Office Action 4: Final Rejection (2020-07-01)
Rejection types: 103 - Obviousness

CLAIMS:
Amendments to the Claims:

Please amend the claims as follows.

1\. (Currently Amended) A method for droplet formation, the method comprising the steps of:
providing a plurality of aqueous fluids in a plurality of sample inlet channels, providing an immiscible carrier fluid in a main channel, wherein each of the sample inlet channels is in fluid communication with the main channel;
forming a first plurality of droplets comprising a first aqueous fluid from a first inlet channel, wherein the first plurality of droplets are formed surrounded by the immiscible carrier fluid in the main channel;
applying a pressure to flow the immiscible carrier fluid in the main channel; and
adjusting pressure in one or more of the sample inlet channels, thereby to produce a second plurality of droplets comprising a second aqueous fluid 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, further comprising coaleascing one of the first plurality of droplets and one of the second plurality of droplets at a coalescence module downstream of the main channel .
5\. (Currently Amended) The method of claim 4 \[\[1\]\], wherein a plurality of droplets comprising a first aqueous fluid and a plurality of droplets comprising a second fluid are coalesced . SVG {EMBED HERE} 0.16 0.83

6-11. (Cancelled) 710

12\. (Currently Amended) The method of claim 1, wherein the aqueous fluids comprise\[\[s\]\] nucleic acid and reagents for conducting an amplification reaction.

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

14\. (Currently Amended) The method of claim 1, wherein each of the first plurality of droplets comprises one or more molecules.

15-18. (Cancelled) 1617

19\. (New) The method of claim 1, wherein the first inlet channel is in fluid communication with the main channel at a junction at which the main channel includes a nozzle where the first plurality of droplets are formed in the main channel.

20\. (New) 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\. (New) The method of claim 19, wherein the nozzle includes at least one oil line that provides a constant flow of the carrier fluid into the nozzle while the first plurality of droplets are formed.

22\. (New) 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\. (New) 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\. (New) The method of claim 23, wherein the second inlet channel is in fluid communication with the main channel at a second junction at which the main channel includes a second nozzle where the second plurality of droplets are formed in the main channel, wherein the second nozzle includes a second oil line that provides a second flow of the carrier fluid into the second nozzle while the second plurality of droplets are formed and wherein the second flow of carrier fluid provides complete isolation of the nozzle from an aqueous phase of the second aqueous fluid.

25\. (New) The method of claim 20, further comprising coaleascing one of the first plurality of droplets and one of the second plurality of droplets at a coalescence apparatus downstream of the fluidic interconnect.

26\. (New) A method for droplet formation, the method comprising:
providing a carrier fluid in a main channel;
forming a first set of aqueous droplets in the carrier fluid by delivering a first aqueous fluid through a first nozzle from a first inlet channel into the main channel;
providing a second aqueous fluid in a second inlet channel, wherein the second inlet channel is in fluid communication with the main channel through a second nozzle; and
adjusting a pressure within the second inlet channel while an oil line provides a flow of the carrier fluid into the second nozzle and completely isolates the second nozzle from an aqueous phase of the second aqueous fluid to form a second set of aqueous droplets in the carrier fluid.

27\. (New) The method of claim 26, wherein the first nozzle includes a line that flows carrier fluid into the first nozzle to provide complete isolation of the first nozzle from the first aqueous fluid.
28\. (New) The method of claim 26, further wherein the first set of aqueous droplets and the second set of aqueous droplets are interdigitated in the main channel. SVG {EMBED HERE} 0.16 0.83

29\. (New) The method of claim 26, wherein the main channel and at least the second inlet channel define a junction that comprises a fluidic interconnect apparatus that forms a seal between the second inlet channel and the main channel.

30\. (New) The method of claim 26, wherein a design of the second nozzle inhibits the second aqueous fluid from wetting surfaces of the second nozzle or the main channel.

