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 . The preliminary amendment filed 11/14/2022 is acknowledged. Claims 1-26 have been cancelled. Claims 27, 28 and 30 are pending.
Priority
This application is a DIV of 16/615,872 filed 11/22/2019, now US PAT 11,530,436 which is a 371 of PCT/US18/34162 filed 05/23/2018 which claims benefit of 62/509,981 filed 05/23/2017.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/14/2022 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
The drawings were received on 11/14/2022. These drawings are accepted by the Examiner.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 30 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 30 is directed to the method of claim 15. However, the claim 15 was cancelled by amendment filed 11/14/2022. Therefore, the claims 30 fails to further limit the claims 27-29 and appears to not be related to the subject matter of the claims 27-29 recited therein.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
10. Claims 27-29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al (WO 2017015075, citation made of record on IDS filed 11/14/2022) in view of Grunewald et al (US 2014162897).
Regarding Claim 27-29, Chen teaches methods of making nucleic acid fragments described herein utilize a transposase and an RNA polymerase. The transposase is complexed with a transposon DNA including a double stranded transposase binding site and a first nucleic acid sequence including one or more of a barcode sequence, a priming site and an RNA polymerase promoter sequence to form a transposase/transposon DNA complex. The first nucleic acid sequence may be in the form of a single stranded extension or the first nucleic acid sequence may be in the form of a loop with each end connected to a corresponding strand of the double stranded transposase binding site. According to certain aspects, the transposases have the capability to bind to the transposon DNA and dimerize when contacted together, such as when being placed within a reaction vessel, forming a transposase/transposon DNA complex dimer called transposome. The transposome have the capability to bind to target locations along double stranded nucleic acids, such as double stranded genomic DNA, forming a complex including the transposome and the double stranded genomic DNA. The transposases in the transposome cleave the double stranded genomic DNA, with one transposase cleaving the upper strand and one transposase cleaving the lower strand. The transposon DNA in the transposome is attached to the double stranded genomic DNA at the cut site. According to certain aspects, a plurality of transposase/transposon DNA complexe dimers bind to a corresponding plurality of target locations along a double stranded genomic DNA, for example, and then cleave the double stranded genomic DNA into a plurality of double stranded fragments with each fragment having transposon DNA attached at each end of the double stranded fragment. According to one aspect, the transposon DNA is attached to the double stranded genomic DNA and a single stranded gap exists between one strand of the genomic DNA and one strand of the transposon DNA. According to one aspect, gap extension is carried out to fill the gap and create a double stranded connection between the double stranded genomic DNA and the double stranded transposon DNA. According to one aspect, the transposase binding site of the transposon DNA is attached at each end of the double stranded fragment. According to certain aspects, the transposase is attached to the transposon DNA which is attached at each end of the double stranded fragment. According to one aspect, the transposases are removed from the transposon DNA which is attached at each end of the double stranded genomic DNA fragments. (page 4).
Chen further teaches according to certain exemplary aspects; a transposition system is used to make nucleic acid fragments for amplification and sequencing as desired. According to one particular aspect, a transposition system is combined with an RNA polymerase for single cell genome amplification. According to one aspect, a transposition system is used to fragment genomic DNA into double stranded genomic DNA fragments. An RNA polymerase is used to make RNA amplicons which are then reverse transcribed into DNA. Complements to the DNA are made and double stranded genomic DNA sequences are formed which are amplicons linearly amplified from the original double stranded genomic DNA fragments. According to certain aspects, the use of an RNA polymerase to make amplicons in a linear manner advantageously achieves high quality amplification of the single-cell genomic DNA (gDNA) reducing or avoiding (1) amplification bias, leading to the noisy single-cell sequencing data that further affect the genome coverage, as well as the low resolution detection of copy number variations (CNVs); (2) amplification errors, causing a high false positive rate in the single-cell sequencing data that further prevents the accurate detection of single nucleotide variations (SNVs); and (3) chimera formation during amplification, which overwhelms the signal of structural variations (SVs) from the original single-cell genomic DNA sample (page 17, second full paragraph). After the transposition reaction, single cell-genomic DHNA will be fragmented with each fragmented tagged by a strong T7 promoter on both ends, enabling linear amplification afterwards through in vitro transcription (page 18, lines 10-12). Chen teaches that the result of using transposon DNA is a genomic DNA fragment with a transposon DNA transposase binding site attached to the 5’ position if an upper strand of the DNA and a transposon DNA transposon binding site attached to the 5’ position of a lower strand. (page 18, lines 24-26). Chen states in vitro transcription (IVT) is used to linearly amplify the single - cell genomic DNA fragments, generating many RNAs that contain the same sequence as the original genomic double stranded DNA template. Finally, by reverse transcription and second strand synthesis, the amplified RNAs are converted back into double -stranded DNA molecules, with the barcode region originally designed in te transposon DNA attached on both ends of each fragment (page 18, last para. to top of page 19, lines 1-3). Chen additionally teaches that IVT advantageously provides linear amplification, with all the copies generated from the original DNA template. The resulting RNA molecules can be reverse transcribed into single stranded DNA followed by complementary strand formation resulting in double stranded DNA, which are amplicons linearly amplified from the original DNA template. As a result of the use of an RNA polymerase for linear amplification, amplification bias is much smaller between different amplicons. Moreover, amplification accuracy is higher since in linear amplification the amplification errors cannot propagate into later stages page 20, lines 10-20).
