Office Action 5: Non-Final Rejection (2020-11-03)
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 \[\[s\]\] in a first inlet channel on a fluidic chip \[\[s\]\], 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; 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\. (Currently Amended) 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 biotinylated surface .

6-11. (Cancelled) 710

12\. (Currently Amended) 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\. (Currently Amended) The method of claim 1, wherein the first and second inlet channels are \[\[is\]\] 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\. (Currently Amended) 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
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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 October 1, 2020 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.
Information Disclosure Statement
The fifteen IDSs filed on October 13, 2020 are proper and are being considered by the Examiner.  The Office notes the fifteen IDS totals over 1,500 publications some of which date back to the 1930s in their publication dates.
All references have been considered and the signed copy of the PTO/SB form are enclosed herewith.
Priority
T effective priority date of the instant application has been restored to the filing date of the parent application, 11/803,104, that is, May 11, 2007.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA  35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 5 is rejected 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. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA  35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a New matter rejection.
Claim 5 has been amended to recite the phrase, “capturing the annealed one of the nucleic acid molecules to a biotinylated surface”.
The specification does not support for capturing the nucleic acid molecules on a biotinylated surface.  Rather, the primers are biotinylated and captured on a streptavidin coated surface, wherein the nucleic acid molecules are annealed to these primers:
“The present invention provides methods to overcome this limitations.  In one embodiment, a Solexa slide can be coated with any of the compound commonly used to permit binding and immobilization (e.g., carboxy-esters, streptavidin, Igg, gold, etc.)  PCR reaction could be performed as described in the instant microfluidic device using primers modified with the appropriate binding moiety (5’ amines, 5’ biotins, 5’ DNPs or 5’ Thiols, respectively) to efficiently amplify PCR products in solution … then be efficiently bound to the Solexa slide for subsequent sequencing” (section [0384])
Therefore, there is no such support for the language of a “biotinylated surface”.
New matter must be removed.
Claim Rejections - 35 USC § 103
The rejection of claims 1-5 and 12-14 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (US 2005/0202489 A1, published September 2005; priority March 2005) 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 July 1, 2020 is withdrawn in view of the Amendment received on October 1, 2020.
The rejection of claims 19-30 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (US 2005/0202489 A1, published September 2005; priority March 2005) 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 July 1, 2020 is withdrawn in view of the amendment received on October 1, 2020.
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, 12, 13, and 19 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).
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.
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 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).
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.
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 Nobile et al. (US 2005/0227264 A1, published October 13, 2005, priority January 28, 2004).
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.:
PNG
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505
1015
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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.
Conclusion
No claims are allowed.
Applicant’s arguments with respect to the previous rejections of record have been considered but are moot based on the present rejections based on new reference and new grounds.
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. Claims 1 and 5 are amended. Claims 31-25 are new. Support for the amendments and new claims may be found throughout parent U.S. application No. 11/803,104 as-filed, for example, at least at line 31 on page 28 line 35 on page 10; lines 21- 23 on page 18; line 20 on page 19; lines 32-35 on page 27; lines 20-22 on page 28; lines 5-6 on page 29, and lines 30-36 on page 77.
No new matter is added.
Rejections under 35 U.
S.
C. 112
The Office Action on page 3 states that claim 5 is rejected under §112 (pre-AIA), first paragraph, where "biotinylated surface" is new matter.
Claim 5 is amended for consistency with the paragraph on the bottom of page 77 of parent U.S. application No. 11/803,104 as-filed. Applicant notes that the prior version, implying that a biotinylated primer could link to a biotinylated surface, was a clerical error. Applicant thanks the Examiner for the comments, and believes the new matter has been removed.
Rejections under 35 U.
S.
C.
§
The Office Action on page 5 states that claims 1-3, 12, 13, and 19 are rejected under §103 over U.S. Pub. 2006/0257893 to Takahashi in view of U.S. Pub. 2005/0227264 to Nobile. At page 10, claim 4 is so rejected over Takahashi and Nobile and further in view of U.S. Pub. 2011/0059435 to Vogelstein. On page 13, claims 20-23 are rejected over Takahashi in view of Nobile.
Applicant submits that the pending 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 immiscible carrier fluid in the main channel by
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.
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
.
Claim 1 cannot be obvious because the only flow mechanisms for joining fluids disclosed in
the asserted prior art are electro-kinetic or applying a vacuum to waste wells for negative displacement, suction-driven flow
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 says that an immiscible nonaqueous fluid (e.g., mineral oil) may be used as a buffer spacer for the initial sample droplets, and is added to each, or between each, sample droplet being drawn by the sipper in order to surround and separate neighboring sample droplets as they travel through a microfluidic inline reaction channel of the invention. Id., [0083]. 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 of droplets surrounded by the immiscible carrier fluid in the main channel. That is, Takahashi does not explicitly state that after merging template fluid and reagent
fluid, the resultant PCR-ready droplets are in an immiscible carrier fluid. In fact, Takahashi states, "Generally, carrier liquid (6) is a water-based liquid that is the same liquid as the sample liquid." Id., [0083].
Additionally, 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 Takashi gives uses a vacuum to suck fluid through the sipper towards the waste wells.
Using a vacuum to suck into the waste well teach away from "applying a pressure to flow the immiscible carrier fluid in the main channel
by positive displacement pressure driven flow
" as recited in amended claim 1.
Nobile 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].
Vogelstein is a U.S. publication of a patent application for BEAMing, which refers to Bead Emulsion Amplification with Magnets. Vogelstein, [0006]. Vogelstein doesn't mention microfluidics whatsoever. In fact, Vogelstein does not even use any of the words "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.
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.
Takahashi only uses a vacuum 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: January 14, 2021
Respectfully submitted,