Page 5 of 10

OFFICE ACTION:
DETAILED ACTION
The present Office Action is responsive to the Amendment received on April 30, 2020.
Preliminary Remark
6-11 and 15-18 are canceled.
Claims 19-30 are new.
Priority
Applicants’ argument regarding the priority based on the prior application 11/803,104 is found persuasive.  However, because Applicants have failed to show by facts where in any of the earlier provisional applications such support is found and have limited their arguments to the ‘104 application, the effective priority date of the instant application has been restored to the filing date of this application, that is, May 11, 2007.
Claim Rejections - 35 USC § 103
The rejection of claims 6-11 and 15-18 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 February 5, 2020 is withdrawn in view of the Amendment received on April 30, 2020, canceling the rejected claims.
Rejection - Maintained
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 1-5 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 February 5, 2020 is maintained for the reasons of record.
Applicants’ arguments presented in the Amendment received on April 30, 2020 have been carefully considered but they are not deemed persuasive for the reasons set forth in the, “Response to Arguments” section.The Rejection:
With regard to claim 1, Cho et al. disclose a method of droplet formation (“creating a plurality of reactant aqueous solution droplets”, section [0020]), the method comprising the steps of:
providing a plurality of aqueous fluids in a plurality of sample inlet channels (see three inlet channels 341 of Figure 5; “inlet channels 341 … may be respectively connected to inlet ports 342 and 344 that communicate with the outside”, section [0094]; “three first inlet channels 341 for introduction of reactant aqueous solutions…”, section [0094]; “aqueous solution containing reactants for nucleic acid amplification (hereinafter, also simply referred to as ‘reactant aqueous solution’)”, section [0070]);
providing an immiscible carrier fluid in a main channel, wherein each of the sample inlet channels is in fluid communication with the main channel (reaction channel 130 of Figure 5; “oil 131 is introduced into the reaction channel 130”, section [0096]; see the three channels 341 is in fluid communication with the main channel 130);
forming a plurality of droplets comprising at least one aqueous fluid surrounded by the immiscible carrier fluid in the main channel (“aqueous solution droplets 332a, 332b, and 332c surrounded by the oil 131”, section [0096]); and
flowing the immiscible carrier fluid in the main channel (“the reactant solution passes through … at a constant flow rate …”, section [0081]).
With regard to claim 5, a plurality of droplets comprising a first aqueous fluid and a plurality of droplets comprising a second fluid (from three separate sample inlets).
With regard to claim 6, the flow carrier fluid is passes at a constant flow rate producing droplets that are substantially uniform in size (see above, also, “aqueous solution droplets 132 created in the reaction channel 130 have a uniform size”, section [0081]).
With regard to claim 9, the immiscible fluid is oil (see above).
With regard to claims 10, 11, and 18, the oil is perfluorinated (section [0021]).
With regard to claim 12, the aqueous fluid comprises nucleic acid and reagents for conducting an amplification reaction (“aqueous solution containing reactants for nucleic acid amplification (hereinafter, also simply referred to as ‘reactant aqueous solution’)”, section [0070]).
With regard to claim 13, the amplification reaction is a PCR reaction (“nucleic acid amplification apparatus according to the third embodiment of the present invention has a construction suitable for multiplex PCR”, section [0094]).
With regard to claim 14, each droplet comprises one or more molecules (evidenced by amplification products).
With regard to claim 15, the reaction is PCR of nucleic acid molecules (see above).
With regard to claim 17, the droplets are of uniform size (see above), and since there is no demarcation in the specification what is deemed “highly uniform” nor does the PTO have the facility to determine whether the uniform size as explicitly taught by Cho et al.’s method deemed, “high”, it is asserted that Cho et al.’s uniform sized droplets are highly uniform.
According to In re Best 195 USPQ 430, 1997, the court stated that, “Patent Office can require applicant to prove that prior art products do not necessarily or inherently possess characteristics of his claimed product wherein claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes; burden of proof is on applicant” (pp. 430).
Cho et al., although explicitly teaching maintaining flow of the immiscible carrier fluid (continuous PCR), the artisans do not explicitly teach that the pressure is used to flow the immiscible carrier fluid, or that this pressure is adjusted to produce a plurality of droplets.
Consequently Cho et al. do not explicitly disclose that the pressure in the main channel is derived from a pressure in a reservoir in fluid communication with the main channel (claim 2), which is a single pressure source (claim 3), which comprises oil (claim 4).
Cho et al. do not disclose that the size of the droplets formed should be detected (claim 8), or that the droplets are of different sizes (claims 7 and 16).
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]).
