DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status and Formal Matters
This action is in response to papers filed 10/13/2025.
Claims 16, 27, 29 have been amended.
Claims 16, 27, 29-30 are pending.
Applicant’s election without traverse of claims 16-17 in the reply filed on 10/8/2021 is acknowledged.
Claims 26-33 are thus withdrawn as non-elected inventions.
Claim 16 is being examined.
Priority
The instant application was filed 09/24/2019 is a national stage entry of PCT/US2018/022664, international filing date: 03/15/2018 PCT/US2018/022664 claims priority from provisional application 62476234, filed 03/24/2017.
Specification
The specification is objected to as failing to provide proper antecedent basis for the claimed subject matter. See 37 CFR 1.75(d)(1) and MPEP § 608.01(o). Correction of the following is required: Claim 1 has been amended to recite, “single reaction mixture. Review and searching of the specification revealed mixture recited 5 times. None of those recitations of mixture are with respect to single. Thus the specification does not provide antecedent basis for this recitation.
Response to Arguments
This is a new grounds of objection necessitated by amendment.
Claim Rejections - 35 USC § 112
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.
Claim 16 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 16 has been amended to recite, ““b) in a single reaction mixture, simultaneously amplifying said plus and minus strand target regions of said converted DNA duplex fragment with a set of four different types of amplification primers
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comprising: i) a plus strand specific barcoded forward primer which comprises a unique identifier (UID),
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ii) a plus strand specific reverse primer, iii) a minus strand specific barcoded forward primer which comprises a unique identifier (UID), and iv) a minus strand specific reverse primer ,thereby forming four different of amplicons from the converted DNA duplex fragment.” The specification asserts 0011, 0062-0063, and 0084-0086 support the amendment. These appear to be referencing the PGPUB.
Paragraph 0011 appears to teach molecular barcodes being added to each strand but provides no guidance with respect to UID or primers not required to have plus or minus strands.
Paragraphs 0062 merely recites, “BiSeqS work flow.” Paragraph 0063 discuss bisulfite conversion, molecular barcoding, and sample barcoding, but do not teach simultaneous amplification to provide 4 amplicons or the 4 primers recited in the claim. While paragraph 0063 cites figure 1 and 4, these figures are cartoons which do not explicitly teach amplification or the use of 4 primers as required by the claim.
While paragraph 0084 states, “Although bisulfite treatment and specially-designed primers have often been used to evaluate methylation in the past for a variety of clinical purposes (41-43), the combination of molecular barcoding with simultaneous amplification of both strands provides unprecedented sensitivity in this type of analysis.” This does not specifically require a plus strand specific barcoded forward primer which comprises a unique identifier (UID), minus strand specific barcoded forward primer which comprises a unique identifier (UID), plus strand specific reverse primer or minus strand specific reverse primer. Further the instant application specifically teaches, “endogenous UIDs” (0008), thus the discussion of molecular barcoding in 0084 does not explicitly require the primer limitations of the claims. Thus the amendment appears to be new matter.
The response of asserts 10/13/2025 asserts, “Claim 16 is further amended to recite that the plus and minus strands are simultaneously amplified in a single reaction mixture. Support can be found at paragraph [58], "Because both strands must be amplified equivalently and in the same reaction, the primers must be chosen so that the same PCR cycling conditions can be used for amplifying both strands in a highly specific manner", and paragraphs [50]-[51], which describe using four different primers as recited in claim 16 in a single reaction mixture (e.g., well)” However paragraph 0058 states:
[58] Another issue confronting amplification of bisulfite converted DNA is that many polymerases will not efficiently copy DNA that contains uracil bases. We tested seven commercially available polymerases and various reaction conditions to optimize efficiency of template use and uniformity of amplification of both strands when four primers were used (Table 1). While a combination of AMPIGene Hot Start Taq Polymerase and iTAQ Polymerase amplified the greatest number of template molecules, their lack of 3'--5' exonuclease activity proved limiting for specificity in that the number of errors during PCR was unacceptably high. Ultimately, we chose Phusion U Hot Start Polymerase, a polymerase that exhibits 3'--5' exonuclease activity, as the enzyme to amplify uracil-containing templates with the highest specificity while maintaining sensitivity.