Finally, Chen teaches that separation techniques following amplification, it may be desirable to separate the amplification, it may be desirable to separate the amplification products of several different lengths from each other, from the template, and from excess primers for the purpose of analysis or more specifically determining whether specific amplification has occurred (example XIII, page 37).
While Chen teaches numerous embodiments in the amplification steps that would inherently result in separating double stranded DNA prior to amplification such as e.g., via denaturation during amplification and separation techniques after amplification (see example XIII), the reference does not expressly teach separating the target double stranded DNAs prior to the separate amplification of each strand, However MPEP 2144.04 states that the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930).
Nonetheless, Grunenwald discloses a method of amplifying two strands of a double stranded nucleic acid sequencing having different primer sites at each end (para. 0017]. Grunenwald teaches the present invention relates to preparation of linear ssDNA fragments or tagged circular ssDNA fragments (and amplification products thereof) from target DNA comprising any dsDNA of interest (including double-stranded cDNA prepared from RNA), from any source, for genomic, subgenomic, transcriptomic, or metagenomic analysis, or analysis of RNA expression.
At para. [0018] In some embodiments, the present invention provides methods for generating a library of tagged DNA fragments of a target DNA, comprising incubating the target DNA with a transposase and a transposon end or transposon end composition comprising a transferred strand that has a tag domain in its 5' portion, under conditions wherein a transposition reaction is catalyzed by the transposase, and wherein the target DNA is fragmented to generate a plurality of target DNA fragments and a transferred strand of the transposon end or transposon end composition is joined to the 5' ends of each of a plurality of the target DNA fragments, to produce a plurality of 5' tagged target DNA fragments. In some embodiments, the methods further comprise incubating the plurality of 5'-tagged target DNA fragments with at least one nucleic acid modifying enzyme under conditions wherein a 3' tag is joined to a 3' end of the 5'-tagged target DNA fragment to produce a comprising di-tagged target DNA fragments. In the Figure 8, Grunenwald et al illustrates an example of the method wherein a DNA polymerase that has strand-displacement DNA polymerase activity and/or that has 5'-to-3' exonuclease activity is used to join the second tag to the 5'-tagged DNA fragments from the in vitro transposition reaction to generate a library of DNA fragments comprising di-tagged DNA fragments. As shown, the strand-displacement and/or 5'-to-3' exonuclease activity of the DNA polymerase displaces or digests the DNA that is annealed downstream of the DNA polymerase extension product and the extension by the DNA polymerase joins a second tag that comprises or consists of a DNA sequence that is complementary to the first tag inserted into the opposite strand. In some embodiments, the di-tagged ssDNA fragment products are PCR amplified using oligonucleotides that are complementary to the different sequences in the respective first or second tags as PCR primers {0066]. At para. [0175] –[0176], Grunenwald teaches In some preferred embodiments wherein a single transposon end composition is used in the in vitro transposition reaction of the method comprising generating the library of tagged DNA fragments comprising di-tagged DNA fragments using a DNA polymerase that has strand-displacement or 5' nuclease activity and further amplifying the di-tagged DNA fragments generated by PCR, two different PCR primers are used, each of which PCR primers exhibits the sequence of at least a portion of the transferred transposon end that composes the transposon end composition. In some preferred embodiments, each PCR primer comprises a 3'-portion and a 5'-portion, wherein the 3'-portion exhibits the respective transferred transposon end sequence and the 5'-portion exhibits the sequence of a respective tag domain for a particular purpose (e.g., a sequencing tag domain or an amplification tag domain, and optionally an address tag domain for next-generation sequencing or amplification).
[0176] In some preferred embodiments of any of the methods comprising generating the library of tagged DNA fragments comprising di-tagged DNA fragments using a DNA polymerase that has strand-displacement or 5' nuclease activity, the at least one transposome in the in vitro transposition reaction comprises or consists of two different transposomes. In some preferred embodiments wherein two different transposomes are used, each of the two transposomes comprises the same transposase but a different transposon end composition. In some preferred embodiments wherein two different transposomes are used, the two different transposomes each comprise the same transposase and the transposon end compositions comprise different transferred strands. In some preferred embodiments wherein two different transposomes are used, each of the two transposomes comprises different transposase enzymes and different transposon end compositions, each of which forms a functional complex with the respective transposase. In some preferred embodiments of the method wherein two different transposon end compositions are used in the in vitro transposition reaction and wherein the library of tagged DNA fragments comprising di-tagged ssDNA fragments is generated using a DNA polymerase that has strand-displacement or 5' nuclease activity, the first tag exhibits the sequence of the transferred strand of one transposon end composition and the second tag exhibits the sequence of the non-transferred strand of the other transposon end composition.
Grunenwald further teaches the following: [0424] In order to generate a non-selective DNA fragment library that can be amplified prior to library preparation, DNA fragments were generated and tagged at the 3'-end and the 5'-end using "ME Transposomes".
[0426] In order to tag the 3' ends of the transposition-generated and 5'-tagged DNA fragments with the transferred transposon end sequence, the reaction products were incubated with a strand-displacing polymerase mix and dNTPs.
[0427] A portion of the transposition-generated and 5'-tagged DNA fragments was diluted 1:10 prior to 3'-end tagging and amplification to characterize amplification of 4 ng of DNA library template. In order to non-selectively amplify the entire population of tagged DNA fragments using a single primer PCR, the following reaction was performed with the METS PCR primer, which hybridizes only to the transposon end sequence and does not contain additional 3' sequence information. In order to tag the 3' ends of the transposition-generated and 5'-tagged DNA fragments with the transferred transposon end sequence, the reaction products were incubated with a strand-displacing polymerase mix and dNTPs prior to the denaturation step (See FIG. 7) followed by sequencing [0433].
[0443] In order to generate a non-selective DNA fragment library that can be used directly in emPCR for Roche/454 FLX Titanium sequencing, amplicon DNA was fragmented and 5'-tagged with ME transposomes. Non-selective adaptor oligonucleotides were used to append the DNA library with Roche/454 FLX Titanium emPCR and sequencing adaptor sequence (FIG. 10).
[0446] In order to non-selectively amplify and append the DNA fragment library with adaptors compatible with Roche/454 FLX Titanium emPCR and sequencing, PCR was performed using adaptor oligos which hybridize to the transposon end sequence and do not contain additional 3' sequence information (FIG. 10).
Grunenwald teaches that the method provides improved means of generating libraries of DNA fragments from target DNA molecules for amplification, including amplification of whole or partial genomes from one organism or from multiple organisms for further analysis by (e.g., by real-time PCR, emulsion PCR, comparative genomic hybridization (CGH), comparative genomic sequencing (CGS), or for preparing DNA-specific labeled probes (e.g., chromosome-specific probes, e.g., chromosome paints, or e.g., gene-specific probes, e.g., for fluorescent in situ hybridization (FISH), for a variety of purposes (e.g., for research, diagnostic, and industrial purposes). [0015].
It would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have been motivated to have modified the teachings of Chen et al to encompass tagging steps as taught by Grunenwald with amplification method steps as taught by Chen for the obvious been of generating DNA fragment libraries with improved representation of sequences for amplification at suggested by Grunenwald.
11. No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681