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 .
Status of the Application
Claims 1-9, 12-29 are pending and under examination
The following Office Action is in response to Applicant's communication dated 7/12/2024.
Claim Rejections - 35 USC § 112(b)
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(s) 8, 9, 12, 14, 20 and 26-29 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 8 and 26, the phrase “the DNA mixture” lacks proper antecedent basis. Claims 1 and 2 recites “target nucleic acid” and “reaction mixture(s)”, but claims 8 and 26 recite “the DNA mixture is deaminated. Neither claim 2 or 1 provides clear antecedent support for “DNA mixture”.
Claims 9 depends from claim 8 and is/are therefore similarly rejected.
Claims 27-29 depends from claim 26 and is/are therefore similarly rejected.
Claim 12 depends on claim 1 and recites “the second polymerase”. However, claim 1 does not recite any “second polymerases”. Thus, the phrase “the second polymerase” lack antecedent basis.
Claim(s) 13 depend from claim 12 and is/are therefore similarly rejected.
Claim 14 contains trademark/trade names Phusion and Q5. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe DNA polymerase class and, accordingly, the identification/description is indefinite.
Claim 20, the phrase “the primers of the first set” lacks proper antecedent basis. Claim 1 and 2 recites “first primer”, i.e. singular primer, while claim 20 recites plural primers. Neither claim 1, 2 nor dependency chain leading to claim 30 provides clear antecedent support for “the first set”.
Claim 29, the phrase “ the deaminated strands” lacks proper antecedent basis. Claim 26 recites deaminating “the DNA mixture”, but does not introduce “deaminated strands”
Claim Interpretation
“Unusual nucleoside” is interpreted as a nucleotide that is functionally or chemically different from the four standard deoxynucleoside triphosphate: deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP), consistent with definition in ¶0014 of Applicant’s specification. The term encompasses nucleoside and nucleoside analogues, modified nucleosides, non-naturally occurring analogue structures.
Claim 18 is interpreted as requiring a specific universal sequence tail assignment strategy, wherein opposing primers that flank a desired sequence lengths comprise 2 different universal tails (each end have a different universal tail), while opposing primers that are too close/far from each other (i.e. undesired length) comprise the same universal tails at both ends.
Claims 21 is interpreted as requiring the second DNA polymerase capable of using synthesized modified complementary strand comprising unusual nucleotide(s) as templates for subsequent amplification. This is consistent with Applicant specification describing second polymerase capable of using the modified complementary strand as template [¶0123].
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Ren et al.
Claim(s) 1, 2, 5, 6, 12, 16, 21, and 22 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ren et al. (Analyst 140.8 (2015): 2671-2678.).
Regarding claim 1, Ren discloses method of processing target nucleic acids comprising (a) providing a reaction mixture(s), each reaction mixture comprising a first polymerase, one or more unusual nucleoside triphosphates and a first primer, wherein the polymerase is capable of extending a primer using the target nucleic acids as templates and incorporating the unusual nucleotide into extension products to produce modified complementary strands, and cannot efficiently make a further copy using the modified complementary strands as templates, wherein the unusual nucleoside triphosphate is distinct from the four standard nucleotides; and (b) performing one pass extension or cycles of extension reactions of the first primer on target nucleic acid template to produce modified complementary strands, which cannot efficiently be served as template for further copying in the reaction using the first polymerase. ; (e.g. 5-Azidomethyl dUTP have been incorporated into DNA by reverse transcription. Reverse transcription reaction comprises reverse transcriptase (M-MuLV RT), azidomethyl dUTP or unmodified dNTP, and DNA primer [Abstract and Experimental section]. Since M-MuLV is a reverse transcriptase, its primary function is to synthesize a complementary DNA (cDNA) strand using an RNA template. Its ability to read and process DNA template is significantly lower than when it is reading an RNA template.)
