DETAILED ACTION
Note that 5’-methylcytosine and 5’-hydroxymethylcytosine are herein abbreviated as 5mC and 5hmC, respectively, for brevity of this 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 .
Information Disclosure Statement
The information disclosure statement(s) (IDS) filed 14 May 2024 and 15 July 2024 are considered, initialed, and attached hereto.
Claim Status
Claims 1-5, 7-8, 10, 12, 14, 16-17, 19, 27-28, 41, 45-46, and 48-52 are pending and under examination.
Claims 6, 9, 11, 13, 15, 18, 20-26, 29-40, 42-44, and 47 are canceled.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code on page 35 line 17 of the specification filed 2 May 2024. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of terms including, but not limited to, Vent, Therminator, TaqMan, ResolveDNA, and others throughout the specification, which are trade names or marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Interpretation
Claim 1 (and all claims dependent upon claim 1) recites “converting” cytosine to uracil and modified cytosine to thymine (see step (b), lines 5-7 of claim 1). Because “converting” is recited as being done to the cytosine or modified cytosine base, this is interpreted as requiring that a cytosine or modified cytosine base is modified by conditions that cause changes including “deamination of the genomic DNA” (claim 1 lines 5-6) such that the cytosine or modified cytosine itself becomes a uracil or thymine, as appropriate. Therefore, this recitation does not include situations where the cytosine or modified cytosine is wholesale removed from the genetic sequence and replaced by a uracil or thymine, as appropriate, or situations where the cytosine or modified cytosine is converted not into uracil or thymine, as appropriate, but into something else that is read by a polymerase such that using the genomic DNA comprising the cytosine or modified cytosine as a template produces amplicons that have uracil or thymine, as appropriate, at the equivalent site in the amplicon as the base in question.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
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.
Claim 3 is 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 3 recites the limitation "the methylated cytosines" in line 1. There is insufficient antecedent basis for this limitation in the claim. This is interpreted as referring to the “5’-methylcytosines or 5’ hydroxymethylcytosines” recited in lines 1-2 of claim 2, but this limitation is unclear because it could alternatively be interpreted to refer narrowly to only 5mC or as broadly as to encompass any cytosine-derivative has a methyl group attached to it.
Claim Rejections - 35 USC § 112(a) - Scope of Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-5, 7-8, 10, 12, 14, 16-17, 19, 27, 28, 41, 45-46, and 48-52 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the method of claim 1 wherein the modified cytosines comprise 5mC, does not reasonably provide enablement for the method of claim 1 wherein the modified cytosines do not include 5mC, such as when the modified cytosines being identified are only 5hmC. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to practice the invention commensurate in scope with these claims.
Scope of the Claims/Nature of the Invention
The claims are drawn to a method of identifying modified cytosines in genomic DNA in a biological sample. In view of the recitation of “modified cytosines” (claim 1 line 1) and the lack of any limiting definition in the claim or the instant specification, claims 1, 8, 10, 12, 14, 16-17, 19, 27, 28, 41, 45-46, and 48-52 encompass identifying any type or combination of types of modified cytosine in genomic DNA in a biological sample. In view of the recitation of “wherein the modified cytosines are 5’-methylcytosines or 5’ hydroxymethylcytosines” (claim 2 lines 1-2) and the lack of any further limiting definition in the claim or the instant specification, claims 2-5 and 7 encompass identifying 5mC, 5hmC, or the combination of 5mC and 5hmC in genomic DNA in a biological sample. Note that claims 4 and 7 do not restrict the modified cytosine to just 5mC or just 5hmC, but rather limit the deamination of step (b) by defining a capability of enzyme(s) used in the deamination with regard to a conversion of one of 5mC or 5hmC. The nature of the invention is therefore a method of identifying ANY modified cytosine (or any of 5mC, 5hmC, or 5mC and 5hmC for claims 2-5 and 7) involving contacting the isolated genomic DNA under conditions that deaminate the genomic DNA to convert at least some of the cytosines to uracil and at least some of the modified cytosines to thymine.
Teachings in the Specification and Examples
In the Examples section, the instant specification teaches exposing isolated DNA to alkaline conditions at 72 degrees to deaminate unmethylated cytosines into uracil that are removed by USER treatment and to deaminate 5mC to thymine (page 36 section on “Deep Sequencing of methyl-PTA cells”, particularly lines 5-8 and 24-27). The remainder of the Examples section does not explicitly teach a method of converting 5hmC to thymine. In the instant specification on page 9 lines 7-10 teaches that deamination includes using an enzyme to convert 5hmC to thymine and provides SMUG1 as one such enzyme, reciting “Enzymes that can be used to convert 5’-hydroxymethylcytosine to thymine include, but are not limited to, Single-Strand-Selective Monofunctional Uracil-DNA Glycosylase 1 (SMUG1)”, but the instant specification provides no further evidence of such activity of SMUG1 or any teaching of how to use SMUG1 to convert 5hmC to thymine.
