DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Applications, Amendments and/or Claims
This action is written in response to applicant's correspondence submitted 12/11/2025. In the paper of 12/11/2025, Applicant amended claims 1, 8, and 22 and newly cancels claim 18.
Claims 1, 3-5, 7-17 and 19-24 are pending. Claims 2 and 6 were previously canceled.
This paper is a Non-Final Office action as it newly addresses the performing of a reverse transcription within a hydrogel as claimed in claims 20-24. The newly presented rejection under 35 U.S.C. 103 below, provides a number of previously cited references which are rearranged to present a new prima facie case of obviousness over the instant method(s). The rejection under 35 U.S.C. 103 below highlights one or more new teachings/citations from the(se) reference(s) not previously indicated.
Response to Arguments
Withdrawn and/or Moot Rejection(s)
The rejection of claim 18 under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form is moot based on the cancellation of claim 18.
The rejection of claims 1, 3, 5, 8-17 and 19 under 35 U.S.C. 103 as being unpatentable over Ziegler et al. (Nov. 13, 2019, PLoS One, 14(11), e0224656, pp 1-14) as evidenced by Bio-Rad Bulletin, Droplet Digital PCR Applications Guide (2019) in view of Blauwkamp et al. (2019, Nature microbiology, 4(4), pp.663-674) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) or Diehl et al. (2005, P.N.A.S., 102(45), pp.16368-16373) or So et al. (2018, NPJ Genomic Medicine, 3(1), 2, pp.1-10) and Shi et al. (2019, Msystems, 4(4), pp.10-1128) is withdrawn in favor of a new rejection under 35 U.S.C. 103 presented below.
The rejection of claims 1, 3-5, 7-17 and 19 under 35 U.S.C. 103 as being unpatentable over Hatori et al. (Epub July 23, 2018, Analytical chemistry, 90(16), pp.9813-9820) in view of McDermott et al. (2013, Analytical chemistry, 85(23), pp.11619-11627), Ziegler et al. (Nov. 13, 2019, PLoS One, 14(11), e0224656, pp 1-14), Hindson et al. (US2014/0378350A1, pub. Dec 2014) and Gosiewski et al., (2017, Eur. Journal of Clin. Microbio & Infectious Diseases, 36, pp.329-336) and Christensen et al., (Epub Jan 30, 2018, Scientific reports, 8(1), pp.1-11) and Shi et al. (2019, Msystems, 4(4), pp.10-1128) is withdrawn in favor of a new rejection under 35 U.S.C. 103 provided below.
The rejection of claim 18 under 35 U.S.C. 103 as being unpatentable over Hatori et al. (Epub July 23, 2018, Analytical chemistry, 90(16), pp.9813-9820) in view of McDermott et al. (2013, Analytical chemistry, 85(23), pp.11619-11627), Ziegler et al. (Nov. 13, 2019, PLoS One, 14(11), e0224656, pp 1-14), Hindson et al. (US2014/0378350A1, pub. Dec 2014) and Gosiewski et al., (2017, Eur. Journal of Clin. Microbio & Infectious Diseases, 36, pp.329-336) and Christensen et al., (Epub Jan 30, 2018, Scientific reports, 8(1), pp.1-11) and Shi et al. (2019, Msystems, 4(4), pp.10-1128) is now moot based on the cancellation of claim 18.
The provisional rejection of claims 1, 3-5 and 7-17 on the ground of nonstatutory double patenting as being unpatentable over claims 1-10, 12-28 of copending Application No. 17/210,737 (reference application) in view of Hatori et al. (Epub July 23, 2018, Analytical chemistry, 90(16), pp.9813-9820) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) is withdrawn as the claims of copending Application No. 17/210,737 (reference application) are amended to change the scope of that invention and has been allowed.
The provisional rejection of claim 18 on the ground of nonstatutory double patenting as being unpatentable over claims 1-10, 12-28 of copending Application No. 17/210,737 (reference application) in view of Hatori et al. (Epub July 23, 2018, Analytical chemistry, 90(16), pp.9813-9820) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) is moot as claim 18 is canceled.
