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 .
EXAMINER’S AMENDMENT
An examiner’s amendment to the record appears below. Should the changes and/or additions be unacceptable to applicant, an amendment may be filed as provided by 37 CFR 1.312. To ensure consideration of such an amendment, it MUST be submitted no later than the payment of the issue fee.
Authorization for this examiner’s amendment was given in an interview with attorney of record Charles Rauch on 08/20/2025.
The title shall be changed to correct a typographical error. The new title shall now recite:
“METHOD FOR PREPARING HIGH-THROUGHPUT SEQUENCING LIBRARY BASED ON NESTED MULTIPLEX PCR AND KIT FOR THE SAME”.
Claim status
Claims 1-13 and 16-22 are pending and under examination. Claims 14-15 were previously cancelled. Claims 1-4 and 12-13 have been amended. Claims 1, 3, and 12 are independent claims.
Response to Arguments
Rejection Withdrawn
The rejection of claims 1-13 and 16-22 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention is withdrawn following the Applicant’s amendments to claims 1-4 and 12-13.
The rejection of claims 1-13 and 16-22 under 35 U.S.C. 103 as being unpatentable over Zheng (WO 2019/023924 A1, of record), in view of Sugino et al. (US 2014/0315211 A1, published Oct. 23, 2014) is withdrawn following the Applicant’s amendments to claims 1-4 and 12-13 and further review of the cited references.
New Rejections
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1 are rejected under 35 U.S.C. 103 as being unpatentable over Del Favero et al. (US . US 2020/0024651 A1, published Jan. 23 , 2020) in view of Zheng (US 2019/0078148 A1, published Mar. 14, 2018, US equivalent of WO 2019/023924 A1, of record).
Del Favero discloses a two-round multiple PCR library preparation method. In the first round, a forward primer (untagged, target-specific) and a reverse primer (target-specific at the 3’-end, with a universal tag at the 5’-end) amplify a targeted region. In the second round, universal primers whose 3’ portion are designed to be “identical (in the 5’ to 3’ direction) to the tags introduced in the first round amplify the tagged product and append sequencing adaptors and optional sample barcodes.
Zheng discloses a target-enrichment method using a first (“outside”) target-specific primer, followed by PCR amplification using a second (“inside”) target-specific primer that is expressly “nested with respect to the outside primer for the locus of interest,” positioned downstream of the outside primer, with a sequences at its 3’-end that specifically anneals to the target nucleotide sequences and a tag at its 5’ end, either a generic universal 5’ tag of at least 13 nucleotides, or a sequence complementary to the sequence of a second sequencing primer compatible with a NGS platform.
In regards to claim 1, Del Favero discloses “A method for preparing a high-throughput sequencing library based on nested multiplex PCR, the method comprising: a first round of PCR amplification: amplifying a targeted region by using a forward primer and a reverse primer, wherein the forward primer is a forward specific sequence binding to the targeted region, and the reverse primer comprises a reverse specific sequence binding to the targeted region at a 3'-end and a first universal sequencing sequence at a 5'-end”. Del Favero discloses “a first forward primer incorporating a primer region that hybridizes with a strand of the target nucleic acid molecule” (untagged) and a “first reverse primer incorporating a primer region that hybridizes with a strand of the target nucleic acid molecule and a nucleic acid tag 5’ of the primer region” (see Del Favero [0027]-[0030]). This reads directly on the claimed forward primer and reverse primer of claim1.
Del Favero further discloses “purification of amplification product: purifying a product of the first round of PCR amplification.” Del Favero teaches that purification is common and well known in the art, and teaches a two-tube embodiment in which the first round product is purified before the second round amplification proceeds in a separate reaction vessel (see Del Favero Fig. 12, [0434]-[0438]).
In regards to the second round of PCR amplification, Del Favero employs only universal primers matching tags introduced in the first round (see Del Favero [0071], [0074], [0078]), and does not disclose a locus-specific primer, downstream of the first forward primer, carrying its own newly introduced 5’ tag.
Zheng teaches this limitation and discloses an inside primer that is “nested with respect to the outside primer for the locus of interest” (see Zheng [0009]), where “the hybridization site of the outside primer is farther away from the locus of interest than the hybridization site of the inside primer (see Zheng [0060]). The inside primer comprises “a sequence at the 3’ end that specifically anneals to the target nucleotide sequence” and at the 5’ end “a sequence identical or complementary to the sequence of a second sequencing primer of an NGS platform” (see Zheng [0151]), reading on the claimed nested primer (second universal sequencing sequence at the 5’ end and specific sequence at the 3’ end)
It would have been obvious to a person of ordinary skill in the art to modify Del Favero’s second-round amplification by substituting, for a locus-specific primer, a nested inner primer bearing its own newly-introduced 5’ tag as taught by Zheng. A person of ordinary skill would have been motivated to make the modification because Zheng expressly states that its nested primer architecture provides “superior efficiency, specificity, and sensitivity” relative to single-round target-specific amplification approaches (see Zheng [0042]), directly addressing the same target-enrichment specificity goal underlying Del Favero’s own multiplex PCR method. This modification would succeed because it is a combination of prior art elements, Del Favero’s tagged first-round pair and second round universal primer architecture and Zheng’s nested inner primer design, according to known methods, each performing its already established function, to yield the predictable results of more specific second-round amplification. See KSR Int’l Co. v. Teleflex Inc. (KSR), 550 U.S. 398, 82 USPQ2d 1385 (2007). MPEP 2143(I)(A).
Del Favero further teaches “a 3'-end sequence of the first tag primer is identical to the first universal sequencing sequence of the reverse primer at the 5'-end,” Del Favero’s first universal tag comprises “a universal primer region identical (in the 5’ to 3’ direction to a first universal tag incorporated into the amplification produces when the primer pairs are using multiplex amplification (see Del Favero [0074]-[0077]). It would have been obvious to person of ordinary skill having already combined Zheng’s nested primer (with its own 5’ tag) into Del Favero’s second round amplification, to further apply Del Favero’s own established “identical (in the 5’ to 3’ direction) design rule to the other tag, designing a corresponding second universal primer with a 3’ end sequence identical to the nester primer’s second universal sequencing sequence. A person of ordinary skill would have been motivated to do so because Del Favero applies this identical sequence design rule to every tag-matching primer pair in the reference without exception, and a person of ordinary skill in the art would have had no reason to depart from that established rule merely because the tag in question originated from a newly-substituted nester primer rather than from one of Del Favero’s own first-round primers. This modification would be likely to succeed because it requires no departure from Del Favero’s own disclosed methodology, only the extension of an already uniform design principle to one additional primer pair, and a person of ordinary skill in the art would have had a reasonable expectation of success given that Zheng independently confirms that its inside primer tag is likewise designed to be “identical to… the sequence of a second sequencing primer” (see Zheng [0151]), consistent with the same identical sequence matching principle Del Favero applies throughout.
The combination of Del Favero and Zheng discloses or renders obvious every element of claim 1 as amended and the claim is unpatentable under 35 U.S.C. 103.
In regards to claim 2, as set out in the rejection of claim 1 above, the combination of Del Favero and Zheng renders obvious a second universal primer whose 3’ end sequence is identical to the nested primers second universal sequencing sequence. Del Favero further teaches that this same second universal primer comprises in addition to the universal primer region, an adaptor sequences and an optional sample barcode sequence (see Del Favero [0078]). The optional sample barcode sequences reads on the claimed second tag sequences, and the primer as a whole, comprising both this tag sequences and a 3’ end sequence identical to the second universal sequencing sequence of the nested primer, reads on the claimed second tag primer.
Del Favero’s disclosure of this sample-barcode-bearing structure for the second universal primer is consistent with and confirms the same design principle Del Favero applies to its first universal primer, which likewise comprises an optional sample barcode alongside its tag matching universal primer region (see Del Favero [0074]). A person of ordinary skill in the art combining Del Favero and the teachings of Zheng as set forth above would accordingly have arrived at a second universal primer that is itself a second tag primer bearing a second tag sequences, for the same reasons claim 1: this is simply Del Favero’s own uniformly applied primer architecture extended to the second universal primer matching the newly introduced nested primer tag.
In regards to claim 3, the claim recites an alternative first-round architecture in which the first tag primer participates directly in the first round of PCR amplification alongside the forward and reverse primers, rather than being introduced in the second round as in claim 1. Claim 3’s second round accordingly recites a “first universal primer” whose 3’end sequence is identical to a 5’end sequence of the first tag primer, in place of the first tag primer itself.
In regards to first round of PCR amplification, Del Favero teaches the limitations of the first forward primer that hybridizes to the target nucleic acid molecule without a 5’tag and a first reverse primer comprising a target-specific 3’ region and a 5’ universal tag (see Del Favero [0027]-[0030]).
Del Favero does not teach the first tag primer participating in the first round of PCR amplification together with the forward and reverse primers. Del Favero’s disclosure of structurally corresponding primer describes that the primer as used in a second, temporally distinct amplification reaction following purification of the first round product (see Del Favero [0007], [0074]).
Del Favero does, however, teach that all primers using in the method, including this same tag-matching universal primer, may be combined together in a single reaction mixture from the outset of amplification, without separating the reaction into temporally and physically distinct first and second amplification steps (see Del Favero [0023] disclosing “all of the reagents required for the method (i.e., to generate the further amplification products) are combined before the first amplification is carried out . Thus, the method may be performed in a single reaction vessel.”).
It would have been obvious to a person of ordinary skill in the art to include Del Favero’s tag matching universal primer in the same reaction mixture as the forward and reverse primers from the start of the first round of amplification rather than adding it only after an intervening purification step. A person of ordinary skill would have been motivated to make this modification because Del Favero itself confirms that combining first round target specific primers and tag matching primers together in one reaction mixture is a workable configuration (see Del Favero [0007]-[0009], [0023]). This modification would succeed because a primer lacking a complementary template, here, the tag-matching primer prior to any amplicon incorporating the reverse primer’s tag, does not productively primer early in a PCR reaction and becomes active for amplification only once its complementary sequences has accumulated in the reaction. A person of ordinary skill would have understood that combining this primer with the forwards and reverse primers from the outset does not alter the fundamental amplification chemistry of any of the primers involved, merely the timing of reagent addition. This is a combination of prior art elements according to known methods, yielding the predictable results of a single reaction mixture containing forward, reverse, and tag-matching primers together. See KSR Int’l Co. v. Teleflex Inc. (KSR), 550 U.S. 398, 82 USPQ2d 1385 (2007). MPEP 2143(I)(A).
Del Favero further discloses “purification of amplification product: purifying a product of the first round of PCR amplification.” Del Favero teaches that purification is common and well known in the art, and teaches a two-tube embodiment in which the first round product is purified before the second round amplification proceeds in a separate reaction vessel (see Del Favero Fig. 12, [0434]-[0438]).
In regards to the second round of PCR amplification with the second universal sequencing primer, Del Favero and Zheng as combined above in claim 1, teach this limitation. Zheng discloses an inside primer “nested with respect to the outside primer for the locus of interest”, comprising a 3’end sequence that specifically anneals to the target nucleotide sequence and a 5’end sequence “identical to or complementary to the sequences of a second sequencing primer of an NGS platform” (see Zheng [0009], [0151), reading on the claimed nested primer. The same motivation and rationale set forth in regards to the rejection of claim 1 apply here as well.
Del Favero and Zheng, combined as set forth above, do not expressly disclose a further primer whose 3’end sequences is identical to a 5’end sequences of the first tag primer itself, as distinct from the tag introduced by the reverse primer. Del Favero teaches the underlying design principle from which this limitation follows. Del Favero discloses that its universal primer architecture is defined by a recursive uniformly applied rule, a locus specific or tag matching primer introduces a tag or adaptor sequence at its 5’end and a further “universal primer” is designed with a 3’end sequence “identical (in the 5’ to 3’ direction” to that introduced sequences, in order to append “any further sequences needed for further processing and identification purposes (such as adaptors)” (see Del Favero [0012]). Del Favero applies this exact design rule at least twice within its own basic two-round architecture, establishing it as a general, repeatable methodology rather than a single-use mechanism specific to one primer pair (see Del Favero [0077], [0089]).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to apply this same recursive design rule one additional time, having incorporated Del Favero’s tag matching primer (bearing its own adaptor/tag sequence at its 5’end) as the first tag primer in the first round would have designed a further “first universal primer” with a 3’end sequence identical to that first tag primer’s won 5’end sequence, exactly as Del Favero’s own primers are designed relative to one another throughout the reference. A person of ordinary skill would have been motivated to do so because Del Favero expressly teaches that additional processing and identification sequences (such as sequencing adaptors) may be incorporated through exactly this kind of iterative tag matching primer design (see Del Favero [0012]), and because Del Favero’s adaptor sequences are themselves disclosed as functioning as primer binding sites for downstream processing (see Del Favero [0019], [0082]), confirming that an outermost tag or adaptor sequence introduced by any primer in the scheme is available as a binding site for a further, subsequent primer. This modification would succeed because it applies Del Favero’s own established repeatedly demonstrated design methodology to one additional layer, with a reasonable expectation of success given that no new chemistry or mechanism is introduced beyond what Del Favero already discloses and practices for every other tag-matching primer pair in the reference. The second universal primer is also obvious for the same reasons. Del Favero’s uniform “identical (in the 5’ to 3’ direction)” design rule (see Del Favero [0077], [0081], [0089], [0111], [0113], [0141], and throughout), applied to the nested primer’s tag introduced by Zheng (see Zheng [0151]), renders obvious a second universal primer with a 3’end sequence identical to the nested primers second universal sequencing sequence.
Accordingly, the combination of Del Favero and Zheng discloses or renders obvious every element of claim 3.
In regards to claim 4, Del Favero teaches this limitation for the same reasons set forth in the rejection of claim 2 above. Del Favero’s second universal primer comprises, in addition to the universal primer region identical to the nested primer’s tag, an adaptor sequence and an optional sample barcode sequence (see Del Favero [0078]). The optional sample barcode reads on the claimed second tag sequence, and the primer as a whole reads on the claimed second tag primer, for the same reasons articulated in the rejection of claim 2.
In regards to claim 5, Del Favero teaches that “the various primers of the invention may also be used to include barcodes into the amplification products… for aspects of the invention concerning the target-specific primers used in the first amplification, molecular barcodes are advantageously included in the primers. A molecular barcode is a specific nucleic acid sequence that enables the subsequent amplicon in which it is incorporated to be identified in in post-sequencing, in silico analysis” (see Del Favero [0020]), reading on the “reverse tag primer further comprises a molecular tag sequence.”
In regards to claims 6 and 11, Del Favero discloses that “[i]n specific embodiments each sample barcode is at least 4 , 6 or 8 nucleotides , optionally up to 20 nucleotides , in length” (see Del Favero [0021]). This range substantially overlaps the claimed 8 bp to 24 bp range or 8 bp to 15 bp range. It would have been obvious to one of ordinary skill in the art to modify the length of the barcode needed based on the complexity of the library being used. A person of ordinary skill would have been motivated to do so because barcode length is a result effective variable, routinely optimized in the art to balance sufficient sequence diversity for unique molecular identification against primer length and amplification efficiency constraints. A person of ordinary skill in the art would have understood that minor extension of a disclosed length range, absent evidence of criticality at the extended boundary, involves only routine experimentation. See MPEP 2144.05.
In regards to claims 7 and 8, Del Favero discloses tiling multiple primer pairs across continuous, overlapping target region (see Del Favero [0027] disclosing “the invention provides a set of primers for use in multiplex amplification of overlapping regions of a target nucleic acid molecule”). Del Favero further teaches that this tiling scheme extends to any number of primer pairs and expressly discloses embodiments wherein “the number of primer pairs in the set of primers may be at least 5 , 10 , 15 , 20 , 25 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 150 , 200 , 300 , 400 , 500 , 600 , 700 , 800 , 900 , 1000 , 2000 , 3000 , 4000 , 5000 etc.” (see Del Favero [0042]). Each such primer pair comprises a forward and reverse primer as set forth in the rejection of claim 1 above, reading on the claimed corresponding forward primer and reverse primer for each of the plurality of continuous target regions. The overlap between adjacent regions is expressly recited as Del Favero recites each successive primer pair amplifies a region that “at least partially overlaps with” the immediately preceding region, reading on the claimed amplification overlap between adjacent amplicon regions (see Del Favero [0027], [0037], [0042]).
In regards to claim 9, Del Favero discloses that for the plurality of tiled overlapping primer pairs, neighboring pairs are deliberately arranged with a reciprocal orientation, such that the tag carried by the reverse primer of one targeted region matches the tag carried by the forward primer of the immediately adjacent targeted region, and vice versa (see Del Favero [0044]-[0047]). Del Favero’s working example confirms this reciprocal arrangement directly (see Del Favero [0742]). This reciprocal alternating orientation between the forward and reverse primers of adjacent regions is expressly adopted for the purpose of suppressing aberrant amplification products at the region of overlap between neighboring amplicons (see Del Favero [0044]-[0055]), and reads on the claimed forward primer and reverse primer of adjacent targeted regions being in opposite amplification directions.
In regards to claim 10, Del Favero’s working example discloses that both the first and second round amplifications are each performed for 20 cycles, falling squarely within the claimed 2 to 30 cycle range (see Del Favero [0743]). Zheng further teaches amplification of “In some embodiments, the method comprises at least 2 or more PCR amplification cycles, such as at least about any one of 5, 10, 15, 20, 25, 30 or more iterative PCR amplification cycles. In some embodiments, the method comprises about 2 - 100 PCR amplification cycles, e.g., any one of any one of about 5-50, about 5-30, about 5-20, about 10-20, about, about 10- 5, about 15-30, about 30-50, or about 10-30 PCR amplification cycles” (see Zheng [0104]). It would have been obvious to person of ordinary skill to select a cycle number within the claimed 2 to 30 range for either or both rounds of PCR since cycle number is a routinely-optimized, result effective variable balancing amplification yield against accumulation of non-specific or aberrant products, and both references independently disclose cycle number within or overlapping the claimed range for amplification steps.
In regards to claim 12, the claim recites a kit comprising primer architecture mirroring those used in claim 1 or claim 3. As recited above, the combination of Del Favero and Zheng makes obvious the primer architecture included in claims 1 and 3. In order to perform the method as outlined, one would necessarily have the components needed. Del Favero’s own primer set is disclosed in the alternative as a either a method for performing amplification or a kit for the same purpose (see Del Favero [0066]) and Zheng likewise discloses its nester primer architecture in kit form using the same primer elements relied upon in the method context (see Zheng [0025], [0212]-[0213]). It would have been obvious to one of ordinary skill in the art at the time of filing that any modification obvious for the method is equally obvious as applied to the corresponding kit.
In regards to claim 13, the combination of Del Favero and Zheng renders obvious a second universal primer whose 3’ end sequence is identical to the nested primers second universal sequencing sequence as outlined in the rejections of claims 1-4 above. It would have been obvious to one of ordinary skill in the art at the time of filing that any modification obvious for the method is equally obvious as applied to the corresponding kit.
In regards to claim 16, Del Favero teaches that “the various primers of the invention may also be used to include barcodes into the amplification products… for aspects of the invention concerning the target-specific primers used in the first amplification, molecular barcodes are advantageously included in the primers. A molecular barcode is a specific nucleic acid sequence that enables the subsequent amplicon in which it is incorporated to be identified in in post-sequencing, in silico analysis” (see Del Favero [0020]), reading on the “reverse tag primer further comprises a molecular tag sequence.” It would have been obvious to one of ordinary skill in the art at the time of filing that any modification obvious for the method is equally obvious as applied to the corresponding kit.
In regards to claims 17, and 20-22, Del Favero discloses that “[i]n specific embodiments each sample barcode is at least 4 , 6 or 8 nucleotides , optionally up to 20 nucleotides , in length” (see Del Favero [0021]). This range substantially overlaps the claimed 8 bp to 24 bp range or 8 bp to 15 bp range. It would have been obvious to one of ordinary skill in the art to modify the length of the barcode needed based on the complexity of the library being used. A person of ordinary skill would have been motivated to do so because barcode length is a result effective variable, routinely optimized in the art to balance sufficient sequence diversity for unique molecular identification against primer length and amplification efficiency constraints. Del Favero further discloses “The length of the tag can be readily determined by one skilled in the art to ensure that, in a given amplification reaction, the production of aberrant amplification products is minimized” (see Del Favero [0060]). A person of ordinary skill in the art would have understood that minor extension of a disclosed length range, absent evidence of criticality at the extended boundary, involves only routine experimentation. See MPEP 2144.05
In regards to claim 18 and 19, Del Favero discloses that for the plurality of tiled overlapping primer pairs, neighboring pairs are deliberately arranged with a reciprocal orientation, such that the tag carried by the reverse primer of one targeted region matches the tag carried by the forward primer of the immediately adjacent targeted region, and vice versa (see Del Favero [0044]-[0047]). Del Favero’s working example confirms this reciprocal arrangement directly (see Del Favero [0742]). This reciprocal alternating orientation between the forward and reverse primers of adjacent regions is expressly adopted for the purpose of suppressing aberrant amplification products at the region of overlap between neighboring amplicons (see Del Favero [0044]-[0055]), and reads on the claimed forward primer and reverse primer of adjacent targeted regions being in opposite amplification directions.
Conclusion
No claim is 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.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681
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