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 claims / Response to Amendment
This office action is in response to an amendment filed March 06, 2026.
Claims 1, 5-8, 13-22, 24 and 26-28 were previously pending. Applicant amended claims 1, 7, 19 and 28.
Claims 1, 5-8, 13-22, 24 and 26-28 are currently pending and under consideration.
Applicant's claim amendments overcame the following rejections:
Rejections of Claims 1, 5-8, 13-22, 24 and 26-28 under 35 U.S.C. 112(b);
Rejections of Claims 1, 5-8, 13-22, 24 and 26-28 under 35 U.S.C. 103 as being unpatentable over Samuels, in view of Meacham 1.
Applicant's submission of the specification amendment obviated the previously presented objection.
Terminal Disclaimer: Applicant filed a terminal disclaimer on 03/06/2026 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of U.S. Patent No.11,174,509 has been reviewed and approved by the office. Therefore the following double patenting rejection has been overcome:
Rejections of Claims 1, 7, 13-15, 18-19, 20-22 and 24 on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 4-8, 12-13, and 15-16 of U.S. Patent No.11174509B2.
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment, which changes "without substantially enriching" to "without enriching" in independent claim 1, shown below:
A method comprising the steps of:
providing amplicons of a sample nucleic acid molecule in a first pool and a second pool;
conducting a first amplification reaction in the first pool to enrich for forward strand amplicons comprising a first unique sequence tag without for reverse strand amplicons in the first pool;
conducting a second amplification reaction in the second pool to enrich for reverse strand amplicons comprising a second unique sequence tag without for forward strand amplicons in the second pool;
sequencing the forward strand amplicons to produce forward strand sequence reads and sequencing the reverse strand amplicons to produce reverse strand sequence reads;
finding a variant in the forward strand sequence reads and the reverse strand sequence reads; and
identifying the variant as a true variant.
Thus, this amendment narrows the exclusion from only "substantially enriching for reverse strand amplicons in the first pool" and "substantially enriching for forward strand amplicons in the second pool" to excluding any enriching of "reverse strand amplicons" and "forward strand amplicons" in the first and second pool, respectively. Thus the scope of the claims have been altered. This updated negative limitation was not present in earlier versions of the claims (including cancelled claims) and was not previously considered by the examiner.
Although the Applicant's amendment responds to the 35 U.S.C. 112(b) rejection set forth in the prior Office Action, the specific amendment made was neither anticipated nor suggested by the examiner. This is evidenced by the prior rejection under 35 U.S.C. 112(b) and the examiner's interpretation of the term "without substantially enriching" (Non-Final Rejection - 12/11/2025, pages 7-8). In that rejection, the examiner did not suggest amending "without substantially enriching" to "without enriching," but instead provided a clarifying interpretation of the phrase under BRI so the claims can be examined ꟷ namely, the phrase "enrich for forward strand amplicons comprising a first unique sequence tag without substantially enriching for reverse strand amplicons" is interpreted to mean that reverse strand amplicons are not enriched to a greater extent than forward strand amplicons.
Accordingly, the present amendment could not have been reasonably foreseen by the examiner. Therefore, this Office Action is made FINAL. See MPEP§ 706.07(a).
Priority
For the instant claims 1, 5-8, 13-22, 24 and 26-28 in this application, the applicant claims priority of US provisional application NO. 61/915,435, which has a filling date on 12/12/2013.
Claim Interpretation -- Updated
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
For the purpose applying prior art, claim 1 and its dependent claims recite the term "amplicon," which is not expressly defined in the application's disclosure.
The specification describes amplicon as products of amplification (page 28, lines 30-31 to page 29, line 1):
“The term “amplification” as used herein generally refers to the production of substantially identical copies of a nucleic acid sequence (typically referred to as “amplicons”).”
