Office Action 6: Final Rejection (2021-04-22)
Rejection types: 103 - Obviousness, 112 - Written Description/Enablement

CLAIMS:
Amendments to the Claims:

Please amend the claims as follows.

1\. (Currently Amended) A method for droplet formation for sample preparation for sequencing, the method comprising:
providing a first aqueous fluid in a first inlet channel on a fluidic chip, the first aqueous fluid comprising primers, \[\[,\]\] providing, via a second inlet channel on the fluidic chip, a second aqueous fluid comprising nucleic acid molecules;
providing an immiscible carrier fluid in a main channel in the fluidic chip, wherein each of the inlet channels is in fluid communication with the main channel;
coalescing the first aqueous fluid with the second aqueous fluid in the main channel in the fluidic chip, thereby forming a plurality of droplets surrounded by the immiscible carrier fluid in the main channel;
applying a pressure to flow the immiscible carrier fluid in the main channel by positive displacement pressure driven flow; and
annealing one of the primers to one of the nucleic acid molecules within one of the plurality of droplets in the main channel.

2\. (Original) The method of claim 1, wherein the pressure in the main channel is derived from pressure in a reservoir in fluid communication with the main channel.

3\. (Original) The method of claim 1, wherein the pressure in the main channel is applied by a single pressure source to the main channel.

4\. (Previously Presented) The method of claim 1, wherein each of the primers is linked to a binding moiety.

5\. (Currently Amended) The method of claim 4, wherein the binding moiety is biotin and the method includes capturing the annealed one of the nucleic acid molecules to a streptavidin- coated surface.

6-11. (Cancelled) 710

12\. (Previously Presented) The method of claim 1, wherein the aqueous fluids further comprise reagents for conducting an amplification reaction.

13\. (Original) The method of claim 12, wherein the amplification reaction is a PCR reaction.

14-18. (Cancelled) 1517

19\. (Previously Presented) The method of claim 1, wherein the first and second inlet channels are in fluid communication with the main channel at a junction on the fluidic chip where the plurality of droplets are formed in the main channel.

20\. (Previously Presented) The method of claim 19, wherein the junction comprises a fluidic interconnect apparatus that forms a seal between the first inlet channel and the main channel.

21\. (Previously Presented) The method of claim 19, wherein the junction defines a nozzle and the nozzle includes at least one oil line that provides a constant flow of the carrier fluid into the nozzle while the plurality of droplets are formed.

22\. (Previously Presented) The method of claim 21, wherein the nozzle design inhibits the first aqueous fluid from wetting surfaces of the nozzle or the main channel.

23\. (Previously Presented) The method of claim 21, wherein the flow of carrier fluid into the nozzle provides complete isolation of the nozzle from the aqueous phase of the first aqueous fluid.

24-30. (Cancelled) 2529

31\. (New) The method of claim 1, wherein the immiscible carrier fluid comprises an oil.

32\. (New) The method of claim 31, wherein the oil comprises a fluorocarbon oil.

33\. (New) The method of claim 31, wherein the oil comprises a surfactant.

34\. (New) The method of claim 33, wherein the surfactant is a fluorosurfactant.