State of the Art and Unpredictability of the Art
The claims (except claims 2-5 and 7) broadly encompass methods of identifying modified cytosines in genomic DNA in a biological sample and claims 2-5 and 7 limit the modified cytosines to 5mC, 5hmC, or 5mC and 5hmC. The instant specification, as discussed above regarding page 9 lines 7-10, asserts that 5hmC can be enzymatically converted to thymine and that SMUG1 is an exemplary enzyme capable of this conversion. However, the prior art as would have been understood by one of ordinary skill in the art as of the effective filing date of the claimed invention, as exemplified by the post-effective filing date art reviewing DNA glycosylases of Lirussi and Nilsen (“DNA Glycosylases Define the Outcome of Endogenous Base Modifications”, Int J Mol Sci 24(12), 10307 (18 June 2023)), herein Lirussi, contradicts this assertion.
Lirussi teaches that deamination of 5hmC, as well as other modified cytosines that are not 5mC, such as 5-formylcytosine and 5-carboxylcytosine, when deaminated are converted not into thymine, like 5mC is, but rather into 5-hydroxymethyluracil (5hmU), 5-formyluracil, or 5-carboxyluracil, respectively (Figure 2 page 5). Furthermore, though Lirussi teaches that the TET enzyme can convert 5mC to 5hmC or thymine to 5hmU, Lirussi importantly does not teach any reverse process that can convert 5hmC to 5mC (such a reverse process would enable the claims, since 5hmC could be converted to thymine by first converting to 5mC and then deaminating it to thymine) or that can convert 5hmU to thymine (such a reverse process would enable the claims, since 5hmC could be deaminated to 5hmU and then to thymine by the reverse process) (Figure 2 page 5). Regarding SMUG1, Lirussi does not teach SMUG1 having a capability to convert 5hmC to thymine, but rather teaches that SMUG1 removes 5hmU modified bases from DNA as well as removing 5-formyluracil and 5-carboxyuracil, which are formed by the deamination of other modified cytosines (page 7 section 4 “Uracil DNA Glycosylases and Epigenetic Marks”). This is illustrated by base excision repair in Figure 3, where SMUG1 removes a uracil or 5hmU base from double stranded DNA, which is then replaced by the base complementary to the other strand (in the case of a 5hmU derived from deamination of a 5hmC of a C:G basepair, it would be replaced by C; in the case of a 5hmU derived from TET oxidation of thymine in a T:A basepair, it would be replaced by thymine). Note that though Figure 4 may appear to suggest that SMUG1 converts 5hmU to T, the legend of Figure 4 states that “SMUG1 recognizes 5-hmU on both nucleic acids. It restores the unmodified base on DNA via BER that can be derived from deamination of 5-hmC and oxidation of thymine”, which clarifies that the thymine of the figure is an example of the base that replaces the 5hmU during base excision repair. Since in the context of the claimed method a 5hmU removed by SMUG1 would have been produced by the deamination of an 5hmC from a C:G basepair as the method is for identifying modified cytosines, the complementary G would template for it to be replaced by a C, not by T.
Therefore, the art does not teach a way to convert 5hmC to thymine and teaches that the known activities of SMUG1 do not include the conversion of 5hmC, or a base that can be it be converted into without base excision and replacement, to thymine. Notably, even if “converting” were interpreted to include wholesale base excision and replacement, one of ordinary skill in the art would expect the 5hmC in genomic DNA that is identified in the claimed method would be basepaired to a guanine, so the replacement would be its complementary base, a cytosine, not a thymine, so the art also does not teach a method of performing the claimed conversion of 5hmC to uracil when viewed under a broader interpretation of “converting”.
Quantity of Experimentation
Though the level of skill of one of ordinary skill in the art of genomic analysis of methylation is high, being one with a relevant doctoral degree and laboratory experience, the quantity of experimentation necessary to enable the full scope of the claims is undue. In support of this position, it is noted that the claimed methods encompass identifying not just the specific modified cytosine, 5mC, but ANY type of, or combination of types of, modified cytosines, such as 5hmC (claims 2-5 and 7 being limited but still including the identifying 5hmC or 5mC and 5hmC together). Using the case of the identification of 5hmC as the modified cytosine as an example, in order practice the claimed invention one of ordinary skill in the art would need to conduct screening and verification experiments to identify heretofore unknown chemical or enzymatic processes that include deamination (in the case of claim 7 an enzyme must be involved) and cause a 5hmC base in genomic DNA to be converted to thymine while still maintaining sufficient integrity of the genomic DNA so that it is able to be amplified and sequenced for the eventual identification of the 5hmC base in the genomic DNA. The specification has not done any of the above and the one enzyme, SMUG1, recited as having such capability in the specification is contradicted by teachings in the art that SMUG1 acts to excise uracil and modified uracil bases from DNA rather than converting 5hmC to thymine. The results of such experimentation, screening for enzymatic or chemical reactions that are not known to occur, is highly unpredictable. This would further need to be done for other modified cytosines, such as 5’-carboxylcytosine. Therefore, the amount of experimentation that would be required to practice the full scope of the claimed invention and the amount of time and cost this experimentation would take supports the position that such experimentation is undue (see MPEP §2164.06).
Conclusions
Herein, although the level of ordinary skill in the art is high, given the lack of disclosure in the specification and the prior art and the unpredictability of the art, it would require undue experimentation for one of ordinary skill in the art to make and use the full scope of the invention as claimed in claims 1-5, 7-8, 10, 12, 14, 16-17, 19, 27, 28, 41, 45-46, and 48-52.
Claim Rejections - 35 USC § 103
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-5, 10, 27-28, 46, 49, and 51-52 are rejected under 35 U.S.C. 103 as being unpatentable over Vaisvila et al. (US 2021/0207200, published 8 July 2021), herein Vaisvila, and as evidenced by Spits et al. (“Whole-genome multiple displacement amplification from single cells” Nat Protoc 1(4), pages 1965-1970 (2006)), herein Spits, and in view of Gundry et al. (“Direct, genome-wide assessment of DNA mutations in single cells” Nucleic Acids Res 40(5), pages 2032-2040 (2011)), herein Gundry.
Regarding claim 1, Vaisvila teaches a method of identifying modified cytosines in genomic DNA in a biological sample (“determine the identity and location of unmodified or modified C” [0170]; “The method described above can be employed to analyze genomic DNA from virtually any organism” [0185]), the method comprising the steps of:
isolating, from the biological sample, nucleic acids comprising genomic DNA comprising cytosines and modified cytosines (“Exemplary NAs that can be used in the method include, for example, NA isolated from cells” [0213]; NA = nucleic acid)
contacting the isolated genomic DNA under conditions resulting in deamination of the genomic DNA thereby converting at least some of the cytosines in the genomic DNA to uracil and at least some of the modified cytosines to thymine (“the method may comprise reacting a first portion of a sample that contains relatively long, intact NA fragments […] with a GT and then a cytidine deaminase to produce a first product. This product differentiates C and 5mC from 5hmC as shown in FIG. 1B. A second portion of the sample may be reacted with a methylcytosine dioxygenase (and optionally a GT) as shown in FIG. 1A […] This reaction is followed by a cytidine deaminase reaction to distinguish between unmodified C and modified C” [0170-0171]; Figure 1; note in Figure 1B that deamination leads the C labeled with m, the 5mC, to be converted into a T and for unmodified Cs in the sequence to be converted into Us, so in the method of identifying the modified cytosines 5mC and 5hmC taught by Vaisvila 5mC, which is at least some of the modified cytosines, is converted to thymine);
amplifying the deaminated, isolated genomic DNA using an amplification process to product a genomic DNA library (“After the first and optionally second products are produced, they may be amplified and/or cloned, and then sequenced” [0173]), wherein the amplification process and library production comprises:
contacting the genomic DNA with at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides;
amplifying at least some of the genomic DNA to generate a plurality of amplification products, wherein replication proceeds by strand displacement replication (“The ss DNA product of enzyme reaction or reactions can be amplified by PCR or isothermal methods such […] multiple displacement amplification” [0189]; Spits provides evidence that multiple displacement amplification (MDA) includes contacting the nucleic acid with at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides “MDA […] 12 Prepare a master-mix that contains 9 µL of Reaction Buffer (containing nucleotides and additional random hexamers) and 1 µL of the Enzyme Mix, for each sample to amplify. 13 Pipette 10 µL of this mix into each sample” Spits page 1968 and Figure 1 on Spits page 1965 teaching Phi29 DNA polymerase and the hexamers being primers); and
sequencing the genomic DNA library, wherein the sequencing identifies the modified cytosines in the genomic DNA (“the genome may be fragmented into fragments of less than 1 kb in size to form a library for Next gen sequencing […] the adaptor ligated libraries may be sequenced using an Illumina sequencer. After the sequences of the first and optionally the second product are obtained, the sequences are compared with a reference sequence to determine which C's in the initial NA fragment are modified. A matrix illustrating an embodiment of this part of the method is illustrated in FIG. 1C” [0174]).
However, though Vaisvila teaches the method including the ligation of adaptors to generate a genomic DNA library, Vaisvila does not explicitly teach that this ligation ligates the adaptors to the amplification products as required by step (c)(iii) of claim 1. Instead, Vaisvila teaches that the adaptors may be added to the fragments prior to enzyme treatment (“Pyrrolo-dC modified adaptors may be added to the fragments in the library prior to enzyme treatment according to FIG. 1A-1B” [0174]). This deficiency is made up for in the teachings of Gundry.
Regarding claim 1, Gundry teaches a method of sequencing the genome of a single cell by amplifying the whole genome using multiple displacement amplification followed by Illumina sequencing involving the ligation of adaptors to the amplification products, which has the advantage of being able to sequence a single cell rather than requiring bulk genomes from a population of multiple cells (“Multiple displacement amplification was performed on single cells using the REPLI-g UltraFast Mini kit […] Cells with the highest number of loci present at a sufficient level (relative abundance > 0.25) were chosen for Illumina sequencing […] Up to 5 µg of DNA was used as input to make Illumina libraries […] control DNA or single cell DNA was randomly fragmented […] The fragmented DNA was end-repaired and size-selected […] The size-selected material was A-tailed and ligated to Illumina paired-end adapters […] Libraries were sequenced using 108-bp paired-end sequencing (S2 cells) or 121-bp single-end sequencing (MEFs) on the HiSeq 2000” page 2034 section titled ‘Single cell isolation and WGA’ through end of page 2034 left column).
Regarding claim 2, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Vaisvila further teaches that the modified cytosines are 5’-methylcytosines or 5’ hydroxymethylcytosines (“The position of a 5mC in the initial NA fragment is determined by the presence of a C in the first product or a T in the second product iii. The position of a 5hmC in the initial NA fragment is determined by the presence of a C in the second product only” [0176-0177]; Figure 1).
Regarding claim 3, the combination of Vaisvila and Gundry teach the method of claim 2 (see 35 U.S.C. 103 rejection of claim 2 above), and Vaisvila further teaches that identifying the 5mC or 5hmC in the genomic DNA comprises identifying CpG islands in the genomic DNA (“Present embodiments provide many advantages over existing systems that result from factors that include […] higher coverage in C rich regions and CpG islands” [0191]).
Regarding claim 4, the combination of Vaisvila and Gundry teach the method of claim 2 (see 35 U.S.C. 103 rejection of claim 2 above), and Vaisvila further teaches that deamination comprises contacting the isolated genomic DNA with an enzyme that converts cytosine to uracil and 5’-methylcytosine to thymine (“then a cytidine deaminase to produce a first product. This product differentiates C and 5mC from 5hmC as shown in FIG. 1B” [0170]; Figure 1; note in Figure 1B that deamination by the recited cytidine deaminase enzyme converts C to U and the 5’-methylcytosine on the right end of the sequence to a T).
Regarding claim 5, the combination of Vaisvila and Gundry teach the method of claim 4 (see 35 U.S.C. 103 rejection of claim 4 above), and Vaisvila further teaches that the enzyme is APOBEC3A or other members of the APOBEC family of cytidine deaminases (“As used herein, the term “cytidine deaminase” refers to an enzyme that is capable of deaminating C to form a U. Many cytidine deaminases are known. For example, the APOBEC family of cytidine deaminases is described in U.S. Pat. No. 9,121,061 […] In any embodiment, the deaminase used may have an amino acid sequence that is at least 90% identical to (e.g., at least 95% identical to) the amino acid sequence of GenBank accession number AKE33285.1, which is the human APOBEC3A” [0148]).
Regarding claim 10, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Vaisvila further teaches that the contacting of step (b) occurs over a period of time of between four (4) and twenty four (24) hours (“deaminated with 0.3 mg of APOBEC3A in 100 I reaction volume for 16 hours for 5hmC detection” [0247]).
Regarding claim 27, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Vaisvila further teaches that the biological sample is a low input sample such as a forensic sample or ancient genomic fragments (“The method described above can be employed to analyze genomic DNA from virtually any organism, including, but not limited to, […] archaeological/ancient samples […] The sample may be made from […] scrape or lavage or cells of a forensic sample” [0185]).
Regarding claim 28, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Gundry further teaches that the biological sample is a single cell (“Multiple displacement amplification was performed on single cells” page 2033 right column).
Regarding claim 46, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Gundry further teaches that the method further comprises a step of enrichment PCR, which is carried out in the presence of magnesium (“Approximately 10 ng of the product was used as input for 10 cycles of enrichment PCR with […] 2mM MgSO4” page 2034 right column paragraph 2). Note that the language of the claim reciting “wherein the method is carried out in the presence of magnesium, DTT or both” is given its broadest reasonable interpretation as requiring that any portion of the method be carried out in the presence of magnesium, DTT, or both, rather than requiring that every single step of the method be carried out in the presence of magnesium, DTT or both.
Regarding claim 49, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Spits provides evidence that the “multiple displacement amplification” (Vaisvila [0189]) of the method comprises one or more cycles of amplification, wherein each cycle comprises denaturing, annealing, and extension, and wherein the temperature remains constant during each cycle (“Multiple displacement amplification (MDA) has been described as a new method of WGA that leads to the synthesis of DNA with limited sequence representation bias. This method is based on the annealing of random hexamers to denatured DNA, followed by strand-displacement synthesis at a constant temperature” Spits page 1965 left column paragraph 2).
Regarding claim 51, the combination of Vaisvila and Gundry teach the method of claim 46 (see 35 U.S.C. 103 rejection of claim 46 above), and Gundry further teaches that concentration of the magnesium is 0.6 to 5 mM (“Approximately 10 ng of the product was used as input for 10 cycles of enrichment PCR with […] 2mM MgSO4” page 2034 right column paragraph 2). Examiner notes that this claim does not exclude DTT as recited in claim 46 on which it depends, so a method according to claim 46 with any concentration of DTT would read on claim 51.
Regarding claim 52, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Vaisvila further teaches that the contacting of step (b) occurs over a period of time between 30 minutes and 240 minutes (“cytidine deaminase (0.3 mg/ml) were added (see also Bransteitter et al. PNAS (2003) vol. 100, 4102-4107) and incubated for 3 hours” [0227]).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the order of steps of ligating adapters to amplification products for generation of the DNA library as taught by Gundry for the ligation of adaptors to fragments prior to enzyme treatment in the method taught by Vaisvila (MPEP §2143 I. B.). Additionally, one of ordinary skill in the art would be motivated to combine Gundry with Vaisvila because Gundry teaches that using multiple displacement amplification followed by Illumina library prep and sequencing enables analysis of a sequence from a single cell, allowing the detection of low-abundance events that can be masked in bulk sequencing methods (“This allows only the detection of clonally amplified mutations present in most or all of the cells in a tissue sample and essentially constrains access to low-abundance mutations. One way to circumvent this problem is to sequence genomes of individual cells after whole genome amplification (WGA)” page 2032 right column paragraph 1) (MPEP §2143 I. G.). One of ordinary skill in the art could have performed this substitution and would have found the results of this substitution predictable with a reasonable expectation of success because, firstly, Vaisvila discloses multiple displacement amplification as an option for amplifying the deaminated DNA, and, secondly, Vaisvila only teaches that the ligation of adaptors before enzyme treatment is an option through the word may and does not recite any steps that actually require the adaptors to be ligated before deamination or amplification are done. Therefore, the invention as a whole of claims 1-5, 10, 27-28, 46, 49, and 51-52 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claims 8 and 50 are rejected under 35 U.S.C. 103 as being unpatentable over Vaisvila et al. (US 2021/0207200, published 8 July 2021), herein Vaisvila, and as evidenced by Spits et al. (“Whole-genome multiple displacement amplification from single cells” Nat Protoc 1(4), pages 1965-1970 (2006)), herein Spits, and in view of Gundry et al. (“Direct, genome-wide assessment of DNA mutations in single cells” Nucleic Acids Res 40(5), pages 2032-2040 (2011)), herein Gundry, as applied to claims 1-5, 10, 27-28, 46, 49, and 51-52 above, and further in view of Wang et al. (“Heat- and alkali-induced deamination of 5’methylcytosine and cytosine residues in DNA” Biochim Biophys Acta 697(3), pages 371-377 (1982)), herein Wang 1.
Regarding claim 8, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above). However, neither Vaisvila nor Gundry teach that the contacting of step (b) comprises incubating the genomic DNA at a temperature between 50°C and 100°C or between 50°C and 65°C. This deficiency is made up for in the teachings of Wang 1.
Regarding claim 8, Wang 1 teaches the deamination of cytosine to uracil and 5mC to thymine by incubating DNA in 1 M NaOH at 60°C (“In 1 M NaOH at 60°C, 5-methyldeoxycytidine was converted to deoxythymidine and deoxycytidine to deoxyuridine” page 372 right column paragraph 3; “Deamination of cytosine and 5-methylcytosine residues in DNA Upon incubation of XP-12 DNA or X. oryzae DNA in 1 M NaOH at 60°C, deamination of 5-methylcytosine and cytosines residues was detectable” page 373 right column paragraph 3).
Regarding claim 50, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Wang 1 teaches the deamination of cytosine to uracil and 5mC to thymine in 1 M NaOH at 60°C wherein after 1 hour 1.8% of cytosine were deaminated to uracil and 2.9% of 5mC were deaminated to thymine and after 6 hours 11% of cytosine were deaminated to uracil and 2.9% of 5mC were deaminated to thymine, which along with other results leads Wang to conclude that the rate of conversion of 5mC to thymine is approximately 1.5-times the rate of conversion of cytosine to uracil (Table I page 373; Table II page 374; “Upon incubation of XP-12 DNA or X. oryzae DNA in 1 M NaOH at 60°C, deamination of 5-methylcytosine and cytosine residues was detectable beginning at 10 min (Table II). The rate of conversion of the former residues to thymine residues was approx. 1.5-times the rate of conversion of the latter residues to uracil (Table I)” page 373 right column paragraph 3). Though none of the values taught by Wang 1 explicitly fall in the claimed range of 0.1% to 10% of cytosines being converted to uracil and 10% to 100% of modified cytosines (such as 5mC) being converted to thymine, the difference in the rate of conversion taught by Wang clearly encompasses such embodiments, since, for example a duration yielding 7% conversion of cytosine to uracil would be expected to yield 1.5-fold more (10.5%) conversion of 5mC to thymine. Furthermore, the values of 11% conversion of cytosine to uracil and 16% conversion of 5mC to thymine after 6 hours is very near to the claimed range, which would be achieved by a slightly shorter duration (or lower temperature or decreased NaOH concentration), so one of ordinary skill in the art would be expected to obtain the claimed range within the course of optimization through routine experimentation. Therefore, the claimed ranges would be prima facie obvious in view of the percentages taught by Wang 1. See MPEP §2144.05 II.
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of deamination by using the combined heat and alkaline induction taught by Wang 1 for the deamination by APOBEC family deaminases taught by the method of the combination of Vaisvila and Gundry (MPEP §2143 I. B.). One of ordinary skill in the art could have performed this substitution and would have found the results of this substitution predictable because Vaisvila uses the deaminase enzymes to deaminate cytosine and/or 5mC and Wang 1 teaches that the heat and alkaline conditions they teach deaminate cytosine and 5mC. Therefore, the invention as a whole of claims 8 and 50 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Vaisvila et al. (US 2021/0207200, published 8 July 2021), herein Vaisvila, and as evidenced by Spits et al. (“Whole-genome multiple displacement amplification from single cells” Nat Protoc 1(4), pages 1965-1970 (2006)), herein Spits, and in view of Gundry et al. (“Direct, genome-wide assessment of DNA mutations in single cells” Nucleic Acids Res 40(5), pages 2032-2040 (2011)), herein Gundry, as applied to claims 1-5, 10, 27-28, 46, 49, and 51-52 above, and further in view of Wang et al. (“Characterization of denaturation and renaturation of DNA for DNA hybridization” Eviron Health Toxicol 29, e2014007 (2014)), herein Wang 2, and as evidenced by Qiagen (“REPLI-g® UltraFast Mini Handbook” (2011)), herein Qiagen 1, and Qiagen (“REPLI-G Kits” https://www.qiagen.com/us/products/discovery-and-translational-research/pcr-qpcr-dpcr/preamplification/repli-g-kits/ downloaded from WayBack Machine snapshot dated 28 April 2021), herein Qiagen 2.
Regarding claim 12, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Gundry further teaches the lysis of the cells for obtaining the DNA using the REPLI-g UltraFast Mini kit according to the manufacturer’s instructions (“using the REPLI-g UltraFast Mini kit (Qiagen, Santa Clara, CA, USA) according to the manufacturer’s instructions with some modification. After lysis…” page 2033 right column). Qiagen 1 provides evidence that the lysis according to manufacturer’s instructions of the REPLI-g UltraFast Mini kit would necessarily include contacting the biological sample with a lysis and denaturation buffer (“The sample material is lysed and the DNA is denatured by adding denaturation buffer” Qiagen 1 page 7; “Buffer D1 (denaturation buffer)” Qiagen 1 page 10) and Qiagen 2 provides evidence that the denaturation buffer is an alkaline buffer (“REPLI-g Kits use gentle alkaline incubation, allowing uniform DNA denaturation with very low fragmentation or generation of abasic sites” Qiagen 2 page 4 paragraph 3). However, it is not clear whether the alkaline conditions of the lysis and denaturation taught by Gundry and evidenced by Qiagen 1 and 2 include a pH of between 9 and 14. This deficiency is made up for in the teachings of Wang 2.
Regarding claim 12, Wang 2 teaches using alkaline solutions with pH between 9 to 14 for denaturation of DNA (Figure 2; page 3 right column paragraph 2 labeled ‘Alkaline’; page 6 right column paragraph 2 labeled ‘Alkaline’; note that the taught conditions all fall within this pH range, since 1 mol/L NaOH has a pH of 14, 0.1 mol/L NaOH has a pH of 13, and 0.01 mol/L NaOH has a pH of 12).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the alkaline denaturation solutions with specific pHs taught by Wang 2 for the alkaline denaturation buffer of unspecified pH in the method of the combination of Vaisvila and Gundry as evidenced by Qiagen 1 and 2 (MPEP §2143 I. B.). One of ordinary skill in the art could have performed this substitution and would have found the results of this substitution predictable because both Wang 2 and Qiagen 1 and 2 teach the purpose of the alkaline solutions of the respective references is to denature DNA. Therefore, the invention as a whole of claim 12 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claims 41 and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Vaisvila et al. (US 2021/0207200, published 8 July 2021), herein Vaisvila, and as evidenced by Spits et al. (“Whole-genome multiple displacement amplification from single cells” Nat Protoc 1(4), pages 1965-1970 (2006)), herein Spits, and in view of Gundry et al. (“Direct, genome-wide assessment of DNA mutations in single cells” Nucleic Acids Res 40(5), pages 2032-2040 (2011)), herein Gundry, as applied to claims 1-5, 10, 27-28, 46, 49, and 51-52 above, and further in view of Gawad et al. (US Patent Application Publications Cite No 1 in IDS filed 14 May 2024)(US 2020/0362394, published 19 November 2020), herein Gawad.
Regarding claim 41, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above). However, neither Vaisvila nor Gundry teach that the mixture of nucleotides comprises at least one terminator nucleotide which terminates nucleic acid replication by the polymerase. This deficiency is made up for in the teachings of Gawad.
Regarding claim 41, Gawad teaches a method for nucleic acid amplification, Primar Template-Directed Amplification (PTA), which is contrasted with multiple displacement amplification (MDA) in FIG. 1A and provides the advantage of “increased accuracy and sensitivity of downstream applications” ([0054]) and that uses a mixture of nucleotides comprising at least one terminator nucleotide which terminates nucleic acid replication by the polymerase (“Provided herein are methods of amplifying a target nucleic acid molecule, the method comprising: contacting a sample comprising the target nucleic acid molecule, at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide which terminates nucleic acid replication by the polymerase, and amplifying the target nucleic acid molecule to generate a plurality of terminated amplification products, wherein the replication proceeds by strand displacement replication” [0005]).
Regarding claim 45, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above), and Gawad teaches the alternative method of nucleic acid amplification as described with regard to claim 41 above wherein at least some of the amplification products comprise a cell barcode or a sample barcode (“Further provided herein are methods wherein the at least one amplification primer comprise a barcode. Further provided herein are methods wherein the barcode comprises a cell barcode. Further provided herein are methods wherein the barcode comprises a sample barcode” [0006]).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the method of whole genome amplification taught by Gawad for the method of whole genome amplification taught by Vaisvila and Gundry in the method of the combination of Vaisvila and Gundry (MPEP §2143 I. B.). Furthermore, one of ordinary skill in the art would be motivated to make this substitution because Gawad teaches that their method provides the advantage of increased accuracy and sensitivity compared to the method used by Vaisvila and Gundry (MPEP §2143 I. G.). One of ordinary skill in the art could have performed this substitution and would have found the results of this substitution predictable because both methods are taught as being used for the purpose of whole genome amplification. Therefore, the invention as a whole of claims 41 and 45 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claim 48 is rejected under 35 U.S.C. 103 as being unpatentable over Vaisvila et al. (US 2021/0207200, published 8 July 2021), herein Vaisvila, and as evidenced by Spits et al. (“Whole-genome multiple displacement amplification from single cells” Nat Protoc 1(4), pages 1965-1970 (2006)), herein Spits, and in view of Gundry et al. (“Direct, genome-wide assessment of DNA mutations in single cells” Nucleic Acids Res 40(5), pages 2032-2040 (2011)), herein Gundry, as applied to claims 1-5, 10, 27-28, 46, 49, and 51-52 above, and further in view of Angermueller et al. (“Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity” Nat Methods 13(3), pages 229-232 (2016)), herein Angermueller 1, and as evidenced by Supplementary Figure 1 of Angermueller et al. (“Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity” Nat Methods 13(3), supplementary figure 1 available online (2016)), herein Angermueller 2.
Regarding claim 48, the combination of Vaisvila and Gundry teach the method of claim 1 (see 35 U.S.C. 103 rejection of claim 1 above). However, neither Vaisvila nor Gundry teach that the nucleic acids further comprise mRNA and the method further comprises converting the mRNA to cDNA by reverse transcription, amplifying the cDNA, and sequencing the cDNA. This deficiency is made up for in the teachings of Angermueller 1.
Regarding claim 48, Angermueller 1 teaches a method for parallel single-cell genome-wide methylome and transcriptome sequencing (Abstract) by separating the RNA and DNA so that the processing of the DNA for methylome analysis does not affect the RNA (“physical separation of RNA and DNA, which allows for bisulfite conversion of DNA without affecting the transcriptome” page 229 right column paragraph 1) and further producing cDNA based on the mRNA, amplifying the cDNA, and sequencing the CDNA (“Separation of poly-A mRNA and DNA”, “Reverse transcription and PCR amplification”, “Illumina sequencing” Angermueller 2). Angermueller 1 further teaches that the combined analysis of methylomes and transcriptomes from single cells is advantageous because it is “a powerful tool for dissecting relationships among epigenetic, genomic and transcriptional heterogeneity” (page 229 left column paragraph 1).
One of ordinary skill in the art would be motivated to combine the method of obtaining methylome data and transcriptome data from a single-cell by physical separation of its DNA and RNA as taught by Angermueller with the method of identifying modified cytosines (methylome data) taught by the combination of Vaisvila and Gundry in order to improve the method of Vaisvil and Gundry with the advantageous capability of being able to better dissect the relationship between epigenetic and transcriptional states in single cells (MPEP §2143 I. G.). One of ordinary skill in the art would have a reasonable expectation of success in this combination because the separation of RNA from the DNA before the deamination of genomic DNA would not be expected to prevent the DNA from being able to be deaminated and further analyzed for the identification of modified cytosines. Therefore, the invention as a whole of claim 48 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claims Free of the Prior Art
Claims 7, 14-17, and 19 are free of the prior art.
Claim 7 is free of the prior art because, as discussed with regard to the rejection of claims 1-5, 7-8, 10, 12, 14, 16-17, 19, 27, 28, 41, 45-46, and 48-52 under 35 U.S.C. 112(a) for lacking enablement of the full scope of the claims above, the prior art does not teach any method of converting “5’-hydroxymethylcytosine to thymine”, as required by claim 7.
Claim 14 is free of the prior art because the prior art does not teach a step of contacting deaminated, isolated genomic DNA with an enzyme to remove uracil from the genomic DNA. The nearest prior art such as Song et al. (Foreign Patent Document Cite No 2 in IDS filed 14 May 2024)(WO 2021/005537, published 14 January 2021, effectively filed 8 July 2019) teaches using an enzyme to remove uracil from genomic DNA prior to amplification in a process of identifying modified cytosines (“DHU digestion by USER enzyme”, Fig. 29A). However, this process identifies modified cytosines by converting them, but not unmodified cytosine, to a uracil derivative that is removed and the DNA cleaved at the location of the site of the uracil derivative, allowing the locations of the modified cytosines to be identified based on the cleavage sites. This would not be compatible with the method of Vaisvila that the combination of Vaisvila and Gundry is based upon, since in Vaisvila unmodified cytosine is converted to a uridine (see Figure 1).
Claims 15-17 and 19 are free of the prior art due to their dependency on claim 14.
Citation of Pertinent Prior Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Kohli et al. (US 2025/0283170, effectively filed 12 July 2021) teaches methods of methylation analysis similar to those of Vaisvila.
Glezer (US 2024/0271208, effectively filed 3 September 2021) teaches methods of methylation analysis similar to those of Vaisvila.
Conclusion
Claims 1-5, 7-8, 10, 12, 14, 16-17, 19, 27, 28, 41, 45-46, and 48-52 are rejected. Claims 6, 9, 11, 13, 15, 18, 20-26, 29-40, 42-44, and 47 are canceled. No claims are allowed.
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/JEFFREY BELLAH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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