The provisional rejection of claims 1, 3-5 and 7-17 on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-21 of copending Application No. 17/747,599 (reference application) in view of Hatori et al. (Epub July 23, 2018, Analytical chemistry, 90(16), pp.9813-9820) and Blauwkamp et al. (2019, Nature microbiology, 4(4), pp.663-674) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) is withdrawn as the claims of copending Application No. 17/747,599 (reference application) are amended to change the scope of that invention.
The provisional rejection of claim 18 on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-21 of copending Application No. 17/747,599 (reference application) in view of Hatori et al. (Epub July 23, 2018, Analytical chemistry, 90(16), pp.9813-9820) and Blauwkamp et al. (2019, Nature microbiology, 4(4), pp.663-674) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) is moot as claim 18 is canceled.
The provisional rejection of claims 1, 3-5 and 7-17 on the ground of nonstatutory double patenting as being unpatentable over claims 1-30 of copending Application No. 17/747,645 (reference application) and Blauwkamp et al. (2019, Nature microbiology, 4(4), pp.663-674) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) as copending Application No. 17/747,645 (reference application) has been abandoned.
The rejection of claims 1, 3-5 and 7-17 on the ground of nonstatutory double patenting as being unpatentable over claims 1-14 of US Patent No. 11, 827,936 in view of Blauwkamp et al. (2019, Nature microbiology, 4(4), pp.663-674) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) is withdrawn as claims 1-14 of US Patent No. 11, 827,936 provide each template particle (i.e. hydrogel particle) bound to first capture probes comprising capture sequences and second capture probes comprising template-switching oligos (TSOs) and provides a step of capturing the plurality of distinct mRNA molecules with the first capture probes; while the instant claims provides a capture probe comprising a sequence for binding a complementary portion of a 16S rRNA or 16S rDNA of a target microbial nucleic acid, the probe being attached to hydrogel of a particle via a linker. The scope of the claims of US Patent No. 11,827,936 are different those of the instant claims.
The provisional rejection of claims 1, 3-5 and 7-17 on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of copending Application No. 17/864,930 (reference application) and Blauwkamp et al. (2019, Nature microbiology, 4(4), pp.663-674) and Hindson et al. (US2014/0378350A1, pub. Dec 2014) is withdrawn in favor of a new rejection provided below.
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.
Claims 1, 3-5, 7-17 and 19-24 are rejected under 35 U.S.C. 103 as being unpatentable over Hindson et al. (US2014/0378350A1, pub. Dec 2014: previously cited) in view of Hatori et al. (Epub July 23, 2018, Analytical chemistry, 90(16), pp.9813-9820: previously cited) and Ziegler et al. (Nov. 13, 2019, PLoS One, 14(11), e0224656, pp 1-14: previously cited) as evidenced by Bio-Rad Bulletin, Droplet Digital PCR Applications Guide (2019: previously cited) and Gosiewski et al., (2017, Eur. Journal of Clin. Microbio & Infectious Diseases, 36, pp.329-336: previously cited) and Shi et al. (2019, Msystems, 4(4), pp.10-1128: previously cited).
Hindson et al. (US2014/0378350A1) (claims 1, 3-5, 10-12, 14, 22-24)
Regarding claim 1, Hindson et al. teach a method to detect nucleic acid, the method comprising:
obtaining a sample comprising a target microbial nucleic acid (para [0253], [0438]);
combining particles with the sample in a first fluid and wherein the particles comprise a hydrogel (see Fig. 4A, see reproduction below: para [0183] teaches hydrogel beads 401, (e.g., disulfide cross-linked polyacrylamide gel beads) are pre-functionalized with a first primer 403. The first primer 403 are coupled to the beads via a disulfide linkage 402 with an acrydite moiety bound to the surface of the beads 401. The first primer 403 constitute a universal primer for priming template sequences of oligonucleotides to be attached to the beads and/or may be a primer binding site (e.g., P5) for use in sequencing an oligonucleotide that comprises first primer 403).