Nobile et al. explicitly disclose that these emulsion particles are then subject to PCR reaction (“emulsion is heat stable to allow thermal processing/cycling), wherein the droplets formed range in size of 5 microns to 500 microns, with an explicitly cited advantage that cross-flow emulsion generation, “allows for control of the droplet formation, and uniformity of droplet size” (section [0061]).
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]).
Nobile et al. evidence that the system is programmed to generate a particular size of the emulsion droplets by adjustment of the oil flow rate (via the pump) and the adjustment of the flow rate of the PCR solution (via the pump) (“emulsion oil flow rate was set at 2 ml/min, while the PCR solution flow rate was set at 5 l/min … droplet (microreactor) size range was 80 m to 120m (270 pl to 900 pl) …”, section [0108]).
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 Cho et al. and Nobile et al., thereby arriving at the invention as claimed for the following reasons.
As discussed above, Cho et al. already disclose a method and a microfluidic device comprising a plurality of sample inlets and an inlet for an immiscible carrier fluid into a main channel for performing continuous generation of emulsion products and PCR.
While Cho 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 Cho et al. and Nobile et al., thereby arriving at a method of performing the method of Cho et al. on a system which is configured to deliver programmable amounts of reagents via pumps so as to continuously generate emulsion droplets of desired sizes, which would have naturally required that the artisan devise a detection means so as to measure the emulsion sizes generated from each of the sample inlet channels.  With regard to the limitations regarding the droplets comprising different sizes, droplets of different sizes would have been present when performing the method of Cho et al. in combination of Nobile et al. during the adjustment of the flow rates of the respective reagents for ensuring that all droplets or the droplets generated from each of the sample inlet channel were of uniform sizes.
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.
Applicants state that neither Cho nor Nobile teach or suggest any droplet forming method that proceeds by applying a pressure to flow a carrier fluid in the main channel and adjusting pressure in a second inlet channel to produce a second set of droplets in a main channel within which a first set of droplets is also produced (page 7, Response).
Applicants further state that the references of record do not disclose the insight that controlling pressure in two ways, in the claimed fashion would be useful to produce the second set of aqueous droplets in a main channel along the first set of droplets that is produced, and contends that the element of applying pressure to a carrier fluid in a main channel and adjusting pressure in an inlet channel to form the second additional set of droplets is wholly missing from the asserted prior art (page 7, Response).
Applicants’ contention, in part, is apparently based on the lack of the usage of the word, “pressure” in the disclosure of Cho.1
It is respectfully pointed out any fluid flowing in a constricted space results by a positive force results in pressure.  While Cho may not have used the term, “pressure,” it is clear that the discussion pertaining to the adjustment of the fluid flow speed by which the reagents/fluid are fed through will necessarily require alterations in the pressure generated in order to force the reagent/fluid at said speed.
Such is plainly evidenced in the below-statements made by Cho:
“performed by continuous-flow PCR” (section [0024])
A “continuous-flow” of the PCR in the artisan’s design requires the carrier reagent to push the emulsion droplets in the device through the device’s body space, requiring a positive and continued pressure.  An absence of such pressure would result in the droplets within the channel to stop from flowing, which would be contrary to the entire intent of the device that requires a “continuous flow”.
“For continuous-flow PCR, the reaction vessel may be a serpentine reaction channel, the first inlet channel and the second inlet channel may be connected to a star[t]ing end of the reaction channel, and an outlet port for releasing the fluid and an amplification product may be connected to a terminal end of the reaction channel” (section [0032])
As evidenced above, the continuous-flow PCR device of Cho requires that the fluid is fed from an inlet and the fluid traveling through the device in a continuous manner may be “released” to a terminal end of the reaction channel.  This clearly evidences the presence of a positive pressure which feeds and pushes the reagents throughout the device.
“when the reactant aqueous solution and the fluid, for example an oil 131, are inserted into the reaction channel 130 via the respective inlet channels 141 and 151 at a constant flow rate …” (section [0077])
“When a flow rate of the reactant aqueous solution passing through the first inlet channel 141 is controlled, the aqueous solution droplets 132 can be periodically made up to a constant volume in the reaction channel 130” (section [0078])
Additionally, for a fluid to travel through the channel of Cho’s device at a “constant rate,” there must be a source which pushes the fluid into and through the device’s channel, the necessary “push” is a pressure.
Lastly, Cho actually even teach the ability of the artisan to alter the flowrate necessary to achieve a desired outcome:
The two standard sample inlet channels 245a and 245b and the distilled water inlet channel 247 are respectively connected to inlet ports 246a, 246b, and 248 that communicate with the outside .. adjustment of the flow rates of the standard samples and the distilled water enables easy formation of the standard sample aqueous solution of different concentrations (section [0088])
The artisans actually discuss that the flowrates of the sample and the water introduced from their respective inlets can be varied in order to achieve the desired dilution amounts of sample being emulsified in the device’s main channel flowing the carrier oil.
Information which is well known in the art need not be described in detail in the specification. See, e.g., Hybritech, Inc. v. Monoclonal Antibodies, Inc., 802 F.2d 1367, 1379-80, 231 USPQ 81, 90 (Fed. Cir. 1986).
As discussed above, while the term, “pressure” may not have been recited in the disclosure of Cho, pressure was necessarily present when performing the method discussed by Cho in order to introduce and push the reagents/fluids into the inlet and throughout the device’s reaction space at a desired flowrate.
Applicants’ arguments simply lack merit therefore.
In addition, it is noted herein that Applicants’ statement found on page 7, 2nd paragraph state the below:
“Neither Cho nor Nobile teach or a [sic] suggest any droplet forming method that proceeds by applying a pressure to a flow carrier fluid in the main channel and adjusting pressure in a second inlet channel to produce a second set of droplets in a main channel…”
It is respectfully pointed out that the claims do not recite that the adjustment of pressure is made in a second inlet channel.  Rather, all that the claim requires is that the pressure be adjusted to produce a “second plurality of droplets” from “one or more of the sample inlets, which would embrace the same inlet.  Even if such limitation were to be claimed, such would be well-within the realm of obviousness in generating multiple emulsion products from multiple samples via multiple inlets (as shown by Nobile on Figure 2).
Next, Applicants discuss the teachings of Nobile at length (pages 7, bottom paragraph to page 8, 1st paragraph).
Applicants then contend that the approach of Nobile is in contrast to the “binary control of pressure recited by claim 1 which requires”, application of pressure to a carrier fluid in the main channel and (ii) adjusting the pressure in an inlet channel to form a second set of aqueous droplets within main channel within which a first set of droplets has been formed (page 8, 2nd paragraph, Response).
This arguments are not found persuasive because the obviousness rejection is based on the combination of the relevant portions of teachings provided by the artisans of record.  Arguing that the art of record cannot be combined together in their entirety is not a proper understanding of obviousness rejection.
In KSR International Co v. Teleflex Inc, the Supreme Court clarified such a concept:
“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).
As discussed in the rejection above, the relevant portions relied upon from Nobile’s teachings is whether or not one of ordinary skill in the art would have had the requisite knowledge of providing a system which is capable of being regulated so as to control the flowrate of the reagents/liquids involved in generating emulsion droplets.
To this end, Nobile teach the generation of emulsion products 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), wherein the size of these droplets are adjustable by varying the flow rate and speed of the components involved (“adjusted by varying the flow rate and speed of the components.”, section [0058]).
In addition, Nobile taught 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]), with explicit showing of such a system an exemplary embodiment  (“emulsion oil flow rate was set at 2 ml/min, while the PCR solution flow rate was set at 5 l/min … droplet (microreactor) size range was 80 m to 120m (270 pl to 900 pl) …”, section [0108]).
Therefore, the combination of teachings not only taught a method of generating droplets within a flowing carrier fluid by use of pressure, but also a system which provided a monitoring system so as to regulate the droplet size flowing therein.  Based on the method and system, said one of ordinary skill in the art would have been motivated to flow the emulsion droplets in the device derived from the combination of Cho and Nobile, and adjust the size of the emulsion droplets to optimal and desired size via the regulation mechanism disclosed by Nobile, rendering the invention as claimed prima facie obvious over the cited references.
Applicants’ argument regarding claim 1, “as a whole [being] clearly directed to adding a second plurality of aqueous droplets into a channel in which a first set is formed,” this argument can be appreciated in two different ways.  The first is that the second plurality of droplets formed from the adjustment step is produced in the main channel while the first droplets are still present therein.  The second is that the second plurality of droplets formed from the adjustment step is produced in the same main channel where the first droplets were produced.  The second interpretation allows one to generate the first droplets, let the first droplets flow through, and make the recited adjustment of pressure, then flowing the new batch of droplets (i.e., second droplets) in the same channel.  The method derived from the combination of Cho and Nobile would meet the second interpretation.
