): 1034-1044 and Supplemental Data p. 1045-1048; previously cited)
and further in view of Guerrero-Preston (U.S. 20130071842; cited in the IDS of 28 January 2022).
The teachings of McCord and Eipel are presented above.
In particular, McCord teaches that determining the methylation status of the CpG site at cg00438740 and other target sequences can be performed by treating DNA in the biological sample from a subject with bisulfite, amplifying the bisulfite-treated DNA, and then performing sequencing (e.g., col. 11, line 55-col. 12, line 12; col. 16 lines 22-33).
McCord does not teach that the step of treating the DNA with bisulfite is performed under alkaline conditions.
However, Guerrero-Preston teaches methods for detecting methylation levels of target nucleic acids in biological samples, including saliva samples, wherein the nucleic acids in the sample are treated with bisulfite under alkaline conditions prior to performing PCR, and particularly methylation-specific PCR (e.g., [0021], [0027], [0031], [0047], [0055] and [0069-0070]).
Guerrero-Preston (para [0047]) states:
“Bisulfite ions, for example, sodium bisulfite, convert non-methylated cytosine residues to bisulfite modified cytosine residues. The bisulfite ion treated gene sequence can be exposed to alkaline conditions, which convert bisulfite modified cytosine residues to uracil residues. Sodium bisulfite reacts readily with the 5,6-double bond of cytosine (but poorly with methylated cytosine) to form a sulfonated cytosine reaction intermediate that is susceptible to deamination, giving rise to a sulfonated uracil. The sulfonate group can be removed by exposure to alkaline conditions, resulting in the formation of uracil. The DNA can be amplified, for example, by PCR, and sequenced to determine whether CpG sites are methylated in the DNA of the sample. Uracil is recognized as a thymine by Taq polymerase and, upon PCR, the resultant product contains cytosine only at the position where 5-methylcytosine was present in the starting template DNA. One can compare the amount or distribution of uracil residues in the bisulfite ion treated gene sequence of the test cell with a similarly treated corresponding non-methylated gene sequence. A decrease in the amount or distribution of uracil residues in the gene from the test cell indicates methylation of cytosine residues in CpG dinucleotides in the gene of the test cell.”
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 McCord
so as to have specifically treated the DNA in the saliva sample with bisulfite under alkaline conditions because Guerrero-Preston teaches that this provides the advantage that methylated cytosines can be distinguished from unmethylated cytosines during subsequent analysis.
Secondly, McCord teaches (col. 16, lines 22-33) that the bisulfite treated DNA is amplified prior to sequencing (i.e., “treating the isolated DNA with bisulfite, PCR amplifying the genetic loci using specifically designed primers to produce amplicons corresponding to the genetic loci”), does not specifically recite that the amplifying step comprises performing methylation-sensitive PCR on the bisulfite converted DNA.
However, Guerrero-Preston teaches that methylation levels in the bisulfite-converted DNA can be determined by sequencing methods, including bisulfite genomic sequencing and quantitative PCR sequencing (e.g., para [0022], [0027], [0032], [0034] and [0039]) and by methylations-specific PCR, including quantitative methylation specific PCR (qPCR( (para [0031] and [0069-0070]).
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 McCord so as to have determined the methylation levels of the target CpG sequence by performing methylation-specific (or methylation-sensitive) PCR because Guerrero-Preston teaches that this is an equally effective means for quantitating the methylation level of CpGs following bisulfite treatment of sample DNA.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng (Winston) Shen can be reached on 571-272-0731. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682
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