According to the specification, amplification encompasses linear amplification using only one of forward or reverse primer in a pool, thereby generating single stranded products. Alternatively, both forward and reverse primers are present in the same pool, thereby generating double-stranded products:
"Embodiments of the invention may involve splitting the sample into two or more pools and using linear amplification and primers to create forward and reverse strand products of a nucleic acid comprising a target locus. In one embodiment only one of either the forward or the reverse primer for a given loci in a given pool, but in some cases it may be advantageous to have both primers present in one or more pools in equal or asymmetric abundances." (page 10, lines 11-16)
"In the described embodiments, a linear amplification reaction is carried out in each pool by, for example, using only a one member of a pair of primer species so that a first strand product is produced but no copies of the first strand product are produced. Linear amplification is well known in the art, an example of which may be found in “DNA linear amplification,” Chih Long Liu, Bradley E. Bernstein and Stuart L. Schreiber, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, Mass., 02138, USA." (page 15, lines 24-29)
Thus, in light of the specification, the term "amplicon" encompasses both single-stranded amplification products and double-stranded amplification products.
The common meaning for this term aligns with interpretation from the specification:
"An amplicon is a piece of DNA formed as the product of natural or artificial amplification events. For example, it can be formed via polymerase chain reactions (PCR) or ligase chain reactions (LCR), as well as by natural gene duplication." See Wikipedia (Amplicon - Wikipedia; Archived Feb 28, 2012 on WaybackMachine).
Thus, under BRI and in light of the specification, the term "amplicon" is understood to encompass single stranded and double-stranded products of both natural or artificial amplification events, including genomic DNAs, which are products of natural DNA replication.
Regarding all claims, terms such as "first" and "second" are interpreted as adjectives for identification purposes to distinguish between repeated instances of an element or limitation, and do not impose any additional features, such as any specific temporal limitation, any sequential order of steps, or any structural or composition differences. See 3M Innovative Props. Co. v. Avery Dennison Corp., 350 F.3d 1365 (Fed. Cir. 2003)2.
For the purpose of applying prior art, claim 1 recites "first unique sequence tag" and "second unique sequence tag," which are terms not expressly defined by the application's disclosure. The specification does not provide any definition for the term "unique sequence tag" or any description of structural features, that can distinguish this term from any sequence known in the art.
Thus, under BRI, the term "unique sequence tag" is interpreted to encompass any sequence, such as a barcode sequence.
For the purpose of applying prior art, claim 8 recites "wherein the forward and reverse strand amplicons further comprise a pool identification tag and a first universal portion."
The application's disclosure does not define the terms "pool identification tag" and "first universal portion" with any structural feature or characteristics that could distinguish these terms from any sequences or molecular biology assay elements, that can be comprised by "forward and reverse strand amplicons" (interpreted under BRI as any nucleic acids).
Therefore, under BRI, the terms "pool identification tag" and "first universal portion" are interpreted to encompass any nucleic acid sequences or elements that can be comprised by or attached to a nucleic acid molecule (e.g. biotin tag, linker, etc. )
For the purpose of applying prior art, claim 1 recites:
"conducting a first amplification reaction in the first pool to enrich for forward strand amplicons" and
"conducting a second amplification reaction in the second pool to enrich for reverse strand amplicons."
The application's disclosure does not expressly define the term "enrich." Thus, under BRI and based on the commonly understood meaning by those skilled in the art, the term "enrich" is understood to encompass any approach that increases the presence of targets of interest in a sample. This can be achieved by increase the number of targets, such as through target amplification 3; isolating targets from non-targets, such as via hybridization capture 2; or depleting non-targets to reduce background 4.
New Grounds of 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)(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.
Claims 1, 7, 13-14, 16-18, 20-22, 24, 26 and 28 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Zhang (WO2014043140A1- Accurate genome sequencing of single cells by single-stranded amplification and sequencing ; effective filing date September 10, 2013).