[50] The molecular barcoding PCR cycles included 12.5 pL of 2X Phusion U Hot Start PCR Master Mix (ThermoFisher, Cat. # F533S) in a 25 pL reaction, and optimized concentrations of each forward and reverse primer, ranging from 0.125 pM to 4 pM of each forward and each reverse primer for a total of four primers per well. The following cycling conditions were used: one cycle of 95°C for 3 minutes, 20 cycles of 95°C for 10 seconds, 63°C for 2 minutes, and 72°C for 2 minutes. [51]AMPure XP (Beckman Coulter, Cat. # A63881) was used to remove the primers for UID assignment. 0.025% of the PCR product generated from the UID cycles was used for the well barcoding (WBC) cycles. Primers used for the well barcode step were identical to those described previously and are diagrammed in Fig. 4A and 4B (28). The WBC cycles were performed in 25 pL reactions containing 11.8 pL of water (ThermoFisher UltraPure, Cat. # 10977-023), 5 pL of 5X Phusion HF Buffer (ThermoFisher, Cat. # F518L), 0.5 pL of 10 mM dNTPs (NEB, Cat. # N0447L), and 0.25 pL of Phusion Hot Start II DNA Polymerase (ThermoFisher, Cat. # F549L). The following cycling conditions were used: one cycle of 98°C for 2 minutes, 24 cycles of 98°C for 10 seconds, 65°C for 2 minutes, and 72°C for 2 minutes.
Response to Arguments
The response begins traversing the rejection by citing MPEP 2163. This is noted.
The response continues by citing , “"Primers are designed to be used in sets of at least four so that both strands of the original duplex template are amplified, sequenced, and identifiable." Paragraph [29].” This argument has been thoroughly reviewed but is not considered persuasive as this does not provide support for simultaneously. Further paragraph {29] in the file wrapper states:
[29] Amplification of barcoded sequences generates families of similarly barcoded templates. Each family shares a molecular barcode, denoting that it derives from a single template molecule. Sequencing of the population of amplified templates, including multiple members of a family, permits comparison of nucleotide sequences of multiple members of a single family and assessment of the fraction of members of a family that contain a particular mutation. A high fraction, such as greater than 50, 60, 70, 80, 90, or 95% of families with a particular mutation suggests that the mutation was present in the original sample, prior to amplification. However, some of the identified mutations may still be ones that have been introduced during processing due to in vitro enzymatic errors. Detection of mutations that are due to such errors can be further reduced by comparing sequences obtained from families of two complementary strands. Requiring that a mutation exist on families generated from two strands reduces artifactual apparent mutations significantly.
The response continues by reproducing the following
"Each forward primer contained the sequence necessary for well barcode amplification at the 5' end, followed by a string of 14 random nucleotides that served as the unique identifier (UID), and amplicon-specific primer sequences at the 3' end (FIGS. 4A and 4B). Each reverse primer contained the sequence necessary for well barcode amplification at the 5' end, followed by amplicon-specific primer sequences. To anneal to bisulfite-converted DNA, it is important to replace specific nucleotides in the various wild type amplicon-specific primer sequences. T replaced C in the plus strand forward primer, while A replaced G in the plus strand reverse primer. A replaced G in the minus strandforward primer, and T replaced C in the minus strand reverse primer. The molecular barcoding PCR cycles included 12.5 L of 2x Phusion U Hot Start PCR Master Mix (ThermoFisher, Cat. # F533S) in a 25 L reaction, and optimized concentrations of each forward and reverse primer, ranging from 0.125 M to 4 M of each forward and each reverse primer for a total offour primers per well. The following cycling conditions were used: one cycle of 950 C. for 3 minutes, 20 cycles of 950 C. for 10 seconds, 630 C. for 2 minutes, and 720 C. for 2 minutes." Paragraph [50]-[51].
However the specification states:
[50] The molecular barcoding PCR cycles included 12.5 pL of 2X Phusion U Hot Start PCR Master Mix (ThermoFisher, Cat. # F533S) in a 25 pL reaction, and optimized concentrations of each forward and reverse primer, ranging from 0.125 pM to 4 pM of each forward and each reverse primer for a total of four primers per well. The following cycling conditions were used: one cycle of 95°C for 3 minutes, 20 cycles of 95°C for 10 seconds, 63°C for 2 minutes, and 72°C for 2 minutes. [51]AMPure XP (Beckman Coulter, Cat. # A63881) was used to remove the primers for UID assignment. 0.025% of the PCR product generated from the UID cycles was used for the well barcoding (WBC) cycles. Primers used for the well barcode step were identical to those described previously and are diagrammed in Fig. 4A and 4B (28). The WBC cycles were performed in 25 pL reactions containing 11.8 pL of water (ThermoFisher UltraPure, Cat. # 10977-023), 5 pL of 5X Phusion HF Buffer (ThermoFisher, Cat. # F518L), 0.5 pL of 10 mM dNTPs (NEB, Cat. # N0447L), and 0.25 pL of Phusion Hot Start II DNA Polymerase (ThermoFisher, Cat. # F549L). The following cycling conditions were used: one cycle of 98°C for 2 minutes, 24 cycles of 98°C for 10 seconds, 65°C for 2 minutes, and 72°C for 2 minutes.
Thus the portion cited by the representative does not appear to be consistent with the specification as filed on 9/28/2019. Further review and searching of the specification did not reveal the alleged teachings of the specification. Thus this argument is not persuasive.
The response continues by citing, “"First, four primers must be designed to amplify each region of interest, two primers for each strand. ... Because both strands must be amplified equivalently and in the same reaction, the primers must be chosen so that the same PCR cycling conditions can be used for amplifying both strands in a highly specific manner." Paragraph [58].”
However, paragraph [58] of the specification states:
[58] Another issue confronting amplification of bisulfite converted DNA is that many polymerases will not efficiently copy DNA that contains uracil bases. We tested seven commercially available polymerases and various reaction conditions to optimize efficiency of template use and uniformity of amplification of both strands when four primers were used (Table 1). While a combination of AMPIGene Hot Start Taq Polymerase and iTAQ Polymerase amplified the greatest number of template molecules, their lack of 3'--5' exonuclease activity proved limiting for specificity in that the number of errors during PCR was unacceptably high. Ultimately, we chose Phusion U Hot Start Polymerase, a polymerase that exhibits 3'--5' exonuclease activity, as the enzyme to amplify uracil-containing templates with the highest specificity while maintaining sensitivity.
Thus the portion cited by the representative does not appear to be consistent with the specification as filed on 9/28/2019. Thus this argument is not persuasive.
The response continues by providing: "Example 4 BiSeqS Increases the Specificity of Mutation Calling We selected eight amplicons within prototypic cancer driver genes to assess BiSeqS performance. For each of the eight amplicons, two forward primers and two reverse primers for each strand were synthesized and tested using the principles described above and in the Materials and Methods." Example 4, paragraph [65].
However, the specification states:
[65] For each of the eight amplicons, we compared the specificity of BiSeqS to that of conventional next generation sequencing (NGS) and molecular barcode-assisted sequencing (i.e., SafeSeqS). We considered only those potential mutations that could be discerned in both strands, as described above. There were a total of 608 bp within these amplicons, yielding a total of 1550 single base substitutions possible. Of these 1550 potential SBS, 1252 (80.8%) were scorable as SDMs; the remainder were transitions that were not scorable for the reasons noted above. There were also many possible indels at each position that could have been observed in the sequencing data, all scorable as SDMs.
Thus the portion cited by the representative does not appear to be consistent with the specification as filed on 9/28/2019. Thus this argument is not persuasive.
The response continues by providing "BiSeqS Simultaneously Detects Methylation Status on Both Strands Cytosine bases in 5'-CpG dinucleotides that are methylated are protected from conversion to uracil during bisulfite treatment, allowing BiSeqS to detect the methylation status of the plus and minus strands simultaneously .Although bisulfite treatment and specially-designed primers have often been used to evaluate methylation in the past for a variety of clinical purposes (41- 43), the combination of molecular barcoding with simultaneous amplification of both strands provides unprecedented sensitivity in this type of analysis." Example 6, paragraph [74].
However, the specification states:
74] To demonstrate the ability of BiSeqS to discriminate the methylation status on both strands simultaneously, we evaluated a region of the TP53 gene that contains a known methylated CpG at hgl9 position 7,572,973 to 4. Greater than 90% of the UIDs on both strands were found to be methylated at the C at the plus strand of position 7,572,973 and the C opposite the G on the minus strand at position 7,572,974. Greater than 99.8% of the C residues that were not at 5'-CpG dinucleotides within this amplicon were found to be converted to T's, providing an essential control for interpreting the extent of methylation. We then searched for evidence of double-stranded methylation within all eight amplicons evaluated in this study in normal WBCs. There were two 5'-CpG residues within the 608 bp that could be evaluated. Of these, we found that both CpG's were methylated on both strands, with the fraction of methylated alleles ranging from 92.10% to 96.10% (data not shown).
Thus the portion cited by the representative does not appear to be consistent with the specification as filed on 9/28/2019. Thus this argument is not persuasive
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 16 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a mental step without significantly more. The claim(s) recite(s) the abstract idea or mental step of comparing and identifying . This judicial exception is not integrated into a practical application because no additional step depend from or otherwise integrate the judicial exceptions. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the active steps of the claim are routine and conventional.
Claim analysis
The instant claim 16 is directed towards A method for detecting the methylation status at a selected 5'- cytosine-phosphate-guanine-3' (CpG) dinucleotide in plus and minus strands of an initial DNA duplex fragment, comprising:a) treating an initial DNA duplex fragment with bisulfite to convert non-methylated cytosine bases in the DNA duplex fragment to uracil bases, thereby forming a converted DNA duplex fragment having plus and minus strands, wherein each converted DNA duplex fragment comprises a converted target region which comprises a plus strand target region that comprises one or more uracil bases that were non- methylated cytosine bases prior to said bisulfite treatment, and a minus strand target region that comprises one or more guanine bases mis-matched to said one or more uracil bases; b) in a single reaction mixture, simultaneously amplifying said plus and minus strand target regions of said converted DNA duplex fragment with a set of four different types of amplification primers comprising: i) a plus strand specific barcoded forward primer which comprises a unique identifier (UID),ii) a plus strand specific reverse primer ,iii) a minus strand specific barcoded forward primer which comprises a unique identifier (UID), and iv) a minus strand specific reverse primer,thereby forming four different types of amplicons from the converted DNA duplex fragment in the same reaction mixture;c) subjecting said four different types of amplicons to sequencing reactions; and d) comparing nucleotide sequences of the amplicons, and identifying a cytosine (C) in a CpG dinucleotide in the nucleotide sequence of an amplicon corresponding to the plus or minus strand of the initial DNA duplex fragment as methylated in the initial DNA duplex fragment when the cytosine in the nucleotide sequence of the amplicon is opposite the nucleotide guanine (G) in a complementary amplicon nucleotide sequence..
The comparing step is mental steps or abstract ideas.
The identifying step is mental steps or abstract ideas
The treating and amplifying steps are considered to be an active step requiring the analysis of a sample.
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case the Step 1 requirement is satisfied as the claims are directed towards a process.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract idea.
With regards to claim 16, the claim recites, “subjecting said four different amplicons to sequencing reactions; and d) comparing nucleotide sequences of two of said four different species of amplicons, and identifying a methylated cytosine (C) opposite nucleotide guanine (G) of the CpG dinucleotide.” This is an abstract idea or mental step..
Further the identifying step is also an abstract idea.
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? The answer is no as the claim requires no additional steps.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? No, the claims provide no specific reagents that result in significantly more.