NEXT 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

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 US20110059435A1: Claims:
A method for analyzing nucleotide sequence variations, comprising: amplifying a region of analyte DNA molecules using a high fidelity DNA polymerase to form a set of first amplicons; forming microemulsions comprising said first amplicons and reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the set of first amplicons; amplifying the first amplicons in the microemulsions, whereby product beads are formed which are bound to a plurality of copies of second amplicons; determining a sequence feature of the second amplicons by single base extension of a primer bound to said second amplicons using at least two differentially labeled dideoxyribonucleotides.
amplifying a region of analyte DNA molecules using a high fidelity DNA polymerase to form a set of first amplicons;
forming microemulsions comprising said first amplicons and reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the set of first amplicons;
amplifying the first amplicons in the microemulsions, whereby product beads are formed which are bound to a plurality of copies of second amplicons;
determining a sequence feature of the second amplicons by single base extension of a primer bound to said second amplicons using at least two differentially labeled dideoxyribonucleotides.
The method of claim 1 wherein the microemulsions comprise a thermostable emulsifying agent.
The method of claim 1 wherein the high fidelity polymerase has an error rate of less than 10 −5 errors per basepair per cycle.
The method of claim 1 wherein the high fidelity polymerase has an error rate of less than 5×10 −6 errors per basepair per cycle.
The method of claim 1 wherein the high fidelity polymerase has an error rate of less than 10 −6 errors per basepair per cycle.
The method of claim 1 wherein the first amplicon is less than or equal to 300 bp.
The method of claim 1 wherein the first amplicon is less than or equal to 200 bp.
The method of claim 1 wherein the first amplicon is less than or equal to 100 bp.
The method of claim 1 wherein the labeled dideoxyribonucleotides are fluorescent and flow cytometry is used to detect the labeled dideoxyribonucleotides present on the product beads.
The method of claim 1 further comprising the step of discarding from analysis beads which display two or more differentially labeled dideoxyribonucleotides extended onto primers bound to second amplicons bound to the beads.
The method of claim 1 wherein the analyte DNA molecules are obtained from plasma DNA.
The method of claim 1 wherein the microemulsions are formed with a tissue homogenizer.
The method of claim 1 wherein the microemulsions are formed with a mechanical tissue homogenizer.
The method of claim 1 wherein the microemulsions are formed with a rotor-stator tissue homogenizer.
The method of claim 1 wherein rolling circle amplification of the second amplicons is performed prior to the step of determining.
A method for amplifying a region of analyte DNA molecules, comprising: amplifying a region of analyte DNA molecules using a high fidelity DNA polymerase to form a set of first amplicons; forming microemulsions comprising said first amplicons and reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the set of first amplicons; amplifying the first amplicons in the microemulsions, whereby product beads are formed which are bound to a plurality of copies of second amplicons; breaking the microemulsions; amplifying the second amplicons using rolling circle amplification to form third amplicons.
breaking the microemulsions;
amplifying the second amplicons using rolling circle amplification to form third amplicons.
The method of claim 16 wherein the rolling circle amplification employs a circularizable probe, said pr
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