Fig. 4A and 4B reproduced from Hindson et al. (US2014/0378350A1)
PNG
media_image1.png
532
832
media_image1.png
Greyscale
Further regarding claim 1, Hindson et al. teach:
adding a second fluid that is immiscible with the first fluid to create a mixture (para [0184]-[0185]);
shearing the mixture, thereby partitioning the sample to form a plurality of droplets (para [0185]),
wherein the target nucleic acid is segregated inside one droplet of the plurality of droplets and wherein the one droplet comprises a particle of the particles (para [0185]);
binding, inside the one droplet, the target microbial nucleic acid with a capture probe, wherein the capture probe is attached to the hydrogel of the particle via a linker (para [0185]-[0186]), and
wherein the capture probe binds to a complementary target sequence (para [0184]-[0186]);
amplifying bound target microbial nucleic acid to create an amplicon inside the one droplet (para [0185]-[0186]);
detecting the amplicon to thereby reveal a presence of target nucleic acid (para [0453]-[0455], para [0242], [0244], [0480]);
releasing the amplicon from the one droplet (para [0187], [0189]); and
sequencing the amplicon (para [0400]-[0401], [0426]-[0427], [0438).
Regarding claims 3-5, Hindson et al. teach amplifying comprises a polymerase chain reaction in the presence of a fluorophore, wherein said fluorophore is incorporated into the amplicon, the fluorophore comprises an intercalating dye, wherein detecting comprises sensing a fluorescent signal from the fluorophore, wherein said fluorescent signal is indicative of the amplicon (para [0242], para [0453]-[0455], para [0244], [0480]).
Regarding claims 10-12, Hindson et al. teach sequencing on amplicons that are recovered from droplets after a ddPCR process wherein the sequencing produces a plurality of sequence reads. Hindson et al. teach analyzing the sequence reads to characterize the amplicon. Hindson et al. teach said analyzing step comprises aligning the sequence reads to one or more references sequences (para [0400]-[0401], [0426]-[0427], [0438]).
Regarding claim 14, Hindson et al. teach sample is a blood sample (para [0254]).
Regarding claim 22, Hindson et al. teach providing reverse transcriptase inside the droplet to generate a copy of the 16s rRNA and amplifying the copy to generate an amplicon (para [0364]-[0367], [0434]).
Regarding claim 23, Hindson et al. teach lysing the one droplet to release the amplicon (para [0162], [0453]).
Regarding claim 24, Hindson et al. teach amplifying the amplicon before the sequencing (para [0400]-[0401], [0426]-[0427], [0438]).
Omitted from Hindson et al. (US2014/0378350A1)(claims 1, 8-9, 13, 19)
Regarding claim 1, Hindson et al. do not teach vortexing of a mixture comprising a sample comprising target microbial nucleic acids and particles comprising hydrogel to partition the sample to form a plurality of droplets simultaneously.
Regarding claim 1, Hindson et al. do not expressly teach capture probe binds a complementary portion of a 16s rRNA or a 16s rDNA of the target microbial nucleic acid.
Regarding claim 19, Hindson et al. do not teach amplifying comprises Multiple Displacement Amplification (MDA).
Hatori et al. (claims 1, 3, 5, 7, 19, 20-21)
Regarding claim 1, Hatori et al. teach a particle-templated emulsification Droplet digital PCR (ddPCR) method for detecting nucleic acid of yeast Saccharomyces cerevisiae and/or lambda virus DNA (pg 9813, right col., 2nd para below the abstract and pg 9814, right col., section entitled “ddPCR”).
Hatori et al. teach the target nucleic acid is a microbial nucleic acid (pg 9814, right col., section entitled “ddPCR” and pg 9815, left col., 1st para below Fig. 2 and pg 9815, all text of section entitled “Cell Culture”: wherein target sequence of a lambda virus DNA and yeast DNA are amplified and detected).
The method of Hatori et al. vortexes to generate compartmentalized reactions in monodispersed droplets simultaneously (pg 9813, right col., 2nd para below the abstract and pg 9815, Results and Discussion).
Regarding claims 1 and 20-21, Specifically, the method of Hatori et al. uses monodispersed particles (PEG-, agarose-, hydrogel-, polyacrylamide (PAA)- templated gel beads) to “template” the formation of droplets of similar size under agitation with oil (pg 9813, right col., 2nd para below the abstract and pg 9814, legend of Fig. 1 and pg 9814, left col., 1st para below Fig. 1).
The method of Hatori et al. comprises:
obtaining a sample comprising a target nucleic acid (pg 9815, all text of section entitled “Cell Culture”);
partitioning the sample to form a plurality of droplets simultaneously, wherein the target nucleic acid is segregated inside one of the droplets (pg 9814, right col., section entitled “ddPCR” and pg 9815, left col., 1st para below Fig. 2 and pg 9815, all text of section entitled “Cell Culture”);
binding, inside the droplet, the target nucleic acid with a capture probe (pg 9813, right col., barcoded oligonucleotides provides into droplet or pg 9814, right col, section entitled “ddPCR”, wherein Hatori et al teach Yeast probe, Lambda probe; Hatori et al. also teach template particles (polyacrylamide PAA particle) comprising or tethered to one or a plurality of capture probes that may/may not be attached via reversible crosslinkers (see PAA particles soaked with primers and probes in Fig. 1b on pg 9814 or pg 9816, left col., 1st and 2nd paragraphs; or PAA particles functionalized with oligonucleotides and comprising reversible cross- linkers: see pg 9819, left col., 2nd para);
amplifying bound target nucleic acid to create an amplicon within the droplet (pg 9814, right col., section entitled “ddPCR”); and
detecting the amplicon to thereby detect the target nucleic acid.
Regarding claim 3, Hatori et al. teach the amplifying comprises a polymerase chain reaction in the presence of a fluorophore, wherein said fluorophore is incorporated into the amplicon (pg 9814, right col., section entitled “ddPCR”: wherein yeast Taqman probe comprises a 5’ FAM fluorophore, an internal ZEN quencher and a 3IABkFQ quencher at its 3’ end while the Lambda Probe comprises a Cy5 label at the 5’ end and the 3IAbRQSp quencher at its 3’ end).
Regarding claim 5, Hatori et al. teach detecting comprises sensing a fluorescent signal from the fluorophore, wherein said fluorescent signal is indicative of the amplicon (pg 9814, right col., section entitled “ddPCR”).
Regarding claim 1, Hatori et al. teach a method that further comprises:
combining template particles with the sample in a first fluid (see pg 9814, Figs. 1a-1c, (reproduced below) and pg 9814, right col., section entitled “ddPCR” and pg 9815, all text of section entitled “Cell Culture”);
adding a second fluid that is immiscible with the first fluid to create a mixture (see pg 9814, Fig. 1d (reproduced below) and pg 9814, right col., section entitled “ddPCR” and pg 9815, all text of section entitled “Cell Culture”); and
vortexing the mixture, thereby partitioning the sample to form the plurality of droplets (see pg 9814, Fig. 1e (reproduced below) and pg 9814, right col., section entitled “ddPCR” and pg 9815, all text of section entitled “Cell Culture”).
PNG
media_image2.png
492
770
media_image2.png
Greyscale
Regarding claim 7, Hatori et al. teach template particles template the formation of the droplets and segregate the target nucleic acid inside one of the droplets away from non-target nucleic acids present in the sample (pg 9815, right col., Results and Discussion).
Omitted from Hatori et al. (claims 1, 4, 8-10,14-17, 19, 22-24)
Regarding claim 1, Hatori et al. do not teach sequencing the amplicon to identify the species of microbe.
Hatori et al do not meet the full limitations of claim 4, 8-10, 14-17 and 22-24.
Regarding claims 1 and 8-9 and 22-24, while Hatori et al. teach template particles (polyacrylamide PAA particle) comprising or tethered to one or a plurality of capture probes that may/may not be attached via reversible crosslinkers (see PAA particles soaked with primers and probes in Fig. 1b on pg 9814 or pg 9816, left col., 1st and 2nd paragraphs; or PAA particles functionalized with oligonucleotides and comprising reversible cross- linkers: see pg 9819, left col., 2nd para), Hatori et al. do not teach the capture probe comprises a nucleotide sequence that is complementary to a portion of a 16s rRNA or 16s rDNA of the target microbial nucleic acid.
Regarding claim 19, Hatori et al. do not teach amplifying comprises Multiple Displacement Amplification (MDA).
Regarding claim 22, Hatori et al. do not teach providing reverse transcriptase inside the droplet to generate a copy of the 16s rRNA and amplifying the copy to generate an amplicon.
Regarding claims 23-24, Hatori et al. do not teach lysing the one droplet to release the amplicon, or amplifying the amplicon before the sequencing.
Ziegler et al. (claims 1, 3, 5, 8-9, 14 and 16-17)
Regarding claim 1, Ziegler et al. teach a quantitative droplet digital PCR (ddPCR) method to detect bacterial DNA for monitor bacteria during BSI (see abstract: which discloses the detection of a nuc target sequence of Staphylococcus aureus, a lytA target sequence of Streptococcus pneumoniae, and an uidA target sequence of Escherichia coli.
The method of Ziegler et al. comprises:
obtaining a sample comprising a target nucleic acid (abstract and pg 2, sections of the Materials and Methods entitled “Patients” and “Blood Cultures” and “Serial dilutions of samples with reference strain bacteria” and also pg 3, 1st para and the section entitled “DNA extraction”);
partitioning the sample to form a plurality of droplets, wherein the target nucleic acid is segregated inside one of the droplets (pg 3, section entitled “Droplet digital PCR”);
binding, inside the droplet, the target microbial nucleic acid with a 16S capture probe (see pg 3, section entitled “Droplet digital PCR” and pg 4, Table 1 where the capture primers/probe(s) are disclosed);
amplifying bound target microbial nucleic acid to create an amplicon inside the droplet (see pg 3, all text of section entitled “Droplet digital PCR” and pg 4, Table 1 where the capture primers/probe(s) are disclosed; see also pg 4, 1st and 2nd paragraphs below Table 1); and
detecting the amplicon to thereby reveal the presence of microbial nucleic acid (see abstract: wherein it is disclosed that from blood containing samples of 83 patients with BSI, 16S rDNA and species-specific DNA were detected in 60% and 61%, respectively, at least at one timepoint and the abstract also discloses that 92% of the patients were 16S rDNA-positive and 85% nuc-positive; see also pg 9, Fig. 2A-2C and pg 10, Fig. 3; see also pg 3, 2nd para of section entitled “Droplet digital PCR” wherein Zeiger et al. discloses detection of DNA using QX100 Droplet digital PCR system manufactured by Bio-rad Laboratories, Inc and use of droplet reader to detect PCR positive droplets).
Regarding claim 3, Ziegler et al. teach the amplifying comprises a polymerase chain reaction in the presence of a fluorophore, wherein said fluorophore is incorporated into the amplicon (see pg 4, Table 1 which discloses use of the 5’ end fluorophore FAM).
Regarding claim 5, Ziegler et al. teach detecting comprises sensing a fluorescent signal from the fluorophore, wherein said fluorescent signal is indicative of the amplicon (see pg 4, Table 1 which discloses use of the 5’ end fluorophore FAM and the 3’ end non-fluorescent quencher MGBNFQ; wherein amplicon provides a fluorescent signal due to probe hybridization).
Regarding claim 1, Ziegler et al. teach a method that further comprises:
combining particles with the sample in a first fluid (pg, 3, 1st and 2nd paragraphs and last para of section entitled “Droplet digital PCR”: wherein Ziegler et al. discloses an aqueous PCR mix in UV irradiated water placed into a droplet generator cartridge);
adding a second fluid that is immiscible with the first fluid to create a mixture (pg, 3, 1st and 2nd paragraphs of section entitled “Droplet digital PCR”: wherein Ziegler et al. discloses adding oil to the droplet generator cartridge and the use of a droplet generator to generate 15,000 water-in-oil droplets formed); and
vortexing the mixture, thereby partitioning the sample to form the plurality of droplets (pg, 3, 1st para and 2nd para of section entitled “Droplet digital PCR”: wherein Ziegler et al. discloses 15,000 water-in-oil droplets formed using droplet generator; see also pg 3 of the Bio-Rad bulletin, Droplet Digital PCR Applications Guide, all text of section entitled “Droplet generation”, further reproduced below: wherein Fig. 1.4 illustration above shows a vortex (i.e. fluid flow that revolves around an axis line) of an aqueous sample mixture meeting together with oil at a junction of the AutoDGTM in order to generate a plurality of oil encapsulated droplets).
Regarding claims 1 and 8-9, Ziegler et al. teach 16s rDNA capture probe (see pg 3, 1st para and 2nd para of section entitled “Droplet digital PCR” and also Table 1 on pg 4: wherein Ziegler et al. provides capture probe (i.e. the instant 16s rDNA F and R primers and 16S rDNA P probe of Table 1 on pg 4) that are useful to tether/link the 16S rDNA template/target nucleic acid).
Regarding claim 14, Ziegler et al. teach a blood sample (see abstract and pg 3, section entitled “DNA extraction”).
Regarding claim 16, Ziegler et al. teach the microbial nucleic acid is present in the sample at a concentration of less than 1 picogram per microliter or 0.001 microgram per milliliter (pg 2, all text of section entitled “Serial dilutions of samples with reference strain bacteria” and pg 3, 1st and 2nd paragraphs and pg 3, 2nd and 4th para of section entitled “Droplet digital PCR”: For the ddPCR: 5 µL template DNA is provided in a final volume of 20 µL reaction for a DNA concentration of 1 - 120,000 copies per 20 µL; Ziegler et al. further teach serial dilutions of template DNA of 1:103 and 1:104 and 1:108).
Regarding claim 17, Ziegler et al. teach the method is performed on a subject suspected of suffering from sepsis (abstract and 1st para of pg 2 and pg 10, Fig. 3 and pg 8, 1st and 2nd paragraphs of section entitled “16S rDNA load on days 1-2 in relation to sepsis and mortality”: where it is disclosed that their method enables quantification of bacterial load of bloodstream microorganisms e.g. Staphylococcus aureus and Escherichia coli, so as to enable diagnosis of severity of sepsis).
Omitted from Ziegler et al. (claims 1, 4, 8-10, 15, 19, 22-24)
Regarding claims 1 and 8-9, Ziegler et al. do not teach 16SrDNA/ rRNA capture probe is attached to a hydrogel of the particle via a linker.
Regarding claim 4, Ziegler et al. do not teach fluorophore comprises an intercalating dye.
Regarding claim 10, Ziegler et al. do not teach sequencing produces a plurality of sequence reads.
Regarding claim 15, Ziegler et al. do not teach target microbial nucleic acid comprises cell-free DNA.
Regarding claim 19, Ziegler et al. do not teach amplifying comprises Multiple Displacement Amplification (MDA).
Regarding claim 22, Ziegler et al. do not teach providing reverse transcriptase inside the droplet to generate a copy of the 16s rRNA and amplifying the copy to generate an amplicon.
Regarding claims 23-24, Ziegler et al. do not teach lysing the one droplet to release the amplicon and amplifying the amplicon before the sequencing.
Gosiewski et al. (claims 8-9, 10-14 and 17)
Regarding claims 8-9, Gosiewski et al. is directed to identifying bacteria sequences of a blood sample in cases of sepsis. Gosiewski et al. teach it already a matter of routine practice in the art to detect sepsis by surveillance of cell-free/ blood plasma DNA collected of clinical samples using one or a plurality of 16S rDNA capture probe(s) that are complementary to a portion of a 16s rDNA gene or nucleotide sequences that are complementary to different portions of the 16s rDNA gene to detect bacterial sequences present in the sample (see pg 330, right col., section entitled “16S Library preparation and sequencing”: wherein instant capture probes are the external V3 and V4 16S rDNA primers and adapter modified-internal 16S rDNA primers/probe disclosed on pg 331, Table 1).
Shi et al. (claim 19)
Regarding claim 19, Shi et al. teach it already a matter of routine practice to perform eMDA (emulsion based multiple displacement amplification) (see pg 4, 1st para and pg 11, last para of page).
It would have been prima facie obvious before the effective filing date of the invention to modify the ddPCR method of Hindson et al. by applying teachings from Hatori et al. and Ziegler et al. who each teach equivalent alternative droplet-based detection methods that were already known to the art.