35\. (New) The method of claim 33, wherein the pressure to flow the immiscible carrier fluid in the main channel by positive displacement pressure driven flow is applied using a pump.
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OFFICE ACTION:
DETAILED ACTION
The present Office Action is responsive to the Amendment received on January 14, 2021.
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
Preliminary Remark
Claims 6-11, 14-18, and 24-30 are canceled.
Claims 31-35 are new.
Information Disclosure Statement
The IDS received on January 12, 2021 and April 15, 2021 are proper and are being considered by the Examiner.
The IDS were received with the fee under 37 CFR 1.17(p).
Claim Rejections - 35 USC § 112
The new matter rejection of claim 5 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement, made in the Office Action mailed on November 2, 2020 is withdrawn in view of the Amendment received on January 14, 2021.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA  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, 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.
The rejection of claims 1-3, 12, 13, and 19 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004), made in the Office Action mailed on November 2, 2020 is maintained for the reasons of record.
In addition, claims 5, 31 and 35 are rejected herein as being necessitated by Amendment (its addition).
Applicants’ arguments presented in the Amendment received on January 14, 2021 have been carefully considered but they have not been found persuasive for the reasons set forth in the, “Response to Arguments” section.  The rejection rationale of claims 5, 31 and 35 are set forth in the same section.The Rejection:
With regard to claims 1, 12, 13, and 19 Takahashi discloses a method of forming droplets for sample preparation for sequencing (“present invention provides methods of using inline automated platform of the invention … to prepare, amplify, detect … and/or optionally select for further analysis (e.g., sequence) genomic material isolated…”, section [0038]), the method comprising:
providing a first aqueous fluid in a first inlet channel, the first aqueous fluid comprising primers (see Figure 1A, the first inlet is channel carrying the primers A/B and Markers A/B);
providing via a second inlet channel, a second aqueous fluid comprising nucleic acid molecules (see Figure 1A, where gene is provided);
providing an immiscible carrier in a main channel in a fluidic chip (see element 6, Figure 1A, where “carrier fluid” is provided; “a sample plug is surrounded by an immiscible nonaqueous fluid (e.g., mineral oil)”, section [0050]), wherein each of the inlet channels is in fluid communication with the main channel (see where the inlet containing the gene and the inlet containing the primer are connected to the channel where the carrier liquid flows (element 5);
coalescing the first aqueous fluid with the second aqueous fluid in the main channel in the fluid chip thereby forming a plurality of droplets surrounded by the immiscible carrier fluid in the main channel (see the gene primer become coalesced into a single emulsion, Figure 1A, area 10; “each sample droplet is further prepared at a junction, e.g., a T-shaped junction (10) to form a sample plug by being mixed with a primer plug comprising amplification reagents (e.g., primer(s), nucleotide, polymerase, etc).”, section [0084]);
applying pressure to flow the immiscible carrier fluid in the main channel (“controlling device … such as pumps, valves … may be used to control the movement and timing of each sample plug and/or DNA sample plug .. such controlling devices are well known to those skilled in the art”, section [0115]); and
annealing one of the primers to one of the nucleic acid molecules within one of the plurality of droplets (“mixed with amplification reagents to form a sample plug, it is drawn along the microfluidic inline reaction channel into an amplification area of a device … only DNA sample plugs will comprise DNA that will be amplified”, section [0085]; “low temperature area (13) may be set to a temperature appropriate to effectuate annealing”, section [0088]).
While Takahashi explicitly disclose that carrier fluid is provided for flowing the plugs in the channels, the artisans do not explicitly disclose that the carrier fluid is pressure driven from a reservoir in communication with the main channel through which the carrier fluid flows (claim 2), or that the pressure is applied by a single pressure source to the main channel (claim 3).
While Takahashi explicitly teach and suggests that the amplification products of the invention can be subject to sequencing analysis (“microfluidic devices and instruments of the invention are intended to work together to provide an automated inline platform for either or both PCR and waveform profiling methods and optionally, e.g., sequencing analysis”, section [0047]), the artisans do not explicitly teach that primers are linked to a binding moiety (claim 4), or that the binding moiety is biotin and the method includes capturing the annealed nucleic acid molecules to a biotinylated surface (claim 5).
Nobile et al. disclose a method of generating emulsion particles for nucleic acid amplification reaction in a continuous manner, wherein the artisans disclose an embodiment where in a reservoir comprising the emulsion oil is injected via a pump and a separate reservoir comprising a PCR reaction mixture which is injected via a pump (see Figure 1), wherein emulsion products are generated at a junction where the oil flows on a main channel (the lateral channel) and the PCR reaction mixture containing channels are injected thereto, forming a T-junction (Figure 2).
Nobile et al. explicitly disclose that size of these emulsion droplets (or “microreactors”) may be, “adjusted by varying the flow rate and speed of the components.” (section [0058]), wherein PCR reactions are performed therein (“emulsion is heat stable to allow thermal processing/cycling).
Nobile et al. also state that the apparatus as disclosed is, “controlled by a microprocessor” (section [0073]), which monitors the, “pressure of the emulsion oil flow via the pressure sensor 212, so that the flow rate of the oil may be regulated …” (section [0073]).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Takahashi et al. and Nobile et al., thereby arriving at the invention as claimed for the following reasons.
As discussed above, Takahashi et al. already disclose a method and a microfluidic device comprising a plurality of sample and reagent inlets and an inlet for an immiscible carrier fluid into a main channel for performing continuous generation of emulsion products and PCR.
While Takahashi et al. do not explicitly disclose that the samples and PCR reactants or the oil are provided via a pump, said one of ordinary skill in the art would have been motivated to automate these means so as to provide a system which can be run on a programmed setting.  Such a capability was demonstrated by Nobile et al. who also teach continuous generation of emulsion droplets comprising PCR reaction mixture in a flowing oil, wherein the artisans explicitly teach the use of attached pumps which are programmable to deliver the desired flow rates of the respective reagents in order to achieve the desired emulsion sizes (see above).
Therefore, one of ordinary skill in the art would have been motivated to combine the teachings of Takahashi et al. and Nobile et al., thereby arriving at a method of performing the method of Takahashi et al. on a system which is configured to deliver programmable amounts of reagents via pumps so as to generate emulsion droplets.
In KSR International Co v. Teleflex Inc, the supreme court stated that, “A person of ordinary skill in the art is also a person of ordinary creativity, not an automation” (82 USPQ2d at 1397) and that “in many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle” and take into account, “the inference and creative steps that a person of ordinary skill in the art would employ” (82 USPQ2d at 1396).
Lastly, it is respectfully submitted that such “fitting” of the teachings would not have been “beyond [the] ... skill [level]” of one of ordinary skill in the art
In KSR, the supreme court stated:
“When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one.  If a person of ordinary skill can implement a predictable variation, 103 likely bars its patentability.  For the same reason, if a technique has been used to improve one device, and a person of ordinary skill in the art would recognize that it would improve similar devices in the same way, using the technique is obvious unless its actual application is beyond his or her skill.” (page 13, emphasis added).
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.Response to Arguments:
Applicants traverse the rejection.
Applicants contend that the amended claim 1 now requires that the flow of immiscible carrier fluid in the main channel is by a “positive-displacement pressure-driven flow” and that Takahashi, Nobile and Vogelstein do not teach or suggest the use of positive displacement pressure-driven flow to join an aqueous fluid containing amplification reagents with an aqueous fluid containing target/template on-chip (page 6, Response).
Applicants specifically contend that Takahashi does not clearly teach or suggest an immiscible carrier fluid in a main channel with two aqueous inlet channels as claimed, to form a plurality droplets surrounded by the immiscible carrier fluid in the main channel (page 6, bottom paragraph, Response) and point to a section in Takahashi et al. wherein it is stated, “Generally, carrier liquid (6) is a water-based liquid that is the same liquid as the sample liquid” (page 7, Response).
This argument is not found persuasive nor is the statement entirely correct.
Initially, the carrier fluid employed by Takahashi et al., while is stated as being water-based in an embodiment, it also explicitly discussed as being immiscible to the form of the nucleic acid and primer containing medium:
“the carrier liquid may be an organic-based liquid, e.g., silicone oil … or some other immiscible nonaqueous fluid … separate the sample droplets … in order to separate each sample droplet comprising DNA … from the preceding or following sample droplets as they travel through a microfluidic inline reaction channel of the invention” (section [0083], Takahashi)
The “carrier liquid” to which Takahashi is referring to is identified in Figure 1, as element (6), which is clearly shown therein as the carrier (in this instance, oil) which is driving the droplets through the main channel of the device which connects to the region where amplification is performed.
Therefore, Applicants’ apparent, generalized statement that Takahashi only contemplates using a water-based liquid, is simply incorrect.
As to Applicants’ assertion that Takahashi not teaching or suggesting an immiscible carrier fluid in a main channel with two aqueous inlet channels to form a plurality of droplets surrounded by the immiscible carrier fluid in the main channel, this assertion is also simply untrue.
The figure of Takahashi is reproduced below:
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As seen, the carrier liquid (oil, as discussed above), flow through the main channel (identified in red arrow), wherein there are two aqueous inlet channels (one from gene source; and another from the primer/marker source) that intersect at the main channel carrying the oil wherein droplets are formed surrounded by the oil.
Therefore, Applicants’ assertion is also simply not true.
Next, Applicants reference to Takahashi’s discussion wherein it is stated that, “a vacuum may be applied to waste wells at the end of each microfluidic inline reaction channel and may be used to activate the sipper and move the fluid along the microfluidic inline reaction channels toward the waste wells” (page 7, Response), and thus contend that Takahashi the artisans teach, “only one example of pressure”, that is vacuum pressure (page 7, Response).
This argument is not found persuasive for the following reasons.
Takahashi do teach the use of a sipper for loading the reagents into their respective inlet channels (i.e., solvent A, solvent B, and Base solvent and one gene) and the “waste wells” correspond to sipper mechanism of loading solvents A and B.  However, this is not what Takahashi employs in driving the oil of the main channel, forming the aqueous droplets in the flowing oil of the main channel.
Claim 1 recites that the, “pressure to flow the immiscible carrier fluid in the main channel” is by a positive displacement pressure driven flow.
Therefore, the process of sipper utilizing a negative force to “feed” the reagents is not relevant to the limitation to which Applicants are arguing.
However, as to the movement of oil in the main channel, Takahashi does in fact, specifically suggest the use of any of such well-known means of moving the oil in the main channel:
“microfluidic devices of the invention may be manufactured by methods well known in the art; see, e.g., U.S. Pat. Nos. 6,500,323 and 5,882,465.  Briefly, in designing the microfluidic devices of the invention, a driving force for moving the fluid sample plug(s) (e.g., a DNA sample plug(s)) through at least one microfluidic channel should be chosen” (section [0110], Takahashi)
