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 .
Priority
This application is a 371 of PCT/US2022/077273 filed 09/29/2022. PCT/US2022/077273 has PRO 63/249,653 filed 09/29/2021. All claims are being examined using the earlier filing date of 09/29/2021.
Claim Status
Claims 8-12, 16-21, 24-29, 31-34, and 39-51 were cancelled prior to examination. Claims 1-7, 13-15, 22-23, 30-31, and 35-38 are pending and under examination. Claims 1, 22, and 30 are independent claims.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 22 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 22 recites the limitation "the universal primer region" in step III(a). There is insufficient antecedent basis for this limitation in the claim. Throughout the rest of claim 22, the claims distinguishes “a first universal primer region” and a “second universal primer region”, but this one instance drops the “firs”/ “second” designation. It is unclear whether this refers to the first universal primer region, and is simply missing a word, or is meant as some undefined third term.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Grunenwald et al. (US 2010/0120098 A1, published May 13, 2010, on IDS 04/23/2025).
In regards to claim 1, Grunenwald teaches methods, compositions and kits for using transposase and a transposon end compositions for generating a library of tagged DNA fragments from target DNA. Among the items taught, Grunenwald teaches a kit comprising transposome (in solution without magnesium), a separate reaction buffer containing magnesium (see Grunenwald [0237]). Grunenwald further discloses that the kit may additionally comprise a DNA polymerase (see Grunenwald [0238]). Thus, Grunenwald teaches “a kit comprising: (a) first composition comprising a DNA polymerase.”
Grunenwald does not expressly teach that the transposome composition of the kit comprises both a first synaptic complex and a second synaptic complex as required by claim 1. However, Grunenwald further teaches compositions comprising first and second synaptic complexes. In particular, Grunenwald teaches forming “A-MEDS transposomes” and “B-MEDS transposomes” by preincubating Tn5 transposase with A-MEDS and B-MEDS transposon end compositions, respectively, and combining the A-MEDS and B-MEDS transposomes in equal ratios to form “A/B transposomes” (see Grunenwald [0425]-[0426]). Grunenwald defines a “transposome” as the “hyperactive EZ-Tn5™ Tn5 transposase preincubated with double-stranded transposon DNA under conditions that support non-covalent complex formation” (see Grunenwald [0408]). Accordingly, the A-MEDS transposome corresponds to the claimed first synaptic complex comprising a first transposase and a first adapter oligonucleotide, and B-MEDS transposome corresponds to the claimed second synaptic complex comprising a second transposase and a second adapter oligonucleotide. Grunenwald further teaches that the second composition does not comprise magnesium ions. Specifically, the A/B transposome are diluted in “TSase storage buffer (50 mM Tris chloride pH 7.5, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, 500 mM Sodium chloride, 0.5% w/v NP-40, 0.5% v/v Tween-20)” (see Grunenwald [0426]).
Grunenwald further teaches magnesium ions in a third composition. Specifically, Grunenwald separately provides magnesium containing reaction buffer for use with the A/B transposomes, including 5x TMgCl-DMF Reaction Buffer comprising 25 mM magnesium chloride (see Grunenwald [0408]). Thus, Grunenwald teaches the claimed arrangement in which second composition containing the synaptic complexes does not comprise magnesium ions and magnesium ions are instead provided in a third composition.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to employ Grunewald’s A/B transposome, maintained in the disclosed magnesium free TSase storage buffer, as the transposome composition of Grunenwald’s disclosed kit, while providing magnesium ions separately in the disclosed magnesium-containing reaction buffers. Grunenwald expressly teaches the A/B transposomes as transposome compositions for fragmenting and tagging target DNA and demonstrates their use by combining the separately maintained A/B transposomes with the magnesium-containing reaction buffer for the transposition reaction (see Grunenwald [0425]-[0427]). Accordingly, use of Grunenwald’s expressly disclosed A/B transposome embodiment as the transposome composition in Grunenwald’s kit would have amounted to the predictable use of a disclosed transposome composition according to its established function in the disclosed kit for generating tagged DNA fragments. Further, Grunenwald expressly states “the invention comprises any kit that can be assembled form a combination of any two compositions of the invention and any novel composition that is used in a kit or method of the invention” (see Grunenwald [0233]), which constitutes express disclosure of the claimed kit, since Grunenwald itself instructs the ordinary skilled reader to combine the discloses compositions, including the A/B transposome composition, the DNA polymerase, and the magnesium-containing reaction buffer. A reasonable expectation of success given that Grunenwald discloses each composition and storage buffer that would be needed to assemble a kit.
The combination would result in “a kit comprising: (a) a first composition comprising a DNA polymerase; (b) a second composition comprising: (i) a first synaptic complex comprising a first transposase and a first adapter oligonucleotide; and (ii) a second synaptic complex comprising a second transposase and a second adapter oligonucleotide; wherein the second composition does not comprise magnesium ions; and (c) magnesium ions, wherein the magnesium ions are either in a third composition or in the first composition”, thereby rendering claim 1 obvious over Grunenwald.
Claims 1-5 are rejected under 35 U.S.C. 103 as being unpatentable over Grunenwald et al. (US 2010/0120098 A1, published May 13, 2010, on IDS 04/23/2025) as applied to claim 1 above, included here for reasons supra, and further in view of Gunderson et al. (US 2018/0273933 Al, published Sep. 27, 2018).
In regards to claim 2, Grunenwald discloses the kit of claim 1 for which the claim depends. Grunenwald further teaches the transposon end composition of a transposome can comprise a transferred strand having a 3’ portion comprising the transferred transposon end sequence and a 5’ portion comprising a tag domain for use in next-generation sequencing or amplification (see Grunenwald [0235]-[0237]). Grunenwald does not expressly teach that the first adapter oligonucleotide comprises both a first universal primer region and a first adapter barcode region and that the second adapter oligonucleotide comprises both a second universal primer region and a second adapter barcode region.
However, Gunderson teaches transposon sequences comprising barcodes and specifically teaches that, for transposome comprising two non-contiguous transposon sequences, a first transposon sequences may comprise a first barcode a second transposon sequence may comprise a second barcode (see Gunderson [0052]). Gunderson further teaches that the first and second barcode may be different (see Gunderson [0053]). Gunderson additionally teaches that it is advantageous for template nucleic acids to incorporate universal primer sites, including a first end sequences comprising a first universal primer site and a second end sequence comprising a second universal primer site, and expressly teaches that such universal primer sites may be introduced by insertion of a transposon sequence comprising the universal primer site (see Gunderson [0086]). The universal primer sites are taught as useful for amplification, sequencing, and/or identification of template nucleic acids.
More particularly, Gunderson teaches preparing indexed Tn5 transposomes from a first set of indexed oligonucleotides comprising P5 sequences, a unique 8-base i5 sequence, a universal connector sequence, and a Tn5 mosaic end sequence, and a second set of indexed oligonucleotides comprising P7 sequences, a unique 8-base i7 index sequence, a universal connector sequence, and a Tn5 mosaic end sequence (see Gunderson [0188]-[0190]). Gunderson teaches separately incubating the P5_i5 and P7_i7 transposons with Tn5 transposase to form respective P5_i5 and P7_i7 transposome complexes (see Gunderson [0191]). Thus, Gunderson teaches first and second Tn5 adapter/transposon oligonucleotides comprising respective primer/adapter regions and barcode regions.
It would have been obvious to one of ordinary skill in the art before the effective filling date to modify the first and second adapter oligonucleotides of the A/B Tn5 transposomes taught by Grunewald to comprises respective universal primer regions and adapter barcode regions, as taught by Gunderson, because Gundeson teaches that incorporation of universal primer sites facilitates amplification, and sequencing of the resulting template nucleic acids and that incorporation of barcode sequences permits identification of template nucleic acids. Gunderson expressly describes a two-tier indexing method in which first level indexes are attached by tagmentation as compartment specific indexes contained in the transposons (see Gunderson [0005]). The combination would therefore have predictably provided Grunenwald’s Tn5 transposomes with first and second adapter oligonucleotides comprising respective universal primers regions and barcode regions, thereby providing the kit of claim 2.
In regards to claim 3, Grunenwald discloses the invention of claim 1 as discussed above. Grunenwald further teaches transposon end compositions in which the transferred strand comprises a 3’ portion exhibiting the transferred transposon end sequences and a 5’ portion exhibiting an adapter priming region distinct from any barcode or universal primer region, for use in subsequent PCR amplification with a separate amplifier oligonucleotide homologous to that priming region (see Grunenwald [0278]-[0283], [0405]-[0423]).
To the extent it is argue that Grunenwald does not expressly show a first and a second adapter oligonucleotide each comprising such a priming region in a single transposome composition, Gunderson teaches a “sequencing adaptor” region within a transposon sequence “compris[ing] one or more sites that can hybridize to a primer” (see Gunderson [0042]), and further teaches amplification using “primers that hybridize to primer sites of integrated transposon sequences” (see Gunderson [0082]-[0083]), and that “transposomes… comprises at least a first primer site and a second primer site” (see Gunderson [0042]), confirming that it was known in the art to build a primer-hybridization site into each of a pair of transposon-borne adapters for later amplification. It would have been obvious to combine this teaching with Grunenwald’s two transposome kit for the reasons given above.
In regards to claim 4, Grunewald teaches a kit further comprising first and second amplifier oligonucleotides, each comprising a universal primer region and an amplifier region homologous to the corresponding adapter’s priming region, for amplifying the transposition products by PCR (see Grunenwald [0004], [0241], [0278]-[0283]). Grunenwald’s specific example of amplifier oligonucleotides present a barcode on only one of the paired amplifier oligonucleotides (see Grunenwald [0441]-[0464], [0508]-[0520]). Gunderson teaches amplifying tagmented nucleic acid using “one out of 96 unique combinations of barcoded P5 and P7 PCR primers,” such that both amplification primers carry independent barcodes in combination with a transposon-borne barcode (see Gunderson [0185], [0189]-[0192]), for the purpose of combinatorial multiplexing of a large number of samples using a limited number of physical reagent wells (see Gunderson [0027], [0114], [0187]). It would have been obvious to one of ordinary skill in the art to modify Grunenwald’s kit so that both the first and second amplifier oligonucleotides carrying their own barcode region, as taught by Gunderson, in order to increase the multiplexing capacity of Grunenwald’s library preparation method through combinatorial barcoding, with a predictable increase in the number of distinguishable sample indices.
In regards to claim 5 and 13, Grunenwald in view of Gunderson teaches the kit of claim 2 and claim 4, as set forth above. Grunewald further teaches amplifying a nucleic acid sample using such a kit, e.g., combining size-selected transposition with a DNA polymerase and PCR primers in a single reaction vessel and amplifying by PCR (see Grunenwald [0403]-[0423]), thereby generating sequencing oligonucleotides comprising the recited nucleic acid sequences and its complement. Claims 5 and 13 recites no step other than amplifying the nucleic acid sample using the kit of claims 2 and 4; the “single-pot reaction in a first reaction vessel” language of the preamble is satisfied by performing that amplifying step in a single vessel, as Grunenwald discloses, irrespective of the vessel(s) in which any earlier transposition reaction was performed. The recited generation of a nucleic acid duplex by transposase activity is the ordinary expected result of using the kit as described.
Claims 1-7, 13-15, 22-23, 30-31, and 35-38 are rejected under 35 U.S.C. 103 as being unpatentable over Grunenwald et al. (US 2010/0120098 A1, published May 13, 2010, on IDS 04/23/2025) and Gunderson et al. (US 2018/0273933 Al, published Sep. 27, 2018) as applied to claims 1-5, 13 above, included here for reasons supra, and further in view of Mellor et al. (US 2018/0010120 A1, published Jan. 11, 2018).
In regards to claims 6 and 7, Grunenwald and Gunderson teach the limitations of claim 5 for which claim 6 depends. Grunenwald teaches (ii) generating intermediated nucleic acids comprising the first universal primer region, first tag domain, and homologous sequence of the first nucleic acid fragment, and the second universal primer region, second tag domain, and complement sequence of the first nucleic acid fragment, in a transposition reaction between the nucleic acid sample and two transposomes (see Grunewald Figs. 2 & 7, [0164], [0176]-[0177], [0257]-[0267]); and (iii) generating the sequencing oligonucleotides in a polymerization reaction, wherein the DNA polymerase extends the 3’ end of a nucleic acid duplex comprising a pair of the intermediate nucleic acids (see Grunenwald [0172]-[0173], [0264]). Grunenwald’s disclosed protocol adds the DNA polymerase after the transposition reaction is performed rather than combining the first composition, second composition, magnesium ions, and nucleic acid sample together in the first reaction vessel as recited.
Mellor teaches combining, in a single reaction, a magnesium-containing tagmentation buffer, a “Tagment DNA enzyme” (transposome), a DNA polymerase master mix, and target DNA together before incubating the combined reaction first under transposition conditions (55 °C, reading on option (a) of claim 7), and then under PCR conditions, stating that “tagmentation and sample tagging… were conducted in the same reaction” (see Mellor [0089]-[0090]). It would have been obvious to one of ordinary skill in the art to combine Grunenwald’s first composition, second composition, and magnesium ions with the nucleic acid sample in a single reaction vessel at the outset, as taught by Mellor, rather than adding the polymerase after separate transposition step, in order to simplify the workflow and reduce the number of handling and purification steps, an objective Grunenwald identifies as a goal of the invention (see Grunenwald [0016] disclosing the desire for “methods for generating DNA fragment libraries that do not require specialized instruments, and that are easier, faster, require less hands-on time…”).
In regards to claims 14 and 15, Grunenwald in view of Gunderson teaches or renders obvious the kit components and amplification method of claim 13 as discussed above. Grunenwald teaches combining the first composition a nucleic acid sample with first and second synaptic complexes (the second composition) in a reaction buffer (magnesium ions). In particular, Grunenwald teaches A/B transposomes comprising A-MEDS and B-MEDS transposon end compositions complexed with Tn5 transpose and teaches contacting the A/B transposome with target DNA in a MgCl2 containing reaction buffer to perform tagmentation (see Grunenwald [0425]-[0427]).
Grunenwald further teaches “(ii) generating intermediate nucleic acids comprising nucleic acid sequences of (a) the first adapter priming region and the homologous sequence of the first nucleic acid fragment; and(b) the second adapter priming region and the complement sequence of the first nucleic acid fragment, in a transposition reaction between the nucleic acid sample, the first synaptic complex, and the second synaptic complex.” Specifically, Grunenwald teaches a tagmentation reaction using the A/B transposomes on the nucleic acid sample, having the reaction proceed at 55 °C for 5 minutes (reading on options (a) and (b) of claim 15) (see Grunenwald [0425]-[0427], [0508]-[0520]).
Grunenwald further teaches “(iii) generating the amplicons in a PCR reaction with a pair of the intermediate nucleic acids, DNA polymerase, the first amplifier oligonucleotide, and the second amplifier oligonucleotide.” Grunenwald teaches PCR amplification of the tagged DNA fragments using DNA polymerase and the first and second amplifier oligonucleotides. (see Grunenwald [0508]-[0520]).
Grunenwald does not teach combining “in the first reaction vessel” all of the components for performing the tagmentation reaction and PCR amplification as required by the claim, rather Grunenwald performs the transposition reaction and subsequent PCR amplification in separate reactions.
Mellor teaches combining in a single reaction vessel, a DNA polymerase, a first and second synaptic complex, magnesium ions, a first and second amplifier oligonucleotide, and a nucleic acid sample. Specifically, Mellor’s reaction comprises tagmentation buffer containing MgCl2, Tagment DNA enzyme, Q5 Hot Start DNA polymerase, forward primer, reverse primer, and gDNA sample (see Mellor [0089]). Mellor expressly teaches that “tagmentation and sample-tagging by PCR were conducted in the same reaction” (see Mellor [0090]).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the method of Grunenwald by combining the DNA polymerase, the first and second synaptic complex, magnesium ions, the first and second amplifier oligonucleotide, and a nucleic acid in the first reaction vessel as taught by Mellor, and thereafter performing the transposition reaction and PCR in the first reaction vessel. One of ordinary skill in the art would have been motivated to make such a modification in order to simplify the workflow and reduce the number of handling and purification steps, an objective Grunenwald identifies as a goal of the invention (see Grunenwald [0016] disclosing the desire for “methods for generating DNA fragment libraries that do not require specialized instruments, and that are easier, faster, require less hands-on time…”). Further, Mellor has demonstrated successful tagmentation and PCR in single-reaction vessel and therefore one of ordinary skill would have a reasonable expectation of success.
In regards to claim 22, Grunenwald teaches methods, compositions and kits for using transposase and a transposon end compositions for generating a library of tagged DNA fragments from target DNA. In regards to the specific limitations of claim 22, Grunenwald teaches “A method of generating a library comprising amplicons from a nucleic acid sample in a single-pot reaction in a first reaction vessel.” Grunenwald discloses a method of generating a library of DNA fragments from a nucleic acid sample, including embodiments performed in a single reaction vessel without an intervening purification step (see Grunenwald [0171] disclosing “the library of tagged DNA fragments comprising di-tagged ssDNA fragments is generated from the target DNA in a single tube without performing any intervening purification steps”). Grunewald teaches this generally as a goal of the invention.
“(a) magnesium ions” Grunenwald teaches magnesium ions as a component of its transposition reaction buffers (see Grunenwald [0408]).
“(b) a DNA polymerase” Grunenwald teaches DNA polymerase as a kit and reaction component e.g., FailSafe™ DNA polymerase (see Grunenwald [0238]).
“(c) a first synaptic complex comprising a first transposase and a first adapter oligonucleotide comprising a first adapter priming region; (d) a second synaptic complex comprising a second transposase and a second adapter oligonucleotide comprising a second adapter priming region” Grunenwald teaches transposon end composition in which the transferred strand exhibits the transposon end sequence, used as an adapter priming region for subsequent PCR (see Grunenwald [0430]-[0437], Figs. 7-8, 13).
(e) a first amplifier oligonucleotide comprising a first universal primer region, a first amplifier barcode region, and a first amplifier priming region; and (f) a second amplifier oligonucleotide comprising a second universal primer region, a second amplifier barcode region, and a second amplifier priming region” Grunenwald teaches amplifier/PCR-primer oligonucleotides homologous to the adapter priming region that additionally carry a universal primer region and, in some instances, a barcode region, e.g., A-MID2-METS” comprising a sequencing tag (universal primer region), a barcode (MID2), and a 3’ portion homologous to the METS adapter priming region (see Grunenwald [0395], Fig. 13). Grunenwald does not teach a second amplifier oligonucleotide that itself comprises a second amplifier barcode region. In each of Grunenwald’s disclosed examples, the paired amplifier oligonucleotide carries a universal primer region and an amplifier priming region but no second barcode.
Gunderson teaches that, in a transposome comprising two non-contiguous transposon sequences “the first transposon sequence comprises a first barcode, and the second transposon sequence comprises a second barcode” (see Gunderson [0052]), and teaches amplifying tagmented nucleic acids using “one out of 96 unique combinations of barcoded PS and P7 PCR primers,” such that both amplification primers carry independent barcodes (see Gunderson [0185], [0189]-[0192]). Gunderson teaches a second amplifier oligonucleotide comprising a second amplifier barcode region.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify Grunenwald’s second amplifier oligonucleotide to include its own barcode region, as taught by Gunderson, rather than leaving it unbarcoded. A person of ordinary skill in the art would have been motivated to make this modification because doing so independently indexes both ends of a transposed DNA fragment, increasing the number of samples that can be combinatorially multiplexed and demultiplexed in a single sequencing run, a benefit Gunderson expressly identifies (see Gunderson [0027], [0114], and [0187] disclosing “96x96 combinatorial indexing is used to barcode about 1000 single cells…[t]hroughput can be readily scaled up… using 384x384 combinatorial barcoding… about 10,000 single cells can be individually barcoded in parallel”). A person of ordinary skill would have had a reasonable expectation of success in making this modification because Gunderson demonstrates that adding a second, independent barcode region to the paired oligonucleotide of a two-adapter transposition/PCR scheme is a routine application of known indexing techniques to a structurally similar amplification chemistry, yielding the predictable results of a second, functional barcode without altering the oligonucleotide’s ability to prime amplification.
“wherein the first adapter priming region is homologous to the first amplifier priming region, and the second adapter priming region is homologous to the second amplifier priming region” Grunenwald teaches that the adapter priming region of the transferred transposon end (e.g., METS) is homologous to the amplifier priming region of the corresponding PCR primer (e.g., A-MID2-METS, B-METS), such that the primer anneals to and amplifies across the transposed fragment (see Grunenwald [0508]-[0513] disclosing “PCR was performed using adaptor oligos which hybridize to the transposon end sequence”).
Step I, overarching limitation: “combining in the first reaction vessel” components (a)-(f) together. Grunenwald, as set forth above, teaches each of components (a)-(f) individually, but does not teach combining each of the components in a single reaction vessel before the transposition reaction of step II begins. Grunenwald’s disclosed protocol perform the transposition reaction first, purifies the transposition products, and only then combines the PCR components (polymerase, amplifier oligonucleotides). Mellor teaches combining in a single reaction vessel, a DNA polymerase, a first and second synaptic complex, magnesium ions, a first and second amplifier oligonucleotide, and a nucleic acid sample. Specifically, Mellor’s reaction comprises tagmentation buffer containing MgCl2, Tagment DNA enzyme, Q5 Hot Start DNA polymerase, forward primer, reverse primer, and gDNA sample (see Mellor [0089]). Mellor expressly teaches that “tagmentation and sample-tagging by PCR were conducted in the same reaction” (see Mellor [0090]).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the method of Grunenwald by combining the DNA polymerase, the first and second synaptic complex, magnesium ions, the first and second amplifier oligonucleotide, and a nucleic acid in the first reaction vessel as taught by Mellor, and thereafter performing the transposition reaction and PCR in the first reaction vessel. One of ordinary skill in the art would have been motivated to make such a modification in order to simplify the workflow and reduce the number of handling and purification steps, an objective Grunenwald identifies as a goal of the invention (see Grunenwald [0016] disclosing the desire for “methods for generating DNA fragment libraries that do not require specialized instruments, and that are easier, faster, require less hands-on time…”). Further, Mellor has demonstrated successful tagmentation and PCR in single-reaction vessel and therefore one of ordinary skill would have a reasonable expectation of success.