With regards to claim 16 the claim requires treating, amplifying steps and sequencing. The specification in paragraph 49 teaches the use of commercially available bisulfite conversion kits. The specification teaches sequencing was done by Illumina MiSeq instrument.
Further Vogelstein (WO2012142213), Darst (Curr Protoc Mol Biol. 2010 July ; CHAPTER: Unit–7.917), Li (Bioinformatics (2002) volume 18, pages 1427-1431)Huang (US 2014/0287404 A1), May (2010/0273219) and Kinde (Proceedings national Academy of Sciences (2011) volume 108, pages 9530-9535), Maslow ( Mutation Research 776 (2015) 136–143) Kou (PLOS ONE | DOI:10.1371/journal.pone.0146638 January 11, 2016) teach the active steps of the claim are routine and conventional.
Response to Arguments
The response traverses the rejection asserting the cited prior art does not teach amplification using 4 different primers simultaneously in a single reaction. This argument has been thoroughly reviewed but is not considered persuasive as the claims the prior art does in a single reaction mixture. This argument has been thoroughly reviewed but is not considered persuasive as Example of 2 at least suggests multiplex PCR and this is considered to be routine and conventional.
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 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 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Vogelstein (WO2012142213) and Darst (Curr Protoc Mol Biol. 2010 July ; CHAPTER: Unit–7.917).
The claims have been amended to recite, “four different types of target specific amplification primers.” The specification does not recite and thus does not define, ““four different types of target specific amplification primers.” The claim are drawn to the amplification of a plus strand and minus strand of DNA that has been treated with bisulfite. The claim does not require the target amplified by the plus strand reverse and plus strand forward primer encompass the same sequence as amplified by the minus strand forward or the minus strand reverse. Further the claims do not require the four primers amplify a sequence that contains a cytosine which has been converted due to the bisulfite treatment. The specification in paragraph 0065 of the PGPUB recites, “robust amplification of both strands of every target region attempted.” Thus one interpretation is the limitation it can encompass one set of a forward primer and a reverse primer to a specific target and another set of a forward primer and reverse primer to a second specific primer. Alternatively it can encompass two forward primers and two reverse primers each with a different IUD, amongst other interpretations.
Further while the claims recite, “UID,” the specification does not provide any standard to differentiate a UID from any other sequence. Thus the broadest reasonable interpretation is this encompasses the target specific sequence of the primer which is unique to the target.
While the claim has been amended to require “simultaneously amplifying” the claim does not require it in the same container.
Vogelstein teaches, “The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called "Safe-SeqS" for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant ("super-mutants") if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.” (abstract).
Vogelstein teaches,” UIDs, sometimes called barcodes or indexes, can be assigned to nucleic acid fragments in many ways. These include the introduction of exogenous sequences through PCR (40, 41) or ligation (42, 43). Even more simply, randomly sheared genomic DNA inherently contains UIDs consisting of the sequences of the two ends of each sheared fragment (Fig. 2 and Fig. 5 ). Paired-end sequencing of these fragments yields UID-families that can be analyzed as described above. .” (39).