Lastly, Applicants’ remark regarding “interdigitation” is not applicable to the present rejected claims.
The rejection is maintained therefore.
Rejection – Necessitated by Amendment
Claims 19-30 are rejected 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).
The teachings of Cho et al and Nobile et al. have already been discussed above.
With regard to claim 19, Nobile et al. teach at least a first inlet channel is in fluid communication with the main channel at a junction at which the main channel includes a nozzle where the first plurality of droplets are formed in the main channel (see below):
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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)2.
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.
With regard to claims 24, 26, and 27 the design above is repeated for the at least three inlets shown in Figure 2.
With regard to claim 28, the droplets introduced from the at least two inlets as shown in Figure 2 will result in an interdigitated formation in the main channel.
With regard to claim 25, Cho discloses coalescing of the droplets in a downstream location of their continuous flow through device so as to coalesce the droplets (see Figure 8, section [0116]).
With regard to claim 29, 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 30, 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.
Applicants are advised to consider the configuration of the device as depicted in Figure 3 where oil lines from two opposing sides are flowed toward each other and ejected through the nozzle where the inlet forms a junction.  However, doing so will necessitated new consideration of prior art and an RCE should be filed.
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, and 19-23 are pending, with claim 1 being the only independent claim. Claims 1, 4, 5, 12, 19, and 21 are amended. Support for the amendments may be found throughout parent U.S. application No. 11/803,104 as-filed, for example, at least at lines 9-12 on page 11; line 27 on page 3 through line 13 on page 4; lines 1-3 on page 9; line 36 on page 9 through line 7 on page 10; line 35 on page 39 through line 3 on page 40; lines 19-26 on page 70; line 8 on page 77 through line 12 on page 78 and in particular lines 32-34 on page 77 and lines 1-5 on page 78, as well as in Fig. 1 and Fig. 15, and Fig. 27 No new matter is added.
Rej
ections under 35 U.
S.
C. 103
The Office Action on page 3 states that claims 1-5 and 12-14 are rejected pre-AIA 35 U.S.C. §103(a) over U.S. Pub. 2005/0202489 to Cho in view of U.S. Pub. 2005/0227264 to Nobile. The Office Action on page 16 states that claims 19-30 are so rejected over Cho 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. One of the primers is annealed to one of the nucleic acid molecules within one of the droplets in the main channel.
Prior art fails to show mer
ging
reagent fluid and sample fluid on-chip
The subject matter of amended claim 1 is illustrated in Figs 27, 15, and 1 of the as-filed application. The application shows that amplification reagents and target/template can be separately input in their own separate aqueous fluids. Those fluids undergo coalescence and droplet formation on-chip to form droplets that include template nucleic acid and PCR reagents for amplification for DNA sequencing sample preparation. See Application as-filed, line 19 on page 70-line 19 on page 71 (Example 2) and line 7 on page 77 through line 12 on page 78 (Example 5).
Claim 1 cannot reasonably be said to be obvious over Nobile or Cho because those references do not teach or suggest any mechanism by which to join an aqueous fluid containing amplification reagents with an aqueous fluid containing target/template on-chip.
Cho reports that an aqueous solution containing PCR reactants and an immiscible carrier fluid are injected into a reaction vessel via different inlet channels to form droplets in which amplification is performed. Cho, [0018]. If there is more than one supply of aqueous fluid in respective inlet channels, those are to create distinct amplification droplets, such as for template, a negative control (NTC), and a standard. Id, [0086]. Separate aqueous fluids are never merged or coalesced.
A person of skill in the art reading Cho would only have understood to prepare the complete "reactant aqueous solution" off-chip . Cho, [0070]. Cho is explicit that the reactant aqueous solution that already contains the target DNA, primers, dNTPs, and polymerase is delivered onto a chip. Id., [0076]. Cho does not teach or suggest that the target DNA and any amplification reagents can be inserted into the chip and merged therein to form amplification nanoreactors. Failing teaching any such mechanism, Cho fails to establish any reasonable expectation of success for on-chip merge and droplet formation with distinct aqueous fluids.
Similarly, Nobile does not merge template aqueous fluid with reagent aqueous fluid on- chip.
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.
Both Nobile and Cho presuppose that template nucleic acid and amplification reagents are pre-mixed in some other off-chip aqueous volume that is delivered to the chip for droplet formation and amplification on-chip.
Referring to Fig. 27 of the as-filed application, it is clear that "+PRIMERS, dNTP" (i.e., amplification reagents) are brought in via one inlet while "+TEMPLATE FROM SAