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 .
DETAILED ACTION
Status of the Claims
Claims 21-22, 24-25, 32-33, 35-36, 40-41 and 43-45 are pending and the subject of this FINAL Office Action.
The previous Cover Sheet (PTO-326) mailed 04/23/2026 contained the incorrect statutory period for reply. The attached Cover Sheet corrects this.
Claim Interpretations
The specification discloses digital droplet PCR (single nucleic acid per emulsion) to add barcode adaptors to nucleic acids using the following technique:
PNG
media_image1.png
228
476
media_image1.png
Greyscale
This was known in the art explained below.
The application of this common barcoding scheme to digital droplets just like the digital reactions of MAY is the simple application of familiar techniques to yield familiar results.
Priority
The claims receive a priority date of 04/20/2012 because the priority document filed on that date (US 61/636217) is the first priority document to disclose barcoding adapters in compartments.
Claim Rejections - 35 USC § 102 - Maintained
The following is a quotation of the appropriate paragraphs of 35 U.S.C. § 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 21, 24-25, 33, 40-41 and 43-45 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by MAY (US 2010/0273219).
As to claims 21, 24-25, 33, 40-41 and 43-45, MAY teaches to create aqueous droplets with one target nucleic acid (paras. 0130-37, discussing single-molecule droplet-based digital amplification for sequencing) and the following primers (Figs. 10, 15, 18; para. 0133):
PNG
media_image2.png
444
638
media_image2.png
Greyscale
PNG
media_image3.png
336
486
media_image3.png
Greyscale
PNG
media_image4.png
230
597
media_image4.png
Greyscale
The barcodes are 6 bases (e.g. Table 3).
After amplification, the droplets are pooled, amplified and barcoded nucleic acids released for downstream analyses such as sequencing (paras. 0130-42). One sequence read is used to read the barcode sequence (Figs. 10, 15 18; para. 0282). By dint of the fact that each droplet has only one nucleic acid, the primers (more than one, and each primer always used in concentrations that yield multiple primers for each primer) necessarily are present at higher concentration than single nucleic acid.
Contrary to Applicants’ arguments in the Reply 10/15/2024, May very clearly teaches “Genomic DNA samples (BioChain, USA) at 100 and 0 ng/ml (negative control [“NTC”]) were amplified for 25 cycles 7900HT Fast Real-Time PCR System (Applied Biosystems, USA) with the following primer pairs at 200 nM per primer: 1) 454 tails; 2) A5 specific primers; and 3) the three primers shown in FIG. 10” (para. 0254, emphasis added); “FIG. 15A-15B shows an example of a multi-primer reaction set-up using 4 outer primers with different combinations of primer binding site and nucleotide tags. (Example 5.) A) Two forward barcode primers (454B-BC-Tag8, 454A-BC-Tag8 and two reverse barcode primers (454A-BC-Tag5, 454B-BC-Tag8) are combined with one inner primer pair (Tag8-TSF and Tag5-TSR)” (para. 0056); “FIG. 18 shows the results of PCR reactions of three pools of 10 sets of PCR primers (A, B, C) when the PCR reactions were run for template-specific primers only and in 4-primer mode” (para. 0287, emphasis added). Thus, clearly, all primers were used in the same, single reaction.
“The barcode nucleotide sequence can encode information, such as, e.g., sample origin, about the target nucleotide sequence to which it is attached” (para. 0061).
Response to Arguments
The Examiner maintains the rejection because MAY teaches combining the claimed to allow for downstream molecular detection (e.g. sequencing using UMI). Claim 21 merely requires “contacting a plurality of droplets with the reagent fluid to form a plurality of mixed droplets, wherein one or more mixed droplet contains at least one nucleic acid molecule from a sample, the primer reagents, and the nucleic acid amplification reagents, and wherein the one or more mixed droplet further comprises reverse locus-specific primers that anneal at a target site and include a second tail.” In other words, nothing is specified about the original “plurality of droplets,” much less how they arise or how they are “contacted.” Moreover, the “reagent fluid” is not specified; it can be any fluid form (e.g. bulk, emulsion, other droplets, slug, etc.). Instead, only a resulting “mixed droplet” composition is specified. The resulting droplets in MAY meet this limitation: all primers in MAY were used in the same, single reaction.
Claim Rejection - 35 USC § 103 - Maintained
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
Claims 21-22, 24-25, 32-33, 35-36, 40-41 and 43-45 are rejected under 35 U.S.C. § 103(a) as being unpatentable over MAY (US 2010/0273219), in view of COLSTON (US 2010/0173394), in further view of LINK (US20100137163), LINK2 (US20070003442) and WEITZ (WO2010151776), in further view of LINK3 (US20080003142).
It would have been prima facie obvious to a person of ordinary skill in the art at the time the invention to substitute familiar emulsions with fluorinated oil and use devices that merge aqueous and oil components to create emulsion droplets to achieve the same compartmentalization purpose as in MAY with a reasonable expectation of success. It would also have been incredibly familiar and conventional in the art to use droplet merging techniques such as junctions.
As to claims 21, 24-25, 33-34, 40-41 and 43-47, MAY teaches to create aqueous droplets with one target nucleic acid (paras. 0130-37, discussing single-molecule droplet-based digital amplification for sequencing) and the following primers (Figs. 10, 15, 18; para. 0133):
PNG
media_image2.png
444
638
media_image2.png
Greyscale
PNG
media_image3.png
336
486
media_image3.png
Greyscale
PNG
media_image4.png
230
597
media_image4.png
Greyscale
After amplification, the droplets are pooled, amplified and barcoded nucleic acids released for downstream analyses such as sequencing (paras. 0130-42). One sequence read is used to read the barcode sequence (Figs. 10, 15 18; para. 0282). By dint of the fact that each droplet has only one nucleic acid, the primers (more than one, and each primer always used in concentrations that yield multiple primers for each primer) necessarily are present at higher concentration than single nucleic acid.
Contrary to Applicants’ arguments in the Reply 10/15/2024, May very clearly teaches “Genomic DNA samples (BioChain, USA) at 100 and 0 ng/ml (negative control [“NTC”]) were amplified for 25 cycles 7900HT Fast Real-Time PCR System (Applied Biosystems, USA) with the following primer pairs at 200 nM per primer: 1) 454 tails; 2) A5 specific primers; and 3) the three primers shown in FIG. 10” (para. 0254, emphasis added); “FIG. 15A-15B shows an example of a multi-primer reaction set-up using 4 outer primers with different combinations of primer binding site and nucleotide tags. (Example 5.) A) Two forward barcode primers (454B-BC-Tag8, 454A-BC-Tag8 and two reverse barcode primers (454A-BC-Tag5, 454B-BC-Tag8) are combined with one inner primer pair (Tag8-TSF and Tag5-TSR)” (para. 0056); “FIG. 18 shows the results of PCR reactions of three pools of 10 sets of PCR primers (A, B, C) when the PCR reactions were run for template-specific primers only and in 4-primer mode” (para. 0287, emphasis added). Thus, clearly, all primers were used in the same, single reaction.
MAY does not explicitly teach emulsion-based compartmentalization; coalescing with “destabilizing surfactant”; or droplet merging using junctions.
However, this digital emulsion droplet option was routinely used as a substitute for the other compartmentalization option of digital wells/aqueous droplets as in MAY. In other words, the teaching of COLSTON that emulsion droplets can be created using fluorinated oil and devices that merge aqueous and oil components to create emulsion droplets (Abstract; paras. 0014, 0139 & 0148 (“droplets or other partitions” such as wells/chambers)) was the known substitute for wells/aqueous droplets of MAY.
As to breaking emulsions to release amplicons, then performing pooled sequencing, this is the simple application of the familiar digital-well barcoded primer-adaptor technique of MAY to familiar digital-droplet techniques to yield predictable digital droplets results. For example, as explained above and below, COLSTON, LINK, LINK2 and WEITZ demonstrate the familiar use of emulsion droplets to perform amplification reactions for downstream sequencing (e.g. LINK, paras. 0036, 0322, 0325, 0331, 0342). This includes for 454 sequencing and other next-generation library preparations just like in MAY. In fact, the library preparation technique claimed encompasses familiar 454 library preparation as shown in MAY, which uses first digital amplification with tailed primers to add universal sequences, then a second pooled amplification to add barcoded adaptor-primers (Figs. 10, 15, 18; paras. 0062, 0133). Therefore, a skilled artisan of ordinary creativity would have been motivated to apply the other familiar digital reaction technique (droplets) to the digital-based technique of MAY to achieve similar barcoded adaptor library preparation. To this end, just like the “released” amplicons from the reaction wells/chambers of MAY, a skilled artisan would have known how to break emulsions of the emulsion droplets in the prior art to release amplicons. For example, LINK3 teaches “sample recovery” using “de-stabilizing surfactant, such as a perfluorinated alchohol (e.g., 1H,1H,2H,2H-Perfluoro-1-octanol)” (para. 0277). In sum, the applications of the barcoded primer technique of MAY to droplets instead of reaction wells/chambers is the simple application of familiar droplet techniques to yield familiar digital reaction results.
As to droplet merging using junctions, both LINK and LINK2 demonstrate the very familiar use of these techniques to create single droplets with “reactors” for PCR before sequencing, for example. LINK teaches to coalesce (AKA merge) a droplet with isolated nucleic acids and a droplet with PCR reagents using junctions at “coalescence modules,” which allows “highly efficient droplet coalescence” (Figs, 10, 16 & 19; paras 0009-15, 0038-39, 0322). This PCR can be before sequencing (para. 0015, for example). Finally, “all but the simplest reactions require multiple steps where new reagents are added during each step. In droplet-based microfluidic devices, this can be best accomplished by combining (i.e. coalescing) different droplets, each containing individual reactants” (para. 0038). Electrode can be used to generate electric field (LINK, Abstract, for example). Thus, droplet “coalescence,” or merging of droplets with various reagents (e.g. nucleic acid and PCR reagents) added at various steps (e.g. before PCR) is routinely used with success in the art.
LINK2 teaches similar droplet merging techniques using various channel configurations along with various droplet rupturing techniques to allow merging (Figs. 12-13). Electrode can be used to generate electric field (LINK2, Abstract, for example). A skilled artisan would have readily understood LINK and LINK2 as representative of the very common use, and familiar application of droplet merging techniques to other familiar droplet handling applications.
Finally, as to droplet in second channel that forms bolus that is merged with first droplet, this, too is well-known. For example, WEITZ demonstrate the familiar application of droplet fluid injection system in “[d]roplet microfluidics [which] are useful for a variety of purposes including high-throughput analysis of chemical and biological systems” (pg. 1). This is shown, for example, in Figure 2:
PNG
media_image5.png
282
674
media_image5.png
Greyscale
Weitz teaches a method of forming a mixed droplet, the method comprising forming a droplet of a first fluid (300) and contacting the droplet with a bolus of a fluid (320) wherein a portion of the second fluid segments or protrudes from the second fluid in a channel opening and integrates with the first fluid droplet (pages 15-16 and Fig. 2). Electrode can be used to generate electric field (WEITZ, Abstract, for example).
Thus, it would have been prima facie obvious to a person of ordinary skill in the art at the time of the invention to apply familiar droplet merging to the droplets of the prior art to achieve highly efficient merging of multiple reagents such as nucleic acids and PCR reagents for downstream reactions such as PCR with a reasonable expectation of success.
Response to Arguments
The Examiner maintains the rejection because Applicants arguments rely on unclaimed limitations as explained above.
Additionally, Applicants attack each reference cited for not teaching amplification reaction that uses the claimed primers. However, MAY does. And the additional reference were cited to show that the prior art as a whole demonstrates that it was obvious to substitute familiar emulsions with fluorinated oil and use devices that merge aqueous and oil components to create emulsion droplets to achieve the same compartmentalization purpose as in MAY with a reasonable expectation of success; and it would also have been incredibly familiar and conventional in the art to use droplet merging techniques such as junctions. In other words, per the basic framework of Graham v. John Deere Co., 383 U.S. 1 (1966) and KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398 (2007), after considering the scope and content of the prior art and ascertaining the differences between the claimed invention and the prior art, the Examiner determined, in light of the high skill of a PhD-level biotechnology artisan, that the prior art included each element claimed, although not necessarily in a single prior art reference, with the only difference between the claimed invention and the prior art being the lack of actual combination of the elements in a single prior art reference; PhD-level biotechnology artisan could have combined the elements as claimed by known methods, and that in combination, each element merely performs the same function as it does separately; and PhD-level biotechnology artisan would have recognized that the results of the combination were predictable. Applicants’ arguments fails to address this prima facie case of obviousness in the combination.
The ”reagent fluid” remains unspecified; it can be any fluid form (e.g. bulk, emulsion, other droplets, slug, etc.). Nor does the claim specify the origin of any of the “nucleic acid molecule from a sample, . . . the nucleic acid amplification reagents, and [the] reverse locus-specific primers that anneal at a target site and include a second tail.” These can be included in the “reagent fluid,” or some other source. To this end, combining a “reagent fluid” comprising one portion, or all of reaction components (here, “forward locus-specific primers that anneal at a target site and include a first tail and (ii) secondary primers that anneal to the first tail or a complement thereof and include an adaptor-binding sequence and a barcode sequence,” or this plus “nucleic acid molecule from a sample, . . . the nucleic acid amplification reagents, and [the] reverse locus-specific primers that anneal at a target site and include a second tail”) with another fluid (here, droplet) comprising, at best, another portion of reaction components (here, “nucleic acid molecule from a sample, . . . the nucleic acid amplification reagents, and [the] reverse locus-specific primers that anneal at a target site and include a second tail”), or all reaction components merely formed into a droplet, is the epitome of obviousness. This occurs every day in labs across the world. Applicants have not demonstrated that this simple combination of aqueous components is non-obvious.
Yet, the prior art cited above disclosed innumerable examples of combining reaction components into droplets from “reagent fluids.” As explained above, MAY combines PCR reaction components into single compartments; as do all the other droplet-bases references above. For example, LINK teaches “all but the simplest reactions require multiple steps where new reagents are added during each step. In droplet-based microfluidic devices, this can be best accomplished by combining (i.e. coalescing) different droplets, each containing individual reactants” (para. 0038). Electrode can be used to generate electric field (LINK, Abstract, for example). Thus, droplet “coalescence,” or merging of droplets with various reagents (e.g. nucleic acid and PCR reagents) added at various steps (e.g. before PCR) is routinely used with success in the art.
Applicants argue that
May explicitly requires the skilled artisan to keep the target primers separated from the barcoding primers until after the barcoding primers have been combined with a sample. In May, barcode primers and sample are first combined down one row of inputs after which target-primers are pumped into the sample chambers via different row of inputs. May states, "the initially combined forward and reverse primers are added to the initially combined samples and barcode primers." Id. To serve that principle of operation, May explicitly teaches that the targeting primers (having been combined together) are introduced only after barcode primers have been combined with sample
(Reply, pg. 7). However, Applicants miss the point that all primers are eventually combined before the amplification. In other words, although they are kept separated at some points, they all eventually come out and play so they can bash it up. See The Offspring, Come out and Play (“You gotta keep 'em separated . . . If one guy's colors and the other's don't mix; They're gonna bash it up, bash it up, bash it up, bash it up . . . Hey, come out and play”). How they are combined (which reaction components are combined with which other reaction components) is a matter of reaction setup, how the components are shipped, concentrations required, etc. In other words, these are routinely adjusted based on the application. Applicants have not demonstrated a non-obvious difference in this regard (e.g. unexpected results tied directly to this procedure).
Thus, the claimed invention remains obvious in light of the prior art as a whole.
Prior Art
The following prior art is also pertinent to single-molecule and single-cell barcoding: US 20130261196 (Fig. 4); WO 2012/048341 (single-cell emulsion PCR); WO 2012/083225 (single-cell emulsion PCR); US 20120208724; US 20120289414; US 2013/0116130.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Aaron Priest whose telephone number is (571)270-1095. The examiner can normally be reached 8am-6pm.
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.
/AARON A PRIEST/Primary Examiner, Art Unit 1681
Prosecution Insights