Step “II. generating intermediate nucleic acids comprising nucleic acid sequences of (a) the first adapter priming region and a homologous sequence of a first nucleic acid fragment; and (b) the second adapter priming region and a complement sequence of the first nucleic acid fragment, in a transposition reaction between the nucleic acid sample, the first synaptic complex, and the second synaptic complex, wherein a nucleic acid duplex comprising the first nucleic acid fragment and its complement is generated from the nucleic acid sample by transposase activity” Grunenwald teaches generating intermediate nucleic acids of this structure in a transposition reaction between a target nucleic acid sample and two transposomes, each carrying a different adapter priming region, such that transposase activity fragments the target DNA and joins each adapter priming region to one strand of the resulting duplex (see Grunenwald [0508]-[0520], Fig. 10).
Step “III. generating the amplicons in a PCR reaction involving a pair of the intermediate nucleic acids, the DNA polymerase, the first amplifier oligonucleotide, and the second amplifier oligonucleotide, wherein the amplicons comprise: (a) a nucleic acid sequence comprising the universal primer region, the first amplifier barcode region, the first amplifier priming region, a homologous sequence of the first nucleic acid fragment, the complement sequence of the second amplifier priming region, the complement sequence of the second amplifier barcode region, and the complement sequence of the second universal primer region; and (b) the complement sequence thereof.” Grunenwald teaches generating amplicons in a PCR reaction using a pair of the intermediate nucleic acids, a DNA polymerase, and the first and second amplifier oligonucleotides, such that the resulting amplicon comprises the first universal primer region, the first amplifier priming region, a homologous sequences of the target fragment, and the complement of the second amplifier priming region and second universal primer region, together with the complement sequence thereof (see Grunenwald [0508]-[0520], Fig. 10). Grunenwald does not teach that this amplicon additionally comprises a first amplifier barcode region and the complement of a second amplifier barcode region, for the same reason given above to step I (e)-(f), Grunenwald disclosed amplifier oligonucleotide pairs carry a barcode on only one member of the pair. Gunderson teaches amplification using barcoded primer pairs in which both members carry an independent barcode (see Gunderson [0185], [0189]-[0192]), such that the resulting amplicon incorporates both barcode regions and their complements. It would have been obvious to combine this teaching with Grunenwald’s PCR amplification step for the same reasons given above with respect to step I (e)-(f).
In regards to claim 23, Grunenwald discloses transposition reactions at both 37°C and 55°C (see Grunenwald [0427], [0435], [0511]), depending upon which transposase is used, reading on the claimed range of 25-65°C of option (a).
In regards to claim 30, Grunenwald teaches methods, compositions and kits for using transposase and a transposon end compositions for generating a library of tagged DNA fragments from target DNA. In regards to the specific limitations of claim 30, Grunenwald teaches “A method of generating a library comprising sequencing oligonucleotides from a nucleic acid sample in a single-pot reaction in a first reaction vessel” Grunenwald discloses a method of generating a library of DNA fragments from a nucleic acid sample, including embodiments performed in a single reaction vessel without an intervening purification step (see Grunenwald [0171] disclosing “the library of tagged DNA fragments comprising di-tagged ssDNA fragments is generated from the target DNA in a single tube without performing any intervening purification steps”). Grunewald teaches this generally as a goal of the invention.
“(a) magnesium ions” Grunenwald teaches magnesium ions as a component of its transposition reaction buffers (see Grunenwald [0408]).
“(b) a DNA polymerase” Grunenwald teaches DNA polymerase as a kit and reaction component e.g., FailSafe™ DNA polymerase (see Grunenwald [0238]).
“(c) a first synaptic complex comprising a first transposase and a first adapter oligonucleotide comprising a first universal primer region and a first adapter barcode region” Grunenwald teaches a transposome comprising a transposase and a transposon end composition in which the transferred strand comprises a 3’ portion exhibiting the transferred transposon end sequence and a 5’ potion exhibiting a tag domain, including a sequencing tag domain (universal primer region) and, in some embodiments, an address (barcode) tag domain (see Grunenwald [0103], [0113], [0235]). Grunenwald teaches and adapter oligonucleotide of this structure, e.g., “A-MID2-METS” which comprises a sequencing tag, a barcode, and the transposon end sequence (see Grunenwald [0395], [0433]-[0434]).
“(d) a second synaptic complex comprising a second transposase and a second adapter oligonucleotide comprising a second universal primer region and a second adapter barcode region” Grunenwald teaches forming a reaction from two different transposomes, each comprising a transposase bound toa different transposon end composition (see Grunenwald [0422]-[0429]). Grunenwald teaches that the second transposome adapter oligonucleotide comprises a second universal primer region, e.g., “B-METS”, which comprises a sequencing tag and a transposon end sequence (see Grunenwald [0391]). Grunenwald does not teach that this second adapter oligonucleotide additionally comprises a second adapter barcode region.
Gunderson teaches that, in a transposome comprising two non-contiguous transposon sequences “the first transposon sequence comprises a first barcode, and the second transposon sequence comprises a second barcode” (see Gunderson [0052]), and further teaches assembling transposomes in which both the P5_i5 adapter and the paired P7_i7 adapter independently carry their own barcode (see Gunderson [0189]-[0192]). Gunderson teaches a second adapter oligonucleotide comprising a second adapter barcode region.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify Grunenwald’s second adapter oligonucleotide to include its own barcode region as taught by Gunderson, rather than leaving it unbarcoded. A person of ordinary skill would have been motivated so independently indexes both ends of a transposed DNA fragment, increasing the number of samples that can be combinatorially multiplexed and demultiplexed in a single sequencing run, a benefit Gunderson expressly identifies (see Gunderson [0027], [0114], and [0187] disclosing “96x96 combinatorial indexing is used to barcode about 1000 single cells…[t]hroughput can be readily scaled up… using 384x384 combinatorial barcoding… about 10,000 single cells can be individually barcoded in parallel”). A person of ordinary skill would have had a reasonable expectation of success in making this modification because Gunderson demonstrates that adding a second, independent barcode region to the paired two-adapter transposition is a routine application of known indexing techniques yielding the predictable results of a second, functional barcode without altering the oligonucleotide’s ability to participate in transposition.
Step I, overarching limitation: “combining in the first reaction vessel” components (a)-(f) together. Grunenwald, as set forth above, teaches each of components (a)-(f) individually, but does not teach combining each of the components in a single reaction vessel before the transposition reaction of step II begins. Grunenwald’s disclosed protocol perform the transposition reaction first, purifies the transposition products, and only then combines the PCR components (polymerase, amplifier oligonucleotides). Mellor teaches combining in a single reaction vessel, a DNA polymerase, a first and second synaptic complex, magnesium ions, a first and second amplifier oligonucleotide, and a nucleic acid sample. Specifically, Mellor’s reaction comprises tagmentation buffer containing MgCl2, Tagment DNA enzyme, Q5 Hot Start DNA polymerase, forward primer, reverse primer, and gDNA sample (see Mellor [0089]). Mellor expressly teaches that “tagmentation and sample-tagging by PCR were conducted in the same reaction” (see Mellor [0090]).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the method of Grunenwald by combining the DNA polymerase, the first and second synaptic complex, magnesium ions, the first and second amplifier oligonucleotide, and a nucleic acid in the first reaction vessel as taught by Mellor, and thereafter performing the transposition reaction and PCR in the first reaction vessel. One of ordinary skill in the art would have been motivated to make such a modification in order to simplify the workflow and reduce the number of handling and purification steps, an objective Grunenwald identifies as a goal of the invention (see Grunenwald [0016] disclosing the desire for “methods for generating DNA fragment libraries that do not require specialized instruments, and that are easier, faster, require less hands-on time…”). Further, Mellor has demonstrated successful tagmentation and PCR in single-reaction vessel and therefore one of ordinary skill would have a reasonable expectation of success.
Step “II. generating intermediate nucleic acids comprising nucleic acid sequences of (a) the first universal primer region, the first adapter barcode region, and a homologous sequence of a first nucleic acid fragment; and (b) the second universal primer region, the second adapter barcode region, and a complement sequence of the first nucleic acid fragment, in a transposition reaction between the nucleic acid sample, the first synaptic complex, and the second synaptic complex, wherein a nucleic acid duplex comprising the first nucleic acid fragment and its complement is generated from the nucleic acid sample by transposase activity” Grunenwald teaches generating intermediate nucleic acids of this general structure, comprising the universal priming region, a tag domain, and a homologous or complementary sequence of a target DNA fragment, in a transposition reaction between a target nucleic acid sample and two transposome, wherein transposase activity fragments the target DNA and generates a duplex comprising the first nucleic acid fragment and its complement (see Grunenwald [0164], [0176]-[0177], [0257]-[0267], Fig. 13) As modified by Gunderson to include a barcode region on each adapter oligonucleotide, for the reasons given above with respect to step I (d).
Step “III. generating the sequencing oligonucleotides in a polymerization reaction involving a pair of the intermediate nucleic acids and the DNA polymerase, wherein the polymerization reaction extends the 3' ends of a nucleic acid duplex comprising the pair of intermediate nucleic acids to generate the sequencing oligonucleotides” Grunenwald teaches generating sequencing oligonucleotides in a polymerization reaction in which a DNA polymerase extends the 3’ end of each intermediate nucleic acid using the opposite, paired intermediate nucleic acid as a template (see Grunenwald [0172]-[0173], [0264]).
“wherein the sequencing oligonucleotides comprise:(a) a nucleic acid sequence comprising the first universal primer region, the first adapter barcode region, the homologous sequence of a first nucleic acid fragment, the complement sequence of the second adapter barcode region, and the complement sequence of the second universal primer region; and(b) the complement sequence thereof.” Grunenwald teaches that the resulting sequencing oligonucleotides comprise a nucleic acid sequence including the first universal primer region, a homologous sequence of the target fragment, and the complement of the second universal primer region, together with the complement sequence thereof as the product of the polymerization reaction described above (see Grunenwald Fig. 13, [0264]-[0267]). Grunenwald does not teach that this sequence additionally comprises a first adapter barcode region and the complement of a second adapter barcode region, for the same reason given above as to step I(d). Gunderson teaches transposon sequences in which both members of an adapter pair carry an independent barcode (see Gunderson [0052], [0189]-[0192]), such that the resulting sequencing oligonucleotide incorporates both barcode regions, and their complements once transposed and extended. It would have been obvious to combine this teaching with Grunewald’s adapter oligonucleotides for the same reason as given above with respect to step I(d).
Thus, the combined teachings of Grunenwald, Gunderson, and Mellor teach all of the limitations of claim 30, and the claim is obvious in view of Grunenwald, Gunderson, and Mellor.
In regards to claim 31, Grunenwald discloses transposition reactions at both 37°C and 55°C (see Grunenwald [0427], [0435], [0511]), depending upon which transposase is used, reading on the claimed range of 25-65°C of option (a).
In regards to claims 35 – 37, recite the same kit and method architecture as claims 1, 3, 5, and 22 – a lit comprising two synaptic complexes each with an adapter oligonucleotide comprising and adapter priming region (claim 3, incorporated into claim 35), and a first and second amplifier oligonucleotide each comprising a universal primer region and an amplifier priming region homologous to the corresponding adapter priming region (claim 35), but recite the amplifier oligonucleotides without the amplifier barcode region recited in claim 4/claim 22(e)-(f), and correspondingly recite amplicons/products (claim 36(a)) without the barcode terms recited in claim 22, step III(a). Grunenwald directly teaches this unbarcoded embodiment: “Ti A-METS” and “Ti B-METS”, each comprising a sequencing tag (universal primer region) and a 3’ portion homologous to the METS adapter priming region, with no barcode sequence present in either primer (see Grunenwald Fig. 10, [0396]-[0397], [0494]-[0507]), used to amplify transposed intermediates by PCR into amplicons comprising the recited universal-primer-region/amplifier-priming-region/targe-homolog/complement structure without a barcode term. Because Grunenwald’s example 10 disclosed the unbarcoded amplifier oligonucleotide pair directly, Gunderson’s barcode teaching, relied upon to modify Grunenwald’s amplifier oligonucleotides for claims 4 and 22, is not needed to meet claims 35-37; Gunderson relied upon only for its teaching of an express adapter priming region as a kit-level component, as set forth in the rejection of claim 3 above. Claim 37’s further limitation of combining these components together in the first reaction vessel before transposition is met by Mellor for the same reasons given in the rejection of claim 22.
In regards to claim 38, Grunenwald discloses transposition reactions at both 37°C and 55°C (see Grunenwald [0427], [0435], [0511]), depending upon which transposase is used, reading on the claimed range of 25-65°C of option (a).
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/ Primary Examiner, Art Unit 1681
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