Hindson et al. is directed to a ddPCR detection method, wherein droplets that encapsulate a hydrogel tethered to a capture primer/probe are provided to bind a target sequence for analysis.
Hatori et al. particularly teach an alternative droplet PCR method in which an alternative but equivalent means of generating droplets that encapsulates a hydrogel and additional amplification reagents are provided by using vortexing of two immiscible fluids, rather than using a droplet generator as taught by Hindson et al. and Ziegler et al.
The ordinary skilled artisan would be readily privy to combine and vortex the mixture of an aqueous solution comprising target nucleic acids together with hydrogel particles tethered to a target capture sequence via a linker, and other amplification reagents and an immiscible second fluid so as to generate droplets encapsulating the hydrogel particle and nucleic acids simultaneously, in a manner as taught by Hatori et al.
The ordinary skilled artisan would also have been readily apprised to further modify the capture probe attached to the hydrogel of Hindson to comprise a 16S rDNA or 16S rRNA binding sequence as Ziegler et al. as teach droplets as a suitable container for analyzing and establishing the presence of a 16S rDNA/16S rRNA sequence of a microbe.
The ordinary skilled artisan would have motivated by Hindson et al. teachings to subject the modified emulsion droplets of Hindson et al. comprising hydrogel tethered to a 16S rDNA or 16S rRNA capture probe for binding a 16s rRNA target microbial nucleic acid sequence for downstream sequencing after performing a reverse transcription and/or an amplification so as to identify a species of the microbe from the 16S microbial RNA/DNA captured and amplified within the droplet.
The ordinary skilled artisan would have motivated by Gosiewski et al. teaching of sequencing process to screen clinical samples such as cell free/blood samples and analyze sequence reads so as to establish the identity of the disease causative agents in the samples.
The ordinary skilled artisan would have provided DNA at a concentration of less than 1 picogram per microliter since Hatori teach fluorescence signal from a droplet-based detection is strong even at concentration of 0.009 pg μL–1.
It would also have been obvious to a person of ordinary skill in the art before the effective filing date of the invention to modify the step of droplet generation in the Hindson’s ddPCR detection method by substitute the vortexing step of Hatori et al., and further modify the capture probe attached to the hydrogel of Hindson et al. by providing a 16S rRNA or 16S rDNA target binding sequence so as to capture the 16S target sequence of microbial nucleic acids also encapsulated within each droplet.
Concerning amplifying within the droplet, the ordinary skilled artisan would have been privy to substitute the multiple displacement amplification (eMDA) process taught by Shi et al. as an alternative equivalent method of amplifying to the PCR amplification taught by Hindson et al. as both amplification processes achieve the identical goal of template amplification or library amplification and/or quantitation.
In view of the combined teachings and suggestions of all of the cited prior art references, the instant claims 1, 3-5, 7-17 and 19-24 are prima facie obvious.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 3-5, 7-17 and 22-24 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-7 and 10-22 of U.S. Patent Application No. 17/864,940 in view of Blauwkamp et al. (2019, Nature microbiology, 4(4), pp.663-674: previously cited) and Hindson et al. (US2014/0378350A1, pub. Dec 2014: previously cited) and Ziegler et al. (Nov. 13, 2019, PLoS One, 14(11), e0224656, pp 1-14: previously cited) as evidenced by Bio-Rad Bulletin, Droplet Digital PCR Applications Guide (2019: previously cited).
The instant claims are directed to methods of detecting nucleic acid, the method comprising:
obtaining a sample comprising a target microbial nucleic acid;
combining particles with the sample in a first fluid and wherein the particles comprise a hydrogel;
adding a second fluid that is immiscible with the first fluid to create a mixture;
vortexing the mixture, thereby partitioning the sample to form a plurality of droplets simultaneously, wherein the target microbial nucleic acid is segregated inside one droplet of the plurality of droplets and wherein the one droplet comprises a particle of the particles;
binding, inside the droplet, the target microbial nucleic acid with a capture probe, wherein the capture probe is attached to the hydrogel of the particle via a linker, and wherein the capture probe binds to a complementary portion of a 16s rRNA or a 16s rDNA of the target microbial nucleic acid;
amplifying bound target microbial nucleic acid to create an amplicon; and
detecting the amplicon to thereby detect the target nucleic acid reveal the presence of microbial nucleic acid; releasing the amplicon from the one droplet; and
sequencing the amplicon to identify a species of microbe when the amplicon is detected.