Regarding claim 1, Zhang teaches a method comprising the steps of:
providing amplicons of a sample nucleic acid molecule in a first pool and a second pool (Claim 1; FIG. 4; [0054], “evenly distribute single-stranded DNA molecules to 24 amplification
chambers for independent amplification”; [0067]);
conducting a first amplification reaction in the first pool to enrich for forward strand amplicons comprising a first unique sequence tag without enriching for reverse strand amplicons in the first pool (Claim 1; FIG. 4; [0054], [0067]; [0037] complementary strands are placed in separate solutions for independent amplification, thus the solution comprising forward strand of a DNA would not comprise the reverse strand and does not amplify the reverse strand);
conducting a second amplification reaction in the second pool to enrich for reverse strand amplicons comprising a second unique sequence tag without enriching for forward strand amplicons in the second pool (Claim 1; FIG. 4; [0054], [0067]; [0037]);
sequencing the forward strand amplicons to produce forward strand sequence reads and sequencing the reverse strand amplicons to produce reverse strand sequence reads (Claim 1; FIG. 4; [0054], [0067]; [0037]);
finding a variant in the forward strand sequence reads and the reverse strand sequence reads ([0028] “the mutations are called only when sequencing data from
the two strands are consistent… The concept of obtaining sequence information from both the Watson and Crick strands of a DNA molecule was commonly used”); and
identifying the variant as a true variant ([0026]; [0028]).
Regarding claim 7, Zhang teaches the forward and reverse strand amplicons are produced by a polymerase extension reaction ([0054] multiple displacement amplification).
Regarding claim 13, Zhang teaches variant is associated with a disease ([0057]).
Regarding claim 14, Zhang teaches the disease is cancer ([0057]).
Regarding claim 16, Zhang teaches identifying a second variant as a false variant when the second variant is found among one of, but not the other of, the forward strand sequence reads and the reverse strand sequence reads ([0026] lines 5-8; [0043]).
Regarding claim 17, Zhang teaches the second variant is an amplification or sequencing error ([0043]).
Regarding claims 18 and 24, they are anticipated by Zhang because they do not further limit the claimed method.
Claim 18 recites :"wherein the sample nucleic acid molecule is derived from formalin-fixed, paraffin-embedded tissue."
Claim 24 recites: "wherein the nucleic acid molecule is from a blood sample."
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps. In this instant case, the wherein clauses do not limit the method claim because the base claim 1 does not recite any step of obtaining or deriving nucleic acid molecules. As such, this clause merely describes a source of the nucleic acid molecules, without modifying any step of the claimed method. Therefore, these claim languages are descriptive statements without any associated active steps and do not distinguish the claims from the prior art.
Regarding claim 20, Zhang teaches the sequencing step comprises detecting a third variant ([0043] lines 10-13; [0028] “distinguish dozens of true mutations per genome from thousands to tens of thousands of sequencing errors.”).
Regarding claim 21, Zhang teaches identifying the third variant as a genetic mutation when the third variant is found among the forward strand sequence reads and the reverse strand sequence reads ( [0028] “distinguish dozens of true mutations per genome from thousands to tens of thousands of sequencing errors”; “the mutations are called only when sequencing data from the two strands are consistent.”).
Regarding claim 22, Zhang teaches the sequencing is sequencing-by- synthesis ([0042] Illumina™ sequencing,).
Regarding claim 26, Zhang teaches the first unique sequence tag identifies the forward strand sequence reads and the second unique sequence tag identifies the reverse strand sequence reads (([0067] line 16).
Regarding claim 28, Zhang teaches the forward strand amplicons comprise first double-stranded nucleic acids and wherein the reverse strand amplicons comprise second double-stranded nucleic acids ([0059] lines 1-3; [0041]).
New Grounds of 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.
Claims 5-6, 8, 15, 19 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (WO2014043140A1- Accurate genome sequencing of single cells by single-stranded amplification and sequencing ; effective filing date September 10, 2013), in view of Samuels (WO2012112804A1- Compositions and methods for molecular labeling; Published August 23 2012).
A) The teachings of Zhang are recited above and applied as for base claim 1.