“As described in U.S. Pat. No. 6,500,323, a typical driving force for microfluidic systems, such as automatic inline platforms of the invention, is selected from pressure based systems, or hybrids of the two. Use of pressure-based systems is described in, e.g., U.S. Pat. No. 6,500,323” (section [0111], Takahashi)
It is plainly evidenced that Takahashi suggests that any known prior art means using pressure-driven means for driving the fluid sample plugs can be utilized.  And in doing so, Takahashi happen to reference one patent in particular, having the number 6,500,323.
This patent is issued to Chow et al., having an issue date of December 31, 2002, filed on March 26, 1999.
And in this patent explicitly referenced by Takahashi, with specific references to the means of pressure-flow, the artisans state the below with regard to the types of flow utilized in moving oil in the main channel (see below from column 4, lines 34-42, Chow et al.):
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Therefore, not only does Takahashi suggest that any known means for driving the oil in the main channel can be utilized, but points to a particular patent that also suggest that use of positive or negative pressure as the driving force in fluid transport is obvious and well known.
Therefore, Applicants assertion that Takahashi only contemplates or teaches the use of negative pressure is also simply not true.
With regard to claim 5, the primers are bound to bead, wherein the primer bind their target nucleic acid for amplification (“bead is linked to a large number of a single primer species … Alternatively, the bead is liked to chemical groups (e.g., biotin) that can bind to chemical groups (e.g., streptavidin) …”, section [0043]).
Therefore, it would have been obvious to provide the primer immobilized beads via the well-known biotin/streptavidin interaction as taught by Nobile et al.
With regard to claim 31, the carrier fluid is oil (see above arguments).
With regard to claim 35, the patent (Cho et al.) to which Takahashi references also teach that use of pumps for generating positive or negative pressure is commonly known and employed (“Such pressure sources typically include pressure or vacuum pumps … Such pumps are capable of generating the desired positive or negative pressures within channel segments that are connected to pumps” (column 4, lines 46-55, Cho et al.).
Therefore, Applicants’ arguments are not found persuasive and the rejection is maintained.
The rejection of claim 4 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004) as applied to claims 1-3, 12, 13, and 19 above, and further in view of Vogelstein et al. (US 2011/0059435, published March 2011, priority February 7, 2005), made in the Office Action mailed on November 2, 2020 is maintained for the reasons of record.
Applicants’ arguments presented in the Amendment received on January 14, 2021 do not address the present rejection but rely solely on their arguments presented for the rejection over Takahashi et al. in view of Nobile, which have already been fully addressed above as being unpersuasive.  Since no new arguments have been made for the present rejection, the rejection is maintained for the reasons already of record.
The Rejection:
The teachings of Takahashi et al. and Nobile et al. have already been discussed above.
Takahashi, while explicitly suggesting that their disclosed method be for amplification, detection, as well as for sequencing reaction, do not explicitly all possible sequencing means known at the time the invention was made.
Consequently, the artisans do not explicitly disclose all possible means of sequencing reactions, such as those involving primers which are linked to a biotin (claim 4).
Vogelstein et al. disclose a method of amplifying nucleic acids in emulsion particles (region of analyte DNA molecules is amplified using a high fidelity DNA polymerase … Microemulsions comprising said first amplicons and reagents beads are formed”, section [0008]), wherein the primers are biotinylated (“common oligonucleotide … was synthesized with a dual biotin group … coupled to streptavidin-coated magnetic beads”, section [0058]).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Takahashi et al. and Nobile et al. with the teachings of Vogelstein et al., thereby arriving at the invention as claimed for the following reasons.
As stated above, Takahashi et al. explicitly teach that the amplification products in the emulsion particles of their invention can be employed for further analysis such as amplifying, detecting, and sequencing (see section [0030]), and while the artisans did not explicitly list all possible such “further analysis”, said one of ordinary skill in the art would have had a reasonable expectation of success that any of amplification and analysis means known in the art would have been equally useful and beneficial in the art of molecular analysis/diagnostics.
Given that Vogelstein et al. teach a method of producing emulsion particles comprising therein, amplification products bound on a bead via use of bead-bound primers for the purpose of detecting nucleotide sequence variations, said one of ordinary skill in the art would have been reasonably motivated to adopt the teachings of Vogelstein et al. into the method disclosed by the combination of Takahashi et al. and Nobile et al., thereby arriving at the claimed method of generating emulsion particles comprising bead-bound primers and reagents, and template nucleic acids produced from the coalescing means taught by Takahashi et al.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
The rejection of claims 20-23 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004), made in the Office Action mailed on November 2, 2020 is maintained for the reasons of record.
Applicants’ arguments presented in the Amendment received on January 14, 2021 do not address the present rejection but rely solely on their arguments presented for the rejection over Takahashi et al. in view of Nobile, which have already been fully addressed above as being unpersuasive.  Since no new arguments have been made for the present rejection, the rejection is maintained for the reasons already of record.
The Rejection:
The teachings of Takahashi et al and Nobile et al. have already been discussed above.
Takahashi et al. do not explicitly disclose that the junction comprises a fluidic interconnect apparatus that forms a seal between the inlet and the main channel (carrying the oil) (claim 20), or that junction defines a nozzle which includes an oil line that provides a constant oil flow into the nozzle (claim 21), wherein the design prevents the nozzle or the main channel from becoming wet with the aqueous fluid (claim 22) or that the carrier fluid into the nozzle provides complete isolation of the nozzle from the aqueous phase (claim 23).
While Takahashi et al. do not explicitly disclose the above-discussed features of the junction and nozzle, based on the disclosure provided by Nobile, these features are deemed known and prima facie obvious to one of ordinary skill in the art at the time the invention was made for the reasons set forth below.
Nobile et al. provides the below configuration which graphically represents a junction where the aqueous reagents are disposed into the main channel which carries the oil, which is similar to the configuration disclosed by Takahashi et al.:
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Merriam-Webster defines “nozzle” a short tube with a taper or constriction used to speed up or direct a flow of fluid (see attached)1.
Therefore, the main channel has a nozzle 406 (the narrow portion) found on the main channel wherein the oil flows through and the upper reservoir (412) (or first inlet) is in communication with the main channel at a junction at which the nozzle is found and the droplets are formed therefrom.
With regard to claim 20, the junction formed between the inlet and the nozzle of the main channel is configured to generate a plurality of droplets and while the artisans do not explicitly state that there is a seal between this inlet channel and the main channel, it would have been an obvious to one of ordinary skill in the art to make sure that there was a tight seal where the inlet and main channel intersected so as to contain the emulsion oil flowing through the main channel without escape and the reagents of the reaction flow into the main channel without any escape into a gap therebetween.
With regard to claim 21, the left-side of the narrow junction (i.e., nozzle) is considered to be an oil line and it feeds the carrier oil to the nozzle.
With regard to claim 22, the nozzle design inhibits the first aqueous fluid from wetting the surface of the nozzle of the main channel.  This is because the artisans explicitly state that at the narrow junction (i.e., nozzle), the aqueous reagents from the inlet is introduced and shears off in to a plurality of droplets and the formed droplets continue their travel in the main channel surrounded by the carrier oil (sections [0079] and [0080]).
With regard to claim 23, the nozzle is surrounded by the oil flowing there-through and therefore, isolated from the aqueous phase entering from the inlet.
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Rejection – New Grounds, Necessitated by Amendment
Claims 32-34 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004) as applied to claims 1-3, 12, 13, and 19 above, and further in view of Dorfman et al. (Analytical Chemistry, 2005, vol. 77, pages 3700-3704).
The teachings of Takahashi et al. and Nobile et al. have already been discussed above.
Takahashi et al., while explicitly teach performing PCR in aqueous droplets in surrounded by an immiscible oil medium, do not teach every possible such medium or variations known in the art.  Consequently, the artisans do not explicitly teach that the oil is fluorinated (claim 32), or that the oil comprises fluorosurfactant (claims 33 and 34).
Dorfman et al. disclose a method of performing a continuous PCR reaction in an aqueous droplets surrounded by an immiscible oil medium (“[p]erformign continuous flow PCR for high-throughput clinical and quantitative applications requires encapsulating the sample and preventing contamination between them … this can be accomplished in two ways: either the sample can be a droplet entrained in an immiscible solvent …”, page 3700, 2nd column).
Dorfrman et al. disclose the use of fluorinated oil as the immiscible medium when performing the continuous emulsion PCR (“an improvement system for performing continuous flow PCR using droplets in an immiscible fluorinated solvent …”, page 3701, 1st column, 2nd paragraph; “primed with either the bulk fluorinated oil FC-40”, page 3701, 1st column, 3rd paragraph) or oil comprising a fluorosurfactant (“or FC-40 containing various amounts of fluoro alcohol surfactant”, page 3701, 1st column, 3rd paragraph).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Takahashi et al. and Nobile et al. with the teachings of Dorfman et al., thereby arriving at the invention as claimed for the following reasons.
Dorman et al. discuss potential problems associated with performing emulsion droplet PCR in an immiscible medium:
“wall treatment [of the channel] are necessary to prevent adsorption of DNA to the walls and subsequent transfer between samples … wall contamination problem is a significant drawback … bubbles in the stream may introduce irregularities in the thermal history of the droplets, making the technique less attractive … flowing droplet system does not suffer from either of these drawbacks … However, the initial work on this system suffered from contamination between droplets” (page 3701, 1st column, 1st paragraph).
In solving the drawback so identified, Dorman et al. disclose that immiscible solvent, “prevent[ed] the droplet breakage … thereby eliminating the contamination between droplets” (page 3701, 1st column, 2nd paragraph), producing “adequately reproducible amplification between different drops without any detectable contamination between them” (page 3701, 1st column, 2nd paragraph).
Based on such findings, one of ordinary skill in the art would have been motivated to utilize a fluorinate immiscible oil or oil comprising fluorosurfactants into the teachings of Takahashi et al. and Nobile et al., for the same recognized benefit of producing reproducible amplification reaction between emulsion droplets as well as preventing contamination therebetween.
Given that Takahashi et al., Nobile et al. and Dorfman et al. performed emulsion amplification reactions in a flowing oil medium, said one of ordinary skill in the art would have also expected that addition of fluorosurfactants or using fluorinated oil in the method of Takahashi et al. would have yielded the same predictable benefit as observed in Dorman et al.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
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 extension fee 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 date of this final action.
Inquiries