Claims 1-7 and 10-22 of copending Application No. 17/864,930 are directed to decentralized methods for single cell analysis, the method comprising steps:
(a) partitioning a mixture to generate a plurality of partitions, simultaneously, inside of a vessel, wherein the partitions contain a single cell and a template particle that are isolated from the mixture comprising: an aqueous solution; template particles comprising barcoded oligos; cells; and an oil;
(b) lysing the cells inside the partitions and capturing mRNA of single cells with barcoded oligos of the template particles;
(c) copying the mRNA of the single cells into barcoded cDNA;
(d) amplifying the barcoded cDNA to create amplicons;
(e) preparing sequencing libraries from the amplicons;
(f) sequencing the libraries to produce single cell gene expression data; wherein, step (a) is performed at a research lab and step (f) is performed at a core facility.
Omitted from copending Application No. 17/864,930
Claims 1-7 and 10-22 of copending Application No. 17/864,930 omit recitation(s) of the step of obtaining a sample comprising a target microbial nucleic acid, binding a target nucleic within plurality of droplets with one or a plurality of capture probe(s) attached to a hydrogel particle via a linker, and wherein the capture probe binds to a complementary portion of a 16s rRNA or a 16s rDNA of the target microbial nucleic acid. Claims 1-7 and 10-22 of copending Application No. 17/864,930 omit recitation of identifying the species of microbe from sequencing informaion.
Blauwkamp et al. teach it already a matter of routine practice in the art to perform sequencing for discovery and interrogation of species/strains of organisms that are causative of human disease (see abstract and pg 669, right col., the two para before Discussion). The sequencing processes encompassed in Blauwkamp et al. obtain pluralities of sequence reads, sequences are aligned to reference banks to identify and classify the sequence so as to generate informative clinical reports.
Hindson et al. teach it already a matter of routine practice in the art to obtain and partition a sample mixture to form a plurality of droplets, bind a target nucleic within plurality of droplets with one of a plurality of capture probe(s) attached to a hydrogel particle via a linker, and amplify the target nucleic acid to produce amplicon(s) and to perform sequencing on amplicons that are recovered from droplets after a ddPCR process (see Hindson et al., Fig. 1B and para [0400]-[0401], [0426]-[0427], [0438]).
Ziegler et al. teach the utility of detecting microbial nucleic acids within a droplet via 16S rRNA/ 16S rDNA sequence amplification of the(se), thereby motivating the modification of the capture probe tethered to the hydrogel template particle to comprise 16S rRNA/ 16S rDNA sequence as the capture sequence.
Although the claims at issue are not identical, they are not patentably distinct from each other because both are methods comprise: obtaining a sample, partitioning a sample mixture to form a plurality of droplets, binding a target nucleic within plurality of droplets with one of a plurality of capture probe(s) tethered to a template particle and amplifying the target nucleic acid to produce amplicon(s) and detecting/sequencing the amplicons.
To detect the target microbial nucleic acids, the ordinary skilled would have provided the methods of copending Application No. 17/864,930, bind the microbial target nucleic acids within droplets with a 16S rDNA/ 16S rRNA capture probe tethered to a template particle via a linker and amplify the captured nucleic acids attached on the hydrogel/template particle within droplets, detect the amplicons and perform a sequencing so as to identify and classify the microbial sequences and to generate informative clinical reports in a manner as taught by Hindson et al. and Blauwkamp et al. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claims are currently allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to OLAYINKA A OYEYEMI whose telephone number is (571)270-5956. The examiner can normally be reached Monday -Thursday: 9:00 am - 5:00 pm, EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, GARY Benzion can be reached at 571-272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
OLAYINKA A. OYEYEMI
Examiner
Art Unit 1681
/OLAYINKA A OYEYEMI/Examiner, Art Unit 1681
/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681