Zhang teaches methods for strand-specific amplification and sequencing in microfluidic reactors, in which two complementary strands of a double-stranded nucleic acid are separated for independent amplification and sequencing, thereby enabling discrimination of true mutations from in vitro amplification errors (e.g., [0028]).
Regarding claim 8, Zhang teaches that, following strand-specific amplification, sequencing libraries are prepared for Illumina sequencing, including the addition of pool identification tags ([0067] line 16) to the strand-specific amplicons. Although Zhang does not explicitly describe library preparation in detail, such as further reciting a first universal portion, this feature is implicit in the context of sequencing library generation, which routinely requires the incorporation of sequencing adaptors comprising common sequences shared across all library fragments, this is supported by Samuels.
Samuels teaches methods for analyzing nucleic acids by compartmentalizing the nucleic acid amplicons (e.g., droplet encapsulation) with barcode libraries, followed by analysis approaches such as sequencing (entire document; summary of the invention; FIGs 13-14 for examples).
Samuels teaches that sequencing library preparation protocols, including the addition of adaptors, are well known in the art:
"[S]equencing library preparation protocols well known to those skilled in the art. For example, the amplified genetic material can be sheared/fragmented using methods well known to those of ordinary skill in the art, and adaptors can be ligated onto the ends of the fragments to be utilized, for example in direct sequencing, or in an enrichment process." (page 56, para 3, lines 4-7)
Samuels further teaches the use of a universal barcode library that can be combined with sequencing platform adaptor libraries, such as amplification products can be directly incorporated into the workflow of any given sequencing platform, such as Illumina. This approach can minimize PCR bias:
"A barcode library can also be made to include a sticky-end adapter specific for a sequencing platform. In certain embodiments, a construct is made that includes a sequencing platform N-mer and a sticky-end N-mer. A library of these constructs can be made. Separately, a universal barcode library as discussed above can be made. The, the universal barcode library can be combined with the sequencing platform adapter library by means of the sticky ends in view of a particular application. Thus products of any analysis discussed herein can be adapted to go directly into the workflow of any given sequencing platform (e.g. sticky-ended Illumina adaptors to anneal/ligate onto either the primer library or the output from a targeted sequencing run, so that it could be hybridized directly onto their flow cell. A different sticky-end adaptor set could be used for 454, etc.). This approach can minimize PCR bias." (page 46, para 2)
Accordingly, a skilled artisan would have found it prima facie obvious before the effective filing date of the claimed invention to apply Samuels's teaching of utilizing a universal barcode library to the library preparation step of Zhang, to allow direct compatibility with Illumina sequencing platform and to reduce PCR bias, as suggested by Samuels.
The person of ordinary skill would have had a reasonable expectation of success in combining these teachings because the references are technically compatible and provide complementary teachings in the field of sequencing. Both references teach Illumina sequencing. Specifically, Samuels teaches an improved approach for generating sequencing libraries for Illumina platform, which is used in Zhang. Samuels further teaches that the use of a universal barcode library minimizes PCR bias, which directly addresses a known issue identified in Zhang ꟷ namely, that "false positives generated by polymerase errors alone greatly outnumber the true mutations" ([0019]). Zhang teaches PCR amplification in library generation ([0060]). Thus, a skilled artisan would understand that Samuels's approach that reduces polymerase chain reaction (PCR) bias would have been desirable and applicable in Zhang's methods utilizing DNA polymerase in PCR.
Regarding claims 5-6, they are obvious as Zhang teaches "a variety of amplification techniques can be used" in its methods ([0041]), and Samuels teaches exponential amplification comprises PCR (Figure 14, PCR).
Regarding claim 15, Samuels teaches the forward and reverse strand amplicons further comprise a sequencing adaptor (page 56, para 3, lines 3-7).
Regarding claim 19, Samuels teaches the amplicons are produced by linear amplification (page 55, para 4, lines6-9).