Vogelstein teaches, “The UIDs are in excess of the analyte DNA fragments during amplification.” (08)
Vogelstein teaches,” to make more efficient use of the original templates, we developed a Safe-SeqS strategy that employed a minimum number of enzymatic steps. This strategy also permitted the use of degraded or damaged DNA, such as found in clinical specimens or after bisulfite- treatment for the examination of cytosine methylation (45). As depicted in Fig. 3, this strategy employs two sets of PCR primers. The first set is synthesized with standard phosphonamidite precursors and contained sequences complementary to the gene of interest on the 3' end and different tails at the 5' ends of both the forward and reverse primers. The different tails allowed universal amplification in the next step. Finally, there was a stretch of 12 to 14 random nucleotides between the tail and the sequence-specific nucleotides in the forward primer (40). The random nucleotides form the UlDs. An equivalent way to assign UIDs to fragments, not used in this study, would employ 10,000 forward primers and 10,000 reverse primers synthesized on a microarray. Each of these 20,000 primers would have gene-specific primers at their 3 -ends and one of 10,000 specific, predetermined, non-overlapping UID sequences at their 5'-ends, allowing for 108 (i.e., [104- ]2) possible UID combinations. In either case, two cycles of PCR are performed with the primers and a high-fidelity polymerase, producing a uniquely tagged, double-stranded DNA fragment from each of the two strands of each original template molecule (Fig. 3). The residual, unused UID assignment primers are removed by digestion with a single-strand specific exonuclease, without further purification, and two new primers are added. Alternatively or in addition to such digestion, one can use a silica column that selectively retains larger-sized fragments or one can use solid phase reversible immobilization (SPRI) beads under conditions that selectively retain larger fragments to eliminate smaller, non-specific, amplification artifacts. This purification may potentially help in reducing primer-dimer accumulation in later steps. The new primers, complementary to the tails introduced in the UID assignment cycles, contain grafting sequences at their 5' ends, permitting solid-phase amplification on the Illumina instalment, and phosphorothioate residues at their 3' ends to make PCR, the products are loaded on the Illumina instrument. As shown below, this strategy allowed us to evaluate the majority of input fragments and was used for several illustrative experiments.” Vogelstein teaches, “Populations of primer pairs are used to attach exogenous UIDs. The first pnmer comprises a first portion of l 0-100 nucleotides complementary to the gene or gene portion and a second portion of 10 to l 00 nucleotides comprising a site for hybridization to a third primer. The second primer comprises a first portion of 10-100 nucleotides complernentary to the gene or gene portion and a second portion of l 0 to 100 nucleotides comprising a site fi:.)r hybridization to a fourth primer. interposed between the first portion and the second portion of the second primer is a third portion consisting of 2 to 4,000 nucleotides forming a unique identifier (Ui D). The unique identifiers in the population have at least 4, at least 16, at least 64, at least 256, at least 1,024, at least 4,096, at least 16,384, at least 65,536, at least 262,144, at least 1,048,576, at least 4,194,304, at least 16,777,216, or at least 67,108,864 different sequences. The first and second primers are complementary to opposite strands of the gene or gene portion. A kit can be made containing both the primers for attaching exogenous UIDs as well as amplification prirners, i.e .. the third and fourth primers cornplementary to the second portions of each of the first and second primers. The third and fourth primers can optionally contain additional grafting or indexing sequences. The UID may comprise randomly selected sequences, pre-defined nucleotide sequences, or both randomly selected sequences and pre-defined nucleotides.” ([30] Thus Vogelstein teaches amplification with 4 primers to produce 4 amplification products (amplicons). (figure 3)
Vogelstein teaches, “An equivalent way to assign UIDs to fragments, not used in this study, would employ 10,000 forward primers and 10,000 reverse primers synthesized on a microarray. Each of these 20,000 primers would have gene-specific primers at their 3'-ends and one of 10,000 specific, predetermined, non-overlapping lHD sequences at their 5'-ends, allowing for 108 (i.e., [10'1]2) possible UID combinations. In either case, two cycles of PCR are performed with the primers and a high-fidelity polymerase, producing a uniquely tagged, double-stranded DNA fragment from each of the two strands of each original template molecule (Fig. 3).“[45] thus Vogelstein teaches or suggests in a single reaction.
Vogelstein teaches, “With this technology, both strands of each template molecule are sequenced redundantly after a number of preparative enzymatic steps.”33
Vogelstein does not specifically teach comparing sequences of members of a family to determine a C is methylated and part of a CpG island.
However, Darst teaches:
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Thus Darst teaches determining the whether a C is methylated or not in a CpG island. Darst teaches the use of strand specific primers.
Vogelstein teaches, “A super-mutant is defined as a UID-family in which >95% of family members have the same mutation.”(12). Vogelstein teaches, “With either endogenous or exogenous UIDs, a super-mutant was defined as a UID-family in which ≥95% of members shared the identical mutation.”(61) Vogelstein teaches bisulfite treatment for examination of cytosine methylation ([45].