Regarding claim 2, Ren discloses claim 1 and (a) providing a reaction mixture(s), each reaction mixture comprising a first polymerase, four or more different nucleoside triphosphates including one or more unusual nucleoside triphosphates and a first primer, wherein the polymerase is capable of extending a primer using the target nucleic acids as templates and incorporating the unusual nucleotide into extension products to produce modified complementary strands, and is incapable of efficiently making a further copy using the modified complementary strand as template for extension of primers in the opposite orientation, wherein the unusual nucleoside triphosphate is distinct from the four standard nucleotides (deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP)), and is capable of being incorporated into new strands; (b) performing one pass extension or cycles of extension reactions of the first primer on target nucleic acid template to produce modified complementary strands, (e.g. 5-Azidomethyl dUTP (AM dUTP) have been incorporated into DNA by reverse transcription. Reverse transcription reaction comprises reverse transcriptase (M-MuLV RT), azidomethyl dUTP or unmodified dNTP, and DNA primer [Abstract, Experimental section, and Fig 6]. Since M-MuLV is a reverse transcriptase, its primary function is to synthesize a complementary DNA (cDNA) strand using an RNA template. Its ability to read and process DNA template is significantly lower than when it is reading an RNA template.)
(c) adding a second polymerase which is capable of using the modified complementary strands as templates; and (d) replicating or amplifying the modified complementary strands and/or the original strands using the second polymerase. (e.g. Ren further discloses successful PCR amplification involves DNA polymerase reading AM dUTP containing templates strands while incorporating AM dUTP into newly synthesized daughter strands [Abstract and pages 5-6])
Regarding claim 5, Ren discloses step (c) further comprises adding a second primer which is capable of extension in step (d). (e.g. two primers (P7, P8, Table 1) are used in polymerase chain reaction [page 2])
Regarding claim 6, Ren discloses removing the unusual nucleoside triphosphate and/or primers by purification or an enzymatic reaction. (e.g. newly synthesized cDNA is purified using ethanol precipitation [Experimental section])
Regarding claim 12, Ren discloses second polymerase is a DNA polymerase (e.g. KOD1 [Experimental section])
Regarding claim 16, Ren discloses first primer comprises a set of multiple target specific primers, wherein the primer sequence comprises a 3′ target specific sequence [Experimental and table 1].
Kester et al.
Claim(s) 23-25 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Kester et al. (US20110201524A1, EFD: October 21st 2009).
Regarding claims 23-25, Kester discloses method of preparing a sequencing library for methylation analysis comprising:
(a) providing a reaction mixture(s), each reaction mixture comprising nucleic acids to be sequenced, a first DNA polymerase, unusual nucleoside triphosphates and a first set of primers, wherein the unusual nucleoside triphosphates is 5-Methyl-2′-deoxycytidine-5′-Triphosphate, wherein the polymerase is capable of extending primers using the target nucleic acids as templates and incorporating the unusual nucleotide into extension products which are modified complementary strands, wherein the first set of primers comprise target specific primers, universal primers or random primers; (e.g. A method of sequencing nucleic acid comprising cytosine is provided. The method can include the steps of providing a sample comprising a template nucleic acid; generating a complementary copy of the template nucleic acid, wherein the generating produces a complementary copy of the template nucleic acid such that cytosine residues in the complementary copy are conversion resistant cytosine analogs comprising a moiety that inhibits conversion to another base residue. In certain aspects, the generating is directed by oligonucleotide primer [¶0012]. Cytosine analog can be selected from the group consisting of: 5-ethyl dCTP, 5-methyl dCTP [¶0016]. The polymerase that can copy the template in the 5′ to 3′ direction provided that sufficient quantities of free nucleotides, such as dATP, dGTP, dCTP, 5-methyl dCTP and dTTP are present. Examples of DNA polymerases include, but are not limited to Pyrococcus furiosus (PfU) DNA polymerase [¶0060] (PfU is known to be able to incorporate 5-5-Methyl-dCTP to generate 5-5-Methyl-dCTP containing complementary strand. Primers maybe sequence specific [¶0092]. nucleic acids can also have universal priming site for universal primer [¶0021 and ¶0150])
(b) performing extension reaction of primer on target nucleic acid template to produce modified complementary strands under extension condition, wherein the extension condition comprises buffer, any of four standard nucleoside triphosphates and appropriate temperature; ; (e.g. A method of generating a complementary copy of the template nucleic acid, wherein the generating produces a complementary copy of the template nucleic acid such that cytosine residues in the complementary copy are conversion resistant cytosine analogs comprising a moiety that inhibits conversion to another base residue. [¶0012].)