Regarding claim 27, Samuels teaches the first unique sequence tag and the second unique sequence tag identify the forward and reverse strand sequence reads as corresponding to the sample nucleic acid molecule (page 52, para 2, lines 6-9; FIG 43).
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Strand-bias in sequencing data analysis, which hinders accurate detection of gene variants, is a well-known issue and is commonly attributed to artifacts or errors arising from library preparation or sequencing. See Guo (Guo et al., The effect of strand bias in Illumina short-read sequencing data. BMC Genomics. 2012 Nov 24;13:666. doi: 10.1186/1471-2164-13-666. PMID: 23176052; PMCID: PMC3532123) in background and conclusions.
The principle of comparing both DNA strands to determine whether a variant represents a true biological mutation or an amplification error is well-established in the art, and various strand-specific approaches have been developed based on this principle.
See Kirsch (S. Kirsch, & C.A. Klein, Sequence error storms and the landscape of mutations in cancer, Proc. Natl. Acad. Sci. U.S.A. 109 (36) 14289-14290, doi.org/10.1073/pnas.1212246109 (2012)) in Fig. 1;
See also Kaur (Kaur M, Makrigiorgos GM. Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA. Nucleic Acids Res. 2003 Mar 15;31(6):e26. doi: 10.1093/nar/gng026. PMID: 12626725; PMCID: PMC152880) in pages 2-3, "Amplification of DNA hairpins with non-complementary ends."
Performing linear, strand-specific amplification in separate pools is known in the art, see Kurn (US20110105364A1 - Compositions and methods for targeted nucleic acid sequence selection and amplification ; Published on 2011-05-05) in ([0073]; [0008] lines 11-12, "the selective partitioning is strand-specific"; [0037]; [0120]).
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIAN NMN YU whose telephone number is (703)756-4694. The examiner can normally be reached Monday - Friday 8:30 am - 5:30 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at (571) 272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 For clarity of record, it is noted that Applicant's arguments regarding the previously cited prior art references (Samuels (WO2012112804A1- Compositions and methods for molecular labeling; Published August 23 2012) and Meacham (Meacham et al. Identification and correction of systematic error in high-throughput sequence data. BMC Bioinformatics 12, 451 (2011). doi.org/10.1186/1471-2105-12-451) have been thoroughly reviewed but are not found persuasive.
However, the prior rejections under 35 U.S.C. 103 were withdrawn because they were obviated by the claim amendment, which necessitated further search and consideration. As a result of the additional search, new prior art was identified and is applied in the new grounds of rejection set forth herein.
2 Holding that "first pattern" and "second pattern" is equivalent to "Pattern A" and "Pattern B": The use of the terms "first" and "second" is a common patent-law convention to distinguish between repeated instances of an element or limitation. See, e.g., Anchor Wall Sys., Inc. v. Rockwood Retaining Walls, Inc., 340 F.3d 1298, 1304 (Fed. Cir. 2003) ("first and second sidewall surfaces"); Springs Window Fashions LP v. Novo Indus., L.P., 323 F.3d 989, 992 (Fed. Cir. 2003) ("first and second opposed ends"). In the context of claim 1, the use of the terms "first . . . pattern" and "second . . . pattern" is equivalent to a reference to "pattern A" and "pattern B," and should not in and of itself impose a serial or temporal limitation onto claim 1."
3 see Tewhey et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27, 1025–1031 (2009), (introduction);
See also Ware et al., Next generation sequencing for clinical diagnostics and personalised medicine: implications for the next generation cardiologist. Heart. 2012 Feb;98(4):276-81. doi: 10.1136/heartjnl-2011-300742. Epub 2011 Nov 29. PMID: 22128206. (Fig 2)
4 see Feehery et al. A method for selectively enriching microbial DNA from contaminating vertebrate host DNA. PLoS One. 2013 Oct 28;8(10):e76096. doi: 10.1371/journal.pone.0076096. PMID: 24204593; PMCID: PMC3810253 (entire document).
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