APPLICANT REBUTTAL: Remarks
With entry of this amendment, claims 1-5, 12, 13, 19-23, and 31-35 are pending, with claim 1 being the only independent claim. Applicant has amended claims 1 and 13 herein.
Support for the amendments may be found throughoutparent U.S. application No. 11/803,104 as-filed, for example, at least at page 36, line 20 to page 37, line 24 and lines 3-9 on page 78.
No new matter is added.
Rej
ections under 35 U.
S.
C.
§
The Office Action on page 3 states that claims 1-3, 5, 12, 13, 19, 31, and 35 are rejected under §103 over U.S. Pub. 2006/0257893 to Takahashi in view of U.S. Pub. 2005/0227264 to Nobile. At page 14, claim 4 is so rejected over Takahashi and Nobile and further in view of U.S.
Pub. 2011/0059435 to Vogelstein. On page 16-17, claims 20-23 are rejected over Takahashi in view of Nobile. The Office Action on page 19 states that claims 32-34 are rejected under §103 over Takahashi and Nobile and further in view of Dorman, 2005, Anal Chem 77:3700-3704 Applicant submits that the pending, amended claims are not obvious.
The Patent Office must establish three basic criteria in order to establish a prima facie case of obviousness. There must be a suggestion to modify a reference or to combine references to lead one of ordinary skill in the art to the claimed invention; the cited references must convey a reasonable expectation of success in arriving at the claimed invention; and the suggestion to modify the references must be found in the prior art, not in the applicant's disclosure. In re Vaeck, 947 F.2d 488 (Fed. Cir. 1991). In addition, an obviousness rejection cannot be established in the first place unless each claim element is disclosed in the cited art. In re Royka, 490 F.2d 981 (CCPA 1974).
Claim 1, as amended, recites a method for droplet formation for sample preparation for sequencing. The method includes providing primers in a first aqueous fluid and template nucleic acid in a second aqueous fluid in respective inlet channels on a fluidic chip. The fluids are coalesced and formed into droplets in an immiscible carrier fluid within a main channel in the fluidic chip. Pressure is applied to flow the aqueous fluid with sample nucleic acids and the immiscible carrier fluid in the main channel using positive-displacement pressure-driven flow.
One of the primers is annealed to one of the nucleic acid molecules within one of the droplets in the main channel.
The present application explains that an important benefit of using positive pressures, as recited in claim 1, allows superior regulation and control over the size and periodicity of formed droplets. Application, page 37.
Claim 1 cannot reasonably be said to be obvious over Takahashi, Nobile, and Vogelstein because none of those references teach or suggest using positive displacement pressure-driven flow to join an aqueous fluid containing amplification reagents with an aqueous fluid containing target/template on-chip.
Takahashi reports a device for preparing (e.g., isolating, processing, mixing with reaction reagents), amplifying, and detecting genomic material. Takahashi, [0030]. The device includes a sipper to pick up sample and a microfluidic inline reaction channel that runs through a reagent assembly area, temperature-controlled amplification, and a detection area. Id., [0040]. Detecting includes aspirating a sample droplet into the reaction channel, mixing the sample droplet with a primer plug (which includes amplification reagents) to form a sample plug, and then heating and cooling for amplification and subsequent detection. Id., [0052].
Takahashi does not teach or suggest using positive displacement pressure-driven flow to join an aqueous fluid containing amplification reagents with an aqueous fluid containing target/template on-chip. Takahashi states that thee different methods may be used to move fluids in reaction channels: electro-kinetics, pressure, or a hybrid of the two. Takahashi, [0015]. However, Takahashi gives only one example of pressure, stating that "a vacuum may be applied to waste wells at the ends of each microfluidic inline reaction channel and may be used to activate the sipper and move the fluid along the microfluidic inline reaction channels toward the waste wells." Id. The only pressure-related flow embodiment that Takahashi gives uses a vacuum to suck fluid through the sipper towards the waste wells. Takahashi repeatedly states that fluid is aspirated (i.e., removed under negative pressure) through a sipper (a device the sucks fluid). Conversely there is absolutely no instance in which Takahashi discusses any fluid under a positive pressure. Further, Takahashi explains that aspiration is used to form droplets, and thus essential to the disclosure. Id. [0073].
Using a vacuum to suck into the waste well teach away from using positive pressure as recited in amended claim 1.
Further, Takahashi's incorporation by reference of U.S. Patent No. 6,500,323 is insufficient to read any positively driven pressure into the disclosure. This incorporation by reference is only provided to explain apparatuses used to move fluid. Without any further explanation in Takahashi, the only reasonable interpretation of this incorporation by reference is to provide ways in which negative pressure can be applied in a manner consistent with Takahashi's disclosure.
Nobile, Dorfman, and Vogelstein do not make up the deficiencies of Takahashi.
Nobile only reports methods and for amplifying genetic material in which emulsion oil is pumped into a cross-flow emulsifier to emulsify a PCR reaction mixture at a mixing tee. Id., [0065]. The mixing tee has a conduit with a first inlet (for receiving an emulsion oil); a second inlet (for amplification reaction/bead mixture); and an outlet. Id., [0070]-[0071]. The emulsion oil may flow through multiple injection/mixing tees that each receive PCR/bead mixture from a syringe. Id, [0074]. Nobile does not bring aqueous template nucleic acid and aqueous amplification reagents on-chip via separate inlet channels and merge them into droplets on-chip. In fact, Nobile's only mention of droplet coalescence is as
a risk to be avoided
! Nobile, [0081].
Thus, Nobile cannot be combined with Takahashi to arrive at the claimed methods. Nobile teaches away from any methods in which droplets coalesce. Moreover, because Nobile does not teach coalescing droplets, Nobile does not provide a teaching that droplets can be formed by merging fluids under a positive pressure. Thus, combining Nobile and Takahashi cannot provide the claimed device, as neither reference teaches or discloses merging fluids under a positive pressure.
Vogelstein is a U.S. publication of a patent application for BEAMing, which refers to Bead Emulsion Amplification with Magnets. Vogelstein, [0006]. Vogelstein does not mention microfluidics whatsoever, much less using positive pressure. In fact, Vogelstein does not even use any of the terms "microfluidics" or "channel" or "chip" or any synonyms thereof. Like Nobile, there is not any on-chip mixing of an aqueous sample fluid with an aqueous reagent fluid, using microfluidics for fluid manipulation, merger, and droplet formation and handling.
Dorfman, like Takahashi teaches using aspiration (negative pressure) in a microfluidics system. Dorfman, 3701. Accordingly, Dorfman cannot be combined with Takahashi, or any other cited reference, to arrive at the claimed methods.
Only with the benefit of Applicant's disclosure could a skilled practitioner have understood how to bring amplification reagents on-chip in a first aqueous fluid and target nucleic acid in distinct, second aqueous fluid, and use positive-displacement pressure-driven flow to merge those fluids and form PCR-ready droplets. Vogelstein and Nobile nowhere contemplate merging such aqueous fluids on-chip into droplets. While Takahashi and Dorfman only use a negative pressure to suck fluids down to waste wells, i.e., negative-displacement, suction-driven flow. Given that Vogelstein and Nobile do not even teach or suggest fluid merger and droplet formation, and that Takahashi teach away from the claimed positive-displacement, pressure- driving flow, amended claim 1 cannot reasonably be said to be obvious over the asserted references.
Because the only independent claim is not obvious, none of the claims are obvious.
Applicant believes that the rejections under § 103 are overcome.
Summary
On the basis of the foregoing amendments and reasons, Applicant submits that the pending claims are in condition for allowance, which is respectfully requested.
Dated: July 21, 2021
Respectfully submitted,

NEXT OFFICE ACTION: DETAILED ACTION
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection.  Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.  Applicant's submission filed on July 21, 2021 has been entered.
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
Preliminary Remark
Claims 6-11, 14-18, and 24-30 are canceled.
Claim Rejections - 35 USC § 103
The rejection of claims 1-3, 5, 12, 13, 19, 31, and 35 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004), made in the Office Action mailed on April 22, 2021 is withdrawn.
The rejection of claim 4 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004) as applied to claims 1-3, 12, 13, and 19 above, and further in view of Vogelstein et al. (US 2011/0059435, published March 2011, priority February 7, 2005), made in the Office Action mailed on April 22, 2021 is withdrawn.
The rejection of claims 20-23 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004), made in the Office Action mailed on April 22, 2021 is withdrawn.
The rejection of claims 32-34 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004) as applied to claims 1-3, 12, 13, and 19 above, and further in view of Dorfman et al. (Analytical Chemistry, 2005, vol. 77, pages 3700-3704), made in the Office Action mailed on April 22, 2021 is withdrawn.
Rejection – New Grounds, Necessitated by Amendment
The following is a quotation of pre-AIA  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, 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 1-3, 5, 12, 13, 19, 31, and 35 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Quake et al. (US 2002/0058332 A1, published May 16, 2002).
With regard to claims 1, 12, 13, and 19 Takahashi discloses a method of forming droplets for sample preparation for sequencing (“present invention provides methods of using inline automated platform of the invention … to prepare, amplify, detect … and/or optionally select for further analysis (e.g., sequence) genomic material isolated…”, section [0038]), the method comprising:
providing a first aqueous fluid in a first inlet channel, the first aqueous fluid comprising primers (see Figure 1A, the first inlet is channel carrying the primers A/B and Markers A/B);
providing via a second inlet channel, a second aqueous fluid comprising nucleic acid molecules (see Figure 1A, where gene is provided);
providing an immiscible carrier in a main channel in a fluidic chip (see element 6, Figure 1A, where “carrier fluid” is provided; “a sample plug is surrounded by an immiscible nonaqueous fluid (e.g., mineral oil)”, section [0050]), wherein each of the inlet channels is in fluid communication with the main channel (see where the inlet containing the gene and the inlet containing the primer are connected to the channel where the carrier liquid flows (element 5);
coalescing the first aqueous fluid with the second aqueous fluid in the main channel in the fluid chip thereby forming a plurality of droplets surrounded by the immiscible carrier fluid in the main channel (see the gene primer become coalesced into a single emulsion, Figure 1A, area 10; “each sample droplet is further prepared at a junction, e.g., a T-shaped junction (10) to form a sample plug by being mixed with a primer plug comprising amplification reagents (e.g., primer(s), nucleotide, polymerase, etc).”, section [0084]);
applying pressure to flow the immiscible carrier fluid in the main channel (“controlling device … such as pumps, valves … may be used to control the movement and timing of each sample plug and/or DNA sample plug .. such controlling devices are well known to those skilled in the art”, section [0115]); and
annealing one of the primers to one of the nucleic acid molecules within one of the plurality of droplets (“mixed with amplification reagents to form a sample plug, it is drawn along the microfluidic inline reaction channel into an amplification area of a device … only DNA sample plugs will comprise DNA that will be amplified”, section [0085]; “low temperature area (13) may be set to a temperature appropriate to effectuate annealing”, section [0088]).
While Takahashi explicitly disclose that carrier fluid is provided for flowing the plugs in the channels, the artisans do not explicitly disclose that the carrier fluid is pressure driven from a reservoir in communication with the main channel through which the carrier fluid flows (claim 2), or that the pressure is applied by a single pressure source to the main channel (claim 3).
Takahashi et al. do not teach that a positive displacement pressure driven flow is employed for providing a second aqueous fluid through the second inlet channel.
While Takahashi explicitly teach and suggests that the amplification products of the invention can be subject to sequencing analysis (“microfluidic devices and instruments of the invention are intended to work together to provide an automated inline platform for either or both PCR and waveform profiling methods and optionally, e.g., sequencing analysis”, section [0047]), the artisans do not explicitly teach that primers are linked to a binding moiety (claim 4), or that the binding moiety is biotin and the method includes capturing the annealed nucleic acid molecules to a biotinylated surface (claim 5).
Quake et al. teach the use of positive pressure driven flow to flow an immiscible oil in a main channel, and generating droplets via first and second inlet channels attached to said main channel, wherein said inlet channels are also taught as being positive pressure driven:
“the device of the invention comprises at least two inlet regions, each connecting to the main channel at a droplet extrusion region.  In particular, the device may comprise a first inlet region in communication with the main channel at a first droplet extrusion region, and a second inlet region in communication with the main channel at second droplet extrusion region.  A fluid containing a first biological material may pass through the first inlet region so that droplets of the fluid containing the first biological materials are sheared into the main channel.  A fluid containing a second biological material may pass through the second inlet region so that droplets of the fluid containing the second biological material are sheared into the main channel.  In various aspects, the droplets of the first material may mix or combine with the droplets of the second biological material, and the first and second biological material may interact with each other upon mixing.” (section [0018])
“An inlet region may have an opening such as in the floor of the microfabricated chip, to permit entry of the sample into a device.  The inlet region may also contain a connector adapted to receive a suitable piece of tubing ….through which a sample may be supplied … Such an arrangement facilitates introducing the sample solution under positive pressure in order to achieve a desired pressure at the droplet extrusion region” (section [0081])
“device of the invention may have an additional inlet region, in direct communication with the main channel … through which a pressurized stream or ‘flow’ of a fluid is introduced into the main channel.  Preferably, this fluid is one which is not miscible with the solvent or fluid of the sample …” (section [0082])
“the invention use pressure drive flow control, e.g., utilizing valves and pumps, to manipulate flow of cells virions, particles, molecules, enzymes or reagents in one or more directions and/or into one or more channels of a microfluidic device” (section [0099])
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Takahashi et al. and Quake et al., thereby arriving at the invention as claimed for the following reasons.
As discussed above, Takahashi et al. already disclose a method and a microfluidic device comprising a plurality of sample and reagent inlets and an inlet for an immiscible carrier fluid into a main channel for performing continuous generation of emulsion products and PCR.
While Takahashi et al. do not explicitly disclose that the samples and PCR reactants or the oil are provided via a pump that generates a positive pressure, said one of ordinary skill in the art would have recognized that other prior art known pressure driven system utilized for generating droplets in a channel would have worked equally well.  Such a capability was demonstrated by Quake et al. who also teach generation of emulsion droplets comprising PCR reaction mixture in a flowing oil, wherein the artisans explicitly teach the use of pumps which provide a positive pressure flow to oil and the respective reagents from inlets in order to generate emulsion droplets in the main channel (see above).
Therefore, one of ordinary skill in the art would have been motivated to combine the teachings of Takahashi et al. and Quake et al., thereby arriving at a method of performing the method of Takahashi et al. on a system in which the oil and the reagents are provided into the main channel via a pump that produces positive pressure so as to generate emulsion droplets.
In KSR International Co v. Teleflex Inc, the supreme court stated that, “A person of ordinary skill in the art is also a person of ordinary creativity, not an automation” (82 USPQ2d at 1397) and that “in many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle” and take into account, “the inference and creative steps that a person of ordinary skill in the art would employ” (82 USPQ2d at 1396).
Lastly, it is respectfully submitted that such “fitting” of the teachings would not have been “beyond [the] ... skill [level]” of one of ordinary skill in the art
In KSR, the Supreme Court stated:
“When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one.  If a person of ordinary skill can implement a predictable variation, 103 likely bars its patentability.  For the same reason, if a technique has been used to improve one device, and a person of ordinary skill in the art would recognize that it would improve similar devices in the same way, using the technique is obvious unless its actual application is beyond his or her skill.” (page 13, emphasis added).
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.Response to Arguments:
While the instant rejection is based on a newly applied reference, Applicants’ arguments presented in the Amendment received on July 21, 2021 are addressed herein to the extent applicable in desire of compact prosecution.
Applicants traverse the rejection (page 5, Response).
Applicants contend that the teachings of Takahashi directed toward the use of “sipping” pressure (i.e., negative pressure) cannot teach the presently amended claim limitation of reagents being delivered and driven through the main channel via positive pressure.
Applicants then contend that Nobile does not bring aqueous template nucleic acid and aqueous amplification reagents on-chip via separate inlet channels (page 7, Response).
This argument is no longer applicable because Nobile reference is not relied upon.
However, it is noted herein that Nobile reference was relied upon for teaching the use of a positive pressure driven flow for generating emulsion droplets in a channel.  Therefore, Applicants’ arguments cannot be deemed persuasive as they are arguing teaching which is not relied upon from Nobile in the present rejection.
Regardless of this finding, as discussed above, Quake et al. teach a method of utilizing positive pressure to move a non-aqueous medium (i.e., oil) in a main channel of a microfluidic chip, as well as using positive pressure to introduce at least two different inlets, each of which comprising immiscible medium (i.e., aqueous) into the main channel, and coalescing them together to mix the two contents therein.
Therefore, one of ordinary skill in the art would have recognized that the method disclosed by Takahashi et al. that provide aqueous solution via sipping (i.e., suction) could also be performed via a positive pressure driven system as taught by Quake et al.
Given that both system disclosed by Takahashi et al. and Quake et al. successfully generated emulsion droplets within a channel flowing with an immiscible carrier medium, one of ordinary skill in the art would have expected that the use of positive pressure driven system (of Quake et al.) in the method of Takahashi et al. would have yielded the same predictable outcome.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, Applicants’ arguments are not found persuasive and the rejection is maintained.
Claim 4 is rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Quake et al. (US 2002/0058332 A1, published May 16, 2002), as applied to claims 1-3, 12, 13, and 19 above, and further in view of Vogelstein et al. (US 2011/0059435, published March 2011, priority February 7, 2005).
The teachings of Takahashi et al. and Quake et al. have already been discussed above.
Takahashi, while explicitly suggesting that their disclosed method be for amplification, detection, as well as for sequencing reaction, do not explicitly all possible sequencing means known at the time the invention was made.
Consequently, the artisans do not explicitly disclose all possible means of sequencing reactions, such as those involving primers which are linked to a biotin (claim 4).
Vogelstein et al. disclose a method of amplifying nucleic acids in emulsion particles (region of analyte DNA molecules is amplified using a high fidelity DNA polymerase … Microemulsions comprising said first amplicons and reagents beads are formed”, section [0008]), wherein the primers are biotinylated (“common oligonucleotide … was synthesized with a dual biotin group … coupled to streptavidin-coated magnetic beads”, section [0058]).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Takahashi et al. and Quake et al. with the teachings of Vogelstein et al., thereby arriving at the invention as claimed for the following reasons.
As stated above, Takahashi et al. explicitly teach that the amplification products in the emulsion particles of their invention can be employed for further analysis such as amplifying, detecting, and sequencing (see section [0030]), and while the artisans did not explicitly list all possible such “further analysis”, said one of ordinary skill in the art would have had a reasonable expectation of success that any of amplification and analysis means known in the art would have been equally useful and beneficial in the art of molecular analysis/diagnostics.
Given that Vogelstein et al. teach a method of producing emulsion particles comprising therein, amplification products bound on a bead via use of bead-bound primers for the purpose of detecting nucleotide sequence variations, said one of ordinary skill in the art would have been reasonably motivated to adopt the teachings of Vogelstein et al. into the method disclosed by the combination of Takahashi et al. and Quake et al., thereby arriving at the claimed method of generating emulsion particles comprising bead-bound primers and reagents, and template nucleic acids produced from the coalescing means taught by Takahashi et al.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Claims 20-23 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Quake et al. (US 2002/0058332 A1, published May 16, 2002), as applied to claims 1-3, 12, 13, and 19 above, and further in view of Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004).
The teachings of Takahashi et al and Quake et al. have already been discussed above.
Takahashi et al. do not explicitly disclose that the junction comprises a fluidic interconnect apparatus that forms a seal between the inlet and the main channel (carrying the oil) (claim 20), or that junction defines a nozzle which includes an oil line that provides a constant oil flow into the nozzle (claim 21), wherein the design prevents the nozzle or the main channel from becoming wet with the aqueous fluid (claim 22) or that the carrier fluid into the nozzle provides complete isolation of the nozzle from the aqueous phase (claim 23).
While Takahashi et al. do not explicitly disclose the above-discussed features of the junction and nozzle, these features are deemed known and prima facie obvious to one of ordinary skill in the art at the time the invention was made based on the combination of the teachings of Takahashi et al., Quake et al., and Nobile et al. for the reasons discussed below.
Nobile et al. provides the below configuration which graphically represents a junction where the aqueous reagents are disposed into the main channel which carries the oil, which is similar to the configuration disclosed by Takahashi et al.:
PNG
media_image1.png
505
1015
media_image1.png
Greyscale
Merriam-Webster defines “nozzle” a short tube with a taper or constriction used to speed up or direct a flow of fluid (see attached)1.
Therefore, the main channel has a nozzle 406 (the narrow portion) found on the main channel wherein the oil flows through and the upper reservoir (412) (or first inlet) is in communication with the main channel at a junction at which the nozzle is found and the droplets are formed therefrom.
With regard to claim 20, the junction formed between the inlet and the nozzle of the main channel is configured to generate a plurality of droplets and while the artisans do not explicitly state that there is a seal between this inlet channel and the main channel, it would have been an obvious to one of ordinary skill in the art to make sure that there was a tight seal where the inlet and main channel intersected so as to contain the emulsion oil flowing through the main channel without escape and the reagents of the reaction flow into the main channel without any escape into a gap therebetween.
With regard to claim 21, the left-side of the narrow junction (i.e., nozzle) is considered to be an oil line and it feeds the carrier oil to the nozzle.
With regard to claim 22, the nozzle design inhibits the first aqueous fluid from wetting the surface of the nozzle of the main channel.  This is because the artisans explicitly state that at the narrow junction (i.e., nozzle), the aqueous reagents from the inlet is introduced and shears off in to a plurality of droplets and the formed droplets continue their travel in the main channel surrounded by the carrier oil (sections [0079] and [0080]).
With regard to claim 23, the nozzle is surrounded by the oil flowing there-through and therefore, isolated from the aqueous phase entering from the inlet.
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Claims 32-34 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Takahashi et al. (US 2006/0257893 A1, published November 16, 2006) in view of Quake et al. (US 2002/0058332 A1, published May 16, 2002), as applied to claims 1-3, 12, 13, and 19 above, and further in view of Dorfman et al. (Analytical Chemistry, 2005, vol. 77, pages 3700-3704).
The teachings of Takahashi et al. and Quake et al. have already been discussed above.
Takahashi et al., while explicitly teach performing PCR in aqueous droplets in surrounded by an immiscible oil medium, do not teach every possible such medium or variations known in the art.  Consequently, the artisans do not explicitly teach that the oil is fluorinated (claim 32), or that the oil comprises fluorosurfactant (claims 33 and 34).
Dorfman et al. disclose a method of performing a continuous PCR reaction in an aqueous droplets surrounded by an immiscible oil medium (“[p]erformign continuous flow PCR for high-throughput clinical and quantitative applications requires encapsulating the sample and preventing contamination between them … this can be accomplished in two ways: either the sample can be a droplet entrained in an immiscible solvent …”, page 3700, 2nd column).
Dorfrman et al. disclose the use of fluorinated oil as the immiscible medium when performing the continuous emulsion PCR (“an improvement system for performing continuous flow PCR using droplets in an immiscible fluorinated solvent …”, page 3701, 1st column, 2nd paragraph; “primed with either the bulk fluorinated oil FC-40”, page 3701, 1st column, 3rd paragraph) or oil comprising a fluorosurfactant (“or FC-40 containing various amounts of fluoro alcohol surfactant”, page 3701, 1st column, 3rd paragraph).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Takahashi et al. and Quake et al. with the teachings of Dorfman et al., thereby arriving at the invention as claimed for the following reasons.
Dorman et al. discuss potential problems associated with performing emulsion droplet PCR in an immiscible medium:
“wall treatment [of the channel] are necessary to prevent adsorption of DNA to the walls and subsequent transfer between samples … wall contamination problem is a significant drawback … bubbles in the stream may introduce irregularities in the thermal history of the droplets, making the technique less attractive … flowing droplet system does not suffer from either of these drawbacks … However, the initial work on this system suffered from contamination between droplets” (page 3701, 1st column, 1st paragraph).
In solving the drawback so identified, Dorman et al. disclose that immiscible solvent, “prevent[ed] the droplet breakage … thereby eliminating the contamination between droplets” (page 3701, 1st column, 2nd paragraph), producing “adequately reproducible amplification between different drops without any detectable contamination between them” (page 3701, 1st column, 2nd paragraph).
Based on such findings, one of ordinary skill in the art would have been motivated to utilize a fluorinate immiscible oil or oil comprising fluorosurfactants into the teachings of Takahashi et al. and Quake et al., for the same recognized benefit of producing reproducible amplification reaction between emulsion droplets as well as preventing contamination therebetween.
Given that Takahashi et al., Quake et al. and Dorfman et al. performed emulsion amplification reactions in a flowing oil medium, said one of ordinary skill in the art would have also expected that addition of fluorosurfactants or using fluorinated oil in the method of Takahashi et al. would have yielded the same predictable benefit as observed in Dorman et al.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
Inquiries

CITED REFERENCE SUMMARIES: Ref US20060257893A1: Claims:
A microfluidic device, comprising: at least one sipper; at least one fluid reservoir connected to at least one microfluidic inline reaction channel, wherein the at least one microfluidic inline reaction channel runs through a reagent assembly area, an amplification area within a first temperature-controlled area, and a detection area within a second temperature-controlled area; and at least one metal trace for heating of and/or fluid movement within the microfluidic inline reaction channel, wherein detection of amplified DNA products occurs at one or more temperatures.
at least one sipper;
at least one fluid reservoir connected to at least one microfluidic inline reaction channel, wherein the at least one microfluidic inline reaction channel runs through a reagent assembly area, an amplification area within a first temperature-controlled area, and a detection area within a second temperature-controlled area; and
at least one metal trace for heating of and/or fluid movement within the microfluidic inline reaction channel,
wherein detection of amplified DNA products occurs at one or more temperatures.
The microfluidic device of claim 1 , wherein the at least one microfluidic inline reaction channel further comprises a valve downstream of the detection area.
A microfluidic device, comprising: at least one sipper; at least one fluid reservoir connected to at least one microfluidic inline reaction channel, wherein the at least one microfluidic inline reaction channel runs through a reagent assembly area, an amplification area, and a detection area, and wherein the microfluidic inline reaction channel further comprises a valve downstream of the detection area; and at least one metal trace for heating of and/or fluid movement within the microfluidic inline reaction channel.
at least one fluid reservoir connected to at least one microfluidic inline reaction channel, wherein the at least one microfluidic inline reaction channel runs through a reagent assembly area, an amplification area, and a detection area, and wherein the microfluidic inline reaction channel further comprises a valve downstream of the detection area; and
at least one metal trace for heating of and/or fluid movement within the microfluidic inline reaction channel.
An instrument that controls fluid movement within, heating and cooling of, and data acquisition from, the microfluidic device of claim 1 , comprising a cartridge that interfaces between the instrument and the microfluidic device.
An instrument that controls fluid movement within, heating and cooling of, and data acquisition from, the microfluidic device of claim 3 , comprising a cartridge that interfaces between the instrument and the microfluidic device.
An apparatus comprising the microfluidic device of claim 3 and an instrument that controls fluid movement within, heating and cooling of, and data acquisition from, the microfluidic device.
The apparatus of claim 6 , further comprising a cartridge that interfaces between the instrument and the microfluidic device.
A method of determining an organism in a sample, the method comprising the steps of: (a) acquiring the sample; (b) isolating at least one copy of the genomic DNA of the organism, if present in the sample; (c) introducing a first mixture comprising SGP primers, nucleotides, DNA polymerase, and intercalators to the genomic DNA of the organism to form a second mixture; (d) heating the second mixture to a first temperature that will cause the genomic DNA, if present, to denature into a first and second genomic DNA template; (e) cooling the second mixture to a second temperature that will cause the primers to anneal to each genomic DNA template; (f) reheating the second mixture to a third temperature that is between the first and second temperatures to allow the primers to remain annealed to the genomic DNA and the DNA polymerase to elongate nucleic acid polymers originating from the annealed primers; (g) maintaining the third temperature for a first length of time; (h) repeating steps (d)-(g) at least once; (i) repeating steps (d)-(f); (j) maintaining the third temperature for a second length of time equal to about 40-60% of the first length of time; (k) recooling the second mixture to a fourth temperature lower than or equal to that of the second temperature to allow formation of higher-order structures containing intercalators; (l) detecting the resulting higher-order structures; (m) performing melting temperature analysis; (n) detecting a waveform profile; and (o) determining a positive waveform profile from the sample if the sample contained the organism.
(a) acquiring the sample;
(b) isolating at least one copy of the genomic DNA of the organism, if present in the sample;
(c) introducing a first mixture comprising SGP primers, nucleotides, DNA polymerase, and intercalators to the genomic DNA of the organism to form a second mixture;
(d) heating the second mixture to a first temperature that will cause the genomic DNA, if present, to denature into a first and second genomic DNA template;
(e) cooling the second mixture to a second temperature that will cause the primers to anneal to each genomic DNA template;
(f) reheating the second mixture to a third temperature that is between the first and second temperatures to allow the primers to remain annealed to the genomic DNA and the DNA polymerase to elongate nucleic acid polymers originating from the annealed primers;
(g) maintaining the third temperature for a first length of time;
(h) repeating steps (d)-(g) at least once;
(i) repeating steps (d)-(f);
(j) maintaining the third temperature for a second length of time equal to about 40-60% of the first length of time;
(k) recooling the second mixture to a fourth temperature lower than or equal to that of the second temperature to allow formation of higher-order structures containing intercalators;
(l) detecting the resulting higher-order structures;
(m) performing melting temperature analysis;
(n) detecting a waveform profile; and
(o) determining a positive waveform profile from the sample if the sample contained the organism.
The method of claim 8 , wherein the third temperature is maintained for a second length of time about 50% of the first length of time.
The method of claim 8 , wherein the number of times steps (d)-(g) are repeated in step (h) is 20-50 times.
The method of claim 10 , wherein the number of times steps (d)-(g) are repeated in step (h) is 22-24 times.
The method of claim 8 , wherein the method further comprises repeating steps (i)-(j) one or more times prior to step (k).
A method of detecting the absence or presence of an organism in a sample, comprising the steps of: (a) acquiring the sample; (b) isolating at least one copy of the genomic material of the organism, if present in the sample; (c) aspirating at least one sample droplet into a microfluidic reaction channel; (d) forming at least one sample plug by mixing the at least one sample droplet with a primer plug, wherein the primer plug comprises amplification reagents that comprise at least one primer, nucleotides, DNA polymerase, and intercalators; (e) heating the at least one sample plug to a first temperature that will cause each copy of the genomic DNA, if present, to denature into a first and second genomic DNA template; (f) cooling the at least one sample plug to a second temperature that will cause the primers to anneal to each genomic DNA template; (g) reheating the at least one sample plug to a third temperature that is between the first and second temperatures as to allow (1) the primers to remain annealed to the genomic DNA and (2) the DNA polymerase to elongate nucleic acid polymers originating from the annealed primers; (h) maintaining the third temperature for a first length of time; (i) repeating steps (e)-(h) at least once; and (j) detecting the resulting amplified products, wherein at least steps (c)-(j) occur within the apparatus of claim 6 .
(b) isolating at least one copy of the genomic material of the organism, if present in the sample;
(c) aspirating at least one sample droplet into a microfluidic reaction channel;
(d) forming at least one sample plug by mixing the at least one sample droplet with a primer plug, wherein the primer plug comprises amplification reagents that comprise at least one primer, nucleotides, DNA polymerase, and intercalators;
(e) heating the at least one sample plug to a first temperature that will cause each copy of the genomic DNA, if present, to denature into a first and second genomic DNA template;
(f) cooling the at least one sample plug to a second temperature that will cause the primers to anneal to each genomic DNA template;
(g) reheating the at least one sample plug to a third temperature that is between the first and second temperatures as to allow (1) the primers to remain annealed to the genomic DNA and (2) the DNA polymerase to elongate nucleic acid polymers originating from the annealed primers;
(h) maintaining the third temperature for a first length of time;
(i) repeating steps (e)-(h) at least once; and
(j) detecting the resulting amplified products,
wherein at least steps (c)-(j) occur within the apparatus of claim 6 .
The method of claim 13 , wherein the primer plug comprises a PCR primer pair.
The method of claim 14 , wherein the detecting step of step (j) occurs at one temperature.
The method of claim 13 , wherein the primer plug comprises a waveform profiling primer.
The method of claim 16 , wherein the detecting step of step (j) occurs at a range of temperatures.
The method of claim 17 , further comprising after step (i) and before step (j), the steps of: (1) repeating steps (e)-(g); (2) maintaining the third temperature for a length of time equal to about 40-60% of the first length of time; and (3) cooling the at least one sample plug to a fourth temperature lower than or equal to that of the second temperature to allow formation of higher-order structures containing intercalators.
(1) repeating steps (e)-(g);
(2) maintaining the third temperature for a length of time equal to about 40-60% of the first length of time; and
(3) cooling the at least one sample plug to a fourth temperature lower than or equal to that of the second temperature to allow formation of higher-order structures containing intercalators.
The method as in any one of claims 13 - 18 , further comprising a last step of selecting a sample plug for further analysis, wherein the step of selecting occurs at the valve within the apparatus.
A method of screening a sample supply for contamination, comprising the steps of: (a) continuously acquiring sample droplets from the sample supply into at least one microfluidic reaction channel; (b) forming sample plugs by mixing each sample droplet with a primer plug, wherein the primer plug comprises amplification reagents; (c) amplifying DNA in sample plugs comprising genomic material; and (d) detecting the absence or presence of amplified products, wherein steps (a)-(d) occur within the apparatus of claim 6 .
(a) continuously acquiring sample droplets from the sample supply into at least one microfluidic reaction channel;
(b) forming sample plugs by mixing each sample droplet with a primer plug, wherein the primer plug comprises amplification reagents;
(c) amplifying DNA in sample plugs comprising genomic material; and
(d) detecting the absence or presence of amplified products,
wherein steps (a)-(d) occur within the apparatus of claim 6 .
A method of identifying an organism, the method comprising the steps of: (a) preparing at least one DNA sample droplet comprising a DNA molecule isolated from the organism; (b) acquiring the at least one DNA sample droplet into at least one microfluidic inline reaction channel; (c) forming at least one DNA sample plug by mixing at least one DNA sample droplet with a primer plug, wherein the primer plug comprises amplification reagents that comprise at least one known first primer; (d) subjecting the at least one DNA sample plug to at least one amplification cycle such that the at least one DNA sample plug has detectable amplified DNA products; (e) detecting amplified DNA products; (f) identifying the organism based on detection of the amplified products; and (g) optionally repeating steps (a)-(f) with amplification reagents comprising a known primer that is different from the first known primer to increase the accuracy of the identification of the organism, wherein at least steps (b)-(e) occur within the apparatus of claim 6 .
(a) preparing at least one DNA sample droplet comprising a DNA molecule isolated from the organism;
(b) acquiring the at least one DNA sample droplet into at least one microfluidic inline reaction channel;
(c) forming at least one DNA sample plug by mixing at least one DNA sample droplet with a primer plug, wherein the primer plug comprises amplification reagents that comprise at least one known first primer;
(d) subjecting the at least one DNA sample plug to at least one amplification cycle such that the at least one DNA sample plug has detectable amplified DNA products;
(e) detecting amplified DNA products;
(f) identifying the organism based on detection of the amplified products; and
(g) optionally repeating steps (a)-(f) with amplification reagents comprising a known primer that is different from the first known primer to increase the accuracy of the identification of the organism, wherein at least steps (b)-(e) occur within the apparatus of claim 6 .
A method for quantifying the concentration of genomic material in a sample, the method comprising the steps of: (a) diluting the sample using dilution factors such that the concentration of the genomic material is at most approximately one molecule per sample droplet; (b) acquiring sample droplets into at least one microfluidic inline reaction channel; (c) forming at least more than one sample plug by mixing each sample droplet with a primer plug, wherein the primer plug comprises amplification reagents; (d) subjecting each sample plug to amplification cycles such that each sample plug comprising at least one DNA molecule has detectable amplified DNA products, and each sample plug not comprising a DNA molecule will have zero-detection of amplified DNA products; (e) detecting the absence or presence of amplified DNA products in each sample plug; (f) determining the ratio of sample plugs containing detectable amplified DNA products to sample plugs resulting in zero-detection; and (g) using the dilution factor to calculate the original concentration of genomic material in the sample, wherein at least steps (b)-(e) occur within the apparatus of claim 6 .
(a) diluting the sample using dilution factors such that the concentration of the genomic material is at most approximately one molecule per sample droplet;
(b) acquiring sample droplets into at least one microfluidic inline reaction channel;
(c) forming at least more than one sample plug by mixing each sample droplet with a primer plug, wherein the primer plug comprises amplification reagents;
(d) subjecting each sample plug to amplification cycles such that each sample plug comprising at least one DNA molecule has detectable amplified DNA products, and each sample plug not comprising a DNA molecule will have zero-detection of amplified DNA products;
(e) detecting the absence or presence of amplified DNA products in each sample plug;
(f) determining the ratio of sample plugs containing detectable amplified DNA products to sample plugs resulting in zero-detection; and
(g) using the dilution factor to calculate the original concentration of genomic material in the sample,
wherein at least steps (b)-(e) occur within the apparatus of claim 6 .
The method as in any one of claims 20 - 22 , further comprising a last step of selecting a sample plug for further analysis, wherein the step of selecting occurs at the valve within the apparatus.
The method as in any one of claims 13 - 18 and 20 - 22 , further comprising a step wherein the sample plug(s) is surrounded by an immiscible nonaqueous fluid.
Ref US20050227264A1: Claims:
A method for amplifying genetic material comprising: providing a water-in-oil emulsion in a continuous flow wherein the emulsion comprises a plurality of water droplets comprising microreactors, wherein a plurality of the microreactors each include one or more species of nucleic acid templates, and sufficient reagents to amplify the copy number of one of the nucleic acid templates; and thermally processing the emulsion by flowing it across stationary controlled temperature zones to amplify nucleic acid templates by polymerase chain reaction.
providing a water-in-oil emulsion in a continuous flow wherein the emulsion comprises a plurality of water droplets comprising microreactors, wherein a plurality of the microreactors each include one or more species of nucleic acid templates, and sufficient reagents to amplify the copy number of one of the nucleic acid templates; and
thermally processing the emulsion by flowing it across stationary controlled temperature zones to amplify nucleic acid templates by polymerase chain reaction.
The method according to claim 1 , wherein each microreactor contains one or more beads capable of capturing a nucleic acid template.
The method according to claim 1 , wherein one or more of the beads capture amplified copies of the nucleic acid template.
The method according to any of claims 1 - 3 , further comprising breaking the emulsion to retrieve one or more of the amplified nucleic acid templates.
The method according to any of claims 1 - 3 , further comprising filtering the water-in-oil emulsion to collect one or more of the amplified nucleic acid templates.
The method according to claim 1 , wherein the microreactors have an average size of between approximately 50 to approximately 250 Î¼m in diameter.
The method according to claim 1 , wherein the nucleic acid templates are selected from the group consisting of genomic DNA, cDNA, episomal DNA, BAC DNA, and YAC DNA.
The method according to claim 2 , wherein the bead has a diameter of between approximately 2 microns to approximately 100 microns.
The method according to claim 2 , wherein the bead is selected from the group consisting of a sepharose bead, a solid bead and a monodisperse bead.
An apparatus for amplifying genetic material comprising: at least one fluid delivery device; at least one first temperature zone to cycle a plurality of microreactors each including one or more species nucleic acid templates to a first temperature; at least one second temperature zone to cycle the plurality of microreactors to second temperature lower than the first temperature; a first conduit for flowing at least a stream of oil therein from a first reservoir; and a second conduit for flowing at least a water based PCR solution including nucleic acid templates and PCR reagent from a second reservoir out of an orifice and into the first conduit creating a water-in-oil emulsion, wherein the PCR solution upon entering the first conduit comprises a plurality of droplets comprising the microreactors for performing polymerase chain reactions, wherein a plurality of the microreactors each include one or more species of nucleic acid template.
at least one fluid delivery device;
at least one first temperature zone to cycle a plurality of microreactors each including one or more species nucleic acid templates to a first temperature;
at least one second temperature zone to cycle the plurality of microreactors to second temperature lower than the first temperature;
a first conduit for flowing at least a stream of oil therein from a first reservoir; and
a second conduit for flowing at least a water based PCR solution including nucleic acid templates and PCR reagent from a second reservoir out of an orifice and into the first conduit creating a water-in-oil emulsion, wherein the PCR solution upon entering the first conduit comprises a plural
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