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to sequence both sets of amplicons produced simultaneously (in one container or two different primer pairs in different containers in the same PCR run) two set of primer pairs with each primer having an unique UID taught by Vogelstein, align and compare the sequencing read from the sequencing reactions for the separately amplified plus and minus strands and complements to identify cytosines which had not be converted to uracil by bisulfite conversion and complementary to guanine in corresponding strand as part of CpG dinucleotide. The artisan would be motivated to identify CpG dinucleotides to identify methylated genes or sequences in the sample that may be regulated by methylation and Vogelstein specifically identifies identifying sequences with >95% identity as having an allele, in this case methylation or non-methylation.. The artisan would have a reasonable expectation of success as the artisan is merely using the teachings of Vogelstein.
Further Vogelstein teaches, “To make more efficient use of the original templates, we developed a Safe-SeqS strategy that employed a minimum number of enzymatic steps. This strategy also permitted the use of degraded or damaged DNA, such as found in clinical specimens or after bisulfite treatment for the examination of cytosine methylation ( 45). As depicted in Fig. 3, this strategy employs two sets of PCR primers. The first set is synthesized with standard phosphonamidite precursors and contained sequences complementary to the gene of interest on the 3' end and different tails at the 5' ends of both the forward and reverse primers. The different tails allowed universal amplification in the next step. Finally, there was a stretch of 12 to 14 random nucleotides between the tail and the sequence-specific nucleotides in the forward primer ( 40). The random nucleotides form the UIDs. An equivalent way to assign UIDs to fragments, not used in this study, would employ 10,000 forward primers and 10,000 reverse primers synthesized on a microarray. Each of these 20,000 primers would have gene-specific primers at their 3'-ends and one of 10,000 specific, predetermined, non-overlapping UID sequences at their 5'-ends, allowing for 108 (i.e., [10'1]2) possible UID combinations. In either case, two cycles of PCR are performed with the primers and a high-fidelity polymerase, producing a uniquely tagged, double-stranded DNA fragment from each of the two strands of each original template molecule (Fig. 3). The residual, unused UID assignment primers are removed by digestion with a single-strand specific exonuclease, without further purification, and two new primers are added. Alternatively or in addition to such digestion, one can use a silica column that selectively retains larger-sized fragments or one can use solid phase reversible immobilization (SPRI) beads under conditions that selectively retain larger fragments to eliminate smaller, non-specific, amplification artifacts. This purification may potentially help in reducing primer-dimer accumulation in later steps..” (45).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use two primers with UID at one end (plus and minus strand barcoded primer) and two new primers are added as taught by Vogelstein (45) for simultaneous amplification of the sequences to produce four amplicons. The artisan would be motivated as Vogelstein teaches this method is more efficient.” The artisan would have a reasonable expectation of success as the artisan is merely using methods taught by the art.
Alternatively it would have been prima facie obvious to one of skill in the art prior to the effective filing date of the claims to use strand specific primers as taught by Darst in a multiplex PCR amplification. The artisan would be motivated as Vogelstein teaches, “we have demonstrated that the exogenous UIDs strategy can be used to analyze a single amplicon in depth. This technology may not be applicable to situations wherein multiple amplicons must be analyzed from a sample containing a limited number of templates. Multiplexing in the UID assignment cycles (Fig. 3) may provide a solution to this challenge.” The artisan would have a reasonable expectation of success as the artisan is merely using methods taught by the art.
Response to Arguments
The response traverses the rejection asserting Vogelstein does not teach the use of 4 primers for the simultaneous amplification of a single target. This argument has been thoroughly reviewed but is not considered persuasive as the claims do not require simultaneous amplification of a single target as detailed in the beginning of the rejection. Further
The response alleges it is unclear how the rejection is using the Darst reference and alleges Darst teaches the use of strand specific primers are used in different reactions. This argument has been thoroughly reviewed but is not considered persuasive as Darst is only being relied upon for the comparing of sequences. Thus the rejection is maintained.
Summary
No claims are allowed.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. ELTOUKHY (WO2015100427 A1)
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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