(c) deaminating the DNA mixture by either chemical and/or enzymatic processes; (subjecting the template nucleic acid and the complementary copy to conversion treatment to convert cytosine residues in the template nucleic acid into residues comprising the other base (e.g. bisulfite treatment to convert unmethylated cytosine residues in the template nucleic acid into uracil residues) [¶0012-0013]
(d) purifying the DNA mixture; (e.g. obtaining bisulfite-converted template nucleic acid comprising at least one uracil residue; obtaining a non-converted complementary copy of the template nucleic acid [¶0014])
(e) performing amplification of the DNA mixture using a second set of primers and using a second DNA polymerase; (e.g. The bisulfite-treated DNA can subsequently be analyzed by conventional molecular techniques, such as PCR amplification, sequencing, and detection comprising oligonucleotide hybridization. [0052])
(f) processing the products of step (e) to complete the library preparation for massive parallel sequencing. (e.g. Sequencing can be carried out using any suitable sequencing technique, wherein nucleotides are added successively to a free 3′ hydroxyl group, resulting in synthesis of a nucleic acid chain in the 5′ to 3′ direction. The nature of the nucleotide added is preferably determined after each nucleotide addition. Sequencing techniques using sequencing by ligation and techniques such as massively parallel signature sequencing (MPSS) where bases are removed from, rather than added to the strands on the surface are also useful, as are techniques using detection of pyrophosphate release (pyrosequencing).[0131])
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.
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.
Ren et al.
Claim(s) 3 and 4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.)
Regarding claims 3 and 4, Ren does not explicitly disclose repeating the reverse transcription reaction for 2 to 40 cycles. However, as of the application’s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to repeat Ren’s reverse transcription reaction as needed to obtain an appropriate amount of modified cDNA product. Repetition of a known successful synthesis would have predictably produced more modified cDNA product using the same established reaction conditions. This replication represents routine optimization of product yield according to the downstream analysis demands. As set forth in MPEP 2144.05; In re Peterson, 315 F.3d 1325 (Fed. Cir. 2003); "[A] prior art reference that discloses a range encompassing a somewhat narrower claimed range is sufficient to establish a prima facie case of obviousness.")
Ren et al. and Yu et al.
Claim(s) 7-9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of Yu et al. (Arch. Insect Biochem. Physiol., 42: 198-212. ).
Regarding claims 7-9 Ren does not teach the unusual nucleoside triphosphate is 5-Methyl-2'-deoxycytidine-5'-Triphosphate.
Yu discloses M-MuLV reverse transcriptase (used in Ren reverse transcription reaction) able to use 5-methyl-dCTP as substrate to synthesize cDNA [Method and Material section].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Ren’s method by selecting 5-methyl-dCTP as the unusual nucleoside triphosphate to protect the DNA from unwanted endonuclease digestion [Yu method section]. Additionally, skilled artisan would have reasonable expectation of success because Yu demonstrates established reaction conditions to successfully yield 5-methyl-dCTP containing DNA. With respect to “protected from deamination” and “deamination is a chemical conversion by bisulphate” limitations, one of ordinary skill in the art would have understood that the presence of a methyl group at the C5 position of a cytosine base physically blocks the chemical reaction of bisulfite ions. Hence once 5-methyl-dCTP in incorporated to newly synthesized complementary strand, the methylated sites would be predictably protected from bisulfite mediated deamination.
Ren et al. and Sorge et al.
Claim(s) 13-14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of Sorge et al. (US20040081965A1, Disclosed in IDS).
Regarding claim 13 and 14, Ren does not teach first polymerase is an archaeal DNA polymerase, or a modified archaeal DNA polymerase.
Sorge teaches PCR reaction mixture comprising target DNA, primers, dNTPs(e.g. dATP, dTTP, dCTP, dGTP), unusual nucleotide (e.g. dUTP), and DNA polymerases, wherein DNA polymerase incorporate dUTP using two primers and multiple cycles, generating an dUTP containing daughter strand. However, once incorporated, dUTP containing DNA templates cannot be efficiently amplified by DNA polymerases (i.e. dUTP containing nucleic acid sequences cannot efficiently be served as template for further copying in the reaction using DNA polymerases.) [¶0002, ¶0148-0149, and ¶0156-0159]. The polymerase is archaeal DNA polymerases such as Pfu, KOD, Vent, and Deep Vent DNA polymerases [¶0002].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to substitute archaeal DNA polymerases to generate modified complementary strands if the template is DNA rather than RNA. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Ren et al. and Zong et al.
Claim(s) 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of Zong et al. (Science. 2012;338(6114):1622-1626).
Regarding claim 15, Ren does not teach the first primer comprises a set of random primers, wherein the random primers comprise 3' random sequence with or without 5' universal tails, and wherein the first primer is capable of hybridizing to any random regions.
Zong teaches multiple annealing and looping-based amplification cycles (MALBAC), which relies on chimeric primers that hybridize randomly to templates. MALBAC primer is typically about 35 nucleotides long and consists of two essential parts: 5’ universal region (27 nucleotides, act as a adapter/tag for downstream processing) and 3' Variable/Random Region (8 nucleotides, act as a primer that hybridize nonselectively to any region of the target genome) [page 1622].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Ren’s method to use MALBAC primers in place of target specific primers to expand sequencing coverage because MALBAC primer amplification offers high uniformity across the genome, amplified DNA achieves 93% genome coverage, and minimal false positives detected [Zong’s abstract]. Such modification represents substituting target specific primer for random/variable primer in the art to predictably overcome amplification bias/ low genome coverage. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Ren et al. and Fu et al.
Claim(s) 17, 19, and 20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of Fu et al. (US11408025B2, Published: April 16th 2020).
Regarding claim 17, Ren does not teach that primers comprise a random unique molecular identifier, and a 5' universal tail sequence.
Fu teaches that primers comprise 3' target specific sequence, a random unique molecular identifier, and a 5' universal tail sequence [Fig 1A].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Ren’s PCR method to use Fu’s primer deign comprising a random unique molecular identifier, and a 5' universal tail sequence because these are conventional and highly effective tools used in Next-Generation Sequencing (NGS) to accurately track newly synthesize C5-modified dUTP containing molecules. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143, A and C)
Regarding claim 19, Fu teaches set of multiple target specific primers comprise the same sequence of 5' universal tails. (e.g. The universal primers in the forward and reverse reactions may be the same.[column 25, lines 46-48])
Regarding claim 20, the limitation of “second primer comprises a second set of primers that comprises universal primers wherein the universal primers comprise sequence identical or substantially identical to the 5′ tail sequences of the primers of the first set” is interpreted that each universal primer (first and second) has the same 5’ to 3’ sequence as the corresponding universal tail and thus anneals to the complimentary tail sequence in the product. Ren do not teach the second primer comprises a second set of primers that comprises universal primers or/and target specific primers, wherein the universal primers comprise sequence identical or substantially identical to the 5′ tail sequences of the primers of the first set, wherein the target specific primers comprise 3′ target specific sequence and 5′ universal tails.
Fu teaches the first set of target specific primers comprise 3' target specific sequence, a random unique molecular identifier, and a 5' universal tail sequence [Fig 1A]. Fu further teaches subsequent amplifying the products using universal primers corresponding to the universal tail sequences incorporated into products by the first set of primers [Fig 13 and column 37, line 30-column 38 line 7].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to use universal primers that is identical to the 5’ tail sequence of the first primer to amplify the products from first reaction because offers significant advantages in cost-efficiency, workflow simplification. Additionally, using universal sequences are conventional and highly effective tools used in Next-Generation Sequencing (NGS) to accurately track newly synthesize C5-modified dUTP containing molecules. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143, A and C)
Ren et al. and Wang et al.
Claim(s) 18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of Wang et al. (US10221448B2, filed December 30th 2016)
Regarding claim 18, Ren and fu do not disclose the 5′ universal tails comprise at least two different sequences for the opposing primers which flank a desired length of region to be amplified wherein the two opposing primers in proximity which flank an undesired length of region have the same universal tail sequence.
Wang discloses first and second pairs of target specific primers for amplifying overlapping desired target regions. Wang discloses first forward primer F1 comprises first tag t2, first reverse primer R1 comprises second tag t1, a second forward primer F2 comprises first tag t1, second reverse primer R2 comprises third tag t3, wherein t1 and t2 are different tags and t3 may have same sequence as t2. Hence, the opposing primer pairs F1/R1 and F2/R2 that flank respective desired target regions comprise different sequence tag, while the proximal opposing primers F2/R1 that flank the undesired short overlapping region comprise same sequence tag t1 [Fig 2]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to configure Wang’s 5’ tails of target specific primers to permit a scalable multiplex PCR method that can simultaneously amplify overlapping amplicons while reducing preferential amplification of the short overlapping regions between two overlapping amplicons during amplification [Wang’s Background of the invention]. A skilled artisan would have had reasonable expectation of success that the disclosed tail arrangement can improve recovery of the desire amplicon in multiplex reaction. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Ren et al. and Kester et al.
Claim(s) 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of Kester et al. (US20110201524A1, EFD: October 21st 2009).
Regarding claim 21, Ren discloses method of preparing a sequencing library according to claim 1, the method comprising:(a) providing a reaction mixture(s), each reaction mixture comprising nucleic acids to be sequenced, a first DNA polymerase, unusual nucleoside triphosphates and a first set of primers, wherein the polymerase is capable of extending primers using the target nucleic acids as templates and incorporating the unusual nucleotide into extension products which are modified complementary strands, and is incapable of efficiently making a copy using the modified complementary strand as template, wherein the unusual nucleoside triphosphate is distinct from the four standard nucleotides: deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), or deoxycytidine triphosphate (dCTP), and is capable of being incorporated into new strands, wherein the first set of primers comprise target specific primers, universal primers or random primers; (b) performing extension reaction of primer and target nucleic acid template to produce modified complementary strands under extension condition, wherein the extension condition comprises buffer, any of four standard nucleoside triphosphates and appropriate temperature; (e.g. 5-Azidomethyl dUTP (AM dUTP) have been incorporated into DNA by reverse transcription. Reverse transcription reaction comprises reverse transcriptase (M-MuLV RT), azidomethyl dUTP or unmodified dNTP, and DNA primer [Abstract, Experimental section, and Fig 6]. Since M-MuLV is a reverse transcriptase, its primary function is to synthesize a complementary DNA (cDNA) strand using an RNA template. Its ability to read and process DNA template is significantly lower than when it is reading an RNA template.)
(c) optionally removing the nucleoside triphosphate and/or primers by purification or an enzymatic reaction; (e.g. e.g. newly synthesized cDNA is purified using ethanol precipitation [Experimental section])
(d) performing amplification of the modified complementary strands and/or original strands using a second set of primers and using a second DNA polymerase; and(e.g. Ren further discloses successful PCR amplification involves DNA polymerase reading AM dUTP containing templates strands while incorporating AM dUTP into newly synthesized daughter strands [Abstract and pages 5-6])
Ren does not disclose (e) processing the products of step (d) to complete the library preparation for massive parallel sequencing.
Kester discloses processing the products of step (d) to complete the library preparation for massive parallel sequencing. (e.g. Sequencing can be carried out using any suitable sequencing technique, wherein nucleotides are added successively to a free 3′ hydroxyl group, resulting in synthesis of a nucleic acid chain in the 5′ to 3′ direction. The nature of the nucleotide added is preferably determined after each nucleotide addition. Sequencing techniques using sequencing by ligation and techniques such as massively parallel signature sequencing (MPSS) where bases are removed from, rather than added to the strands on the surface are also useful, as are techniques using detection of pyrophosphate release (pyrosequencing).[0131])
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to process Ren’s products of step (d) to complete the library preparation for massive parallel sequencing in light of Kester because is known in the art that massively parallel sequencing is high speed, lower cost per base, and simultaneous analysis of multiple genes. Applying such step in Ren’s workflow would predictably allow processes large number of modified DNA strands at once to detect genetic modification. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Ren et al., Kester et al., and An et al.
Claim(s) 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of Kester et al. (US20110201524A1, EFD: October 21st 2009).
Regarding claim 22, Ren and Kester do not disclose deaminated strands are linearly amplified with or without an unusual nucleotide.
An discloses treating target DNA with bisulfite, then perform one direction asymmetric linear amplification using reagent treated DNA as the template, follow by amplification of linearly amplified DNA using additional primer and universal primer.[Fig. 1]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Ren and Kester method by linearly amplifying deaminated strands before amplification of step d in order to enrich the amount of bisulfite treated target DNA available for subsequent amplification and methylation analysis [An, ¶0048]. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Ren et al., McKerman et al., Vaught et al., and Yu et al.
Claim(s) 26-28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of McKerman et al. (US20100120034A1, EFD: July 6th 2009), Vaught et al. (J. Am. Chem. Soc. 2010, 132, 12, 4141–4151), and Yu et al. (Arch. Insect Biochem. Physiol., 42: 198-212).
Regarding claim 26-28, Ren does not disclose unusual nucleotide is 5-Methyl-2′-deoxycytidine-5′-Triphosphate, wherein after step (b) the DNA mixture is deaminated by chemical and/or enzymatic processes, wherein the modified complementary strands are protected from deamination, and wherein the original strands are deaminated on the sites not methylated.
McKerman discloses the methylation analysis in which 5-methylcytosine (5mC) is incorporated during polymerase mediate DNA synthesis to produce 5mC containing complementary strand. The DNA mixture then subjected to sodium bisulfite treatment, where 5mC containing complementary strand is protected from deamination and original strands are converted [¶0008-0012].
Yu discloses M-MuLV reverse transcriptase (used in Ren reverse transcription reaction) able to use 5mC as substrate to synthesize cDNA [Method and Material section].
Vaught discloses Thermococcus kodakaraensis KOD1 DNA polymerase (same type used in Ren PCR reaction) able to efficiently incorporate 5mC instead of standard dCTP to produce methylated DNA [Experimental Section].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Ren’s method by selecting 5mC) as the unusual nucleoside triphosphate to preserve 5mC containing complimentary copy of target sequence while obtaining methylation information from the bisulfite converted strand. Additionally, a skilled artisan would have had reasonable expectation of success because Yu and Vaught demonstrated M-MuLV reverse transcriptase and KOD1 DNA polymerase used by Ren efficiently incorporated 5mC into the complementary strand during polymerase dependent DNA synthesis. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Ren et al., McKerman et al., Vaught et al., Yu et al., and An et al.
Claim(s) 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ren et al. (Analyst 140.8 (2015): 2671-2678.) in view of McKerman et al. (US20100120034A1, EFD: July 6th 2009), Vaught et al. (J. Am. Chem. Soc. 2010, 132, 12, 4141–4151), Yu et al. (Arch. Insect Biochem. Physiol., 42: 198-212), and An et al. (US20190284608A1, EFD: September 29th 2017).
Regarding claim 29, Ren, McKerman, Vaught, and Yu do not disclose deaminated strands are linearly amplified with or without an unusual nucleotide.
An discloses treating target DNA with bisulfite, then perform one direction asymmetric linear amplification using reagent treated DNA as the template, follow by amplification of linearly amplified DNA using additional primer and universal primer.[Fig. 1]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Ren, McKerman, Vaught, and Yu method by linearly amplifying deaminated strands before amplification of step d in order to enrich the amount of bisulfite treated target DNA available for subsequent amplification and methylation analysis [An, ¶0048]. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Conclusion
Claims 1-9, 12-29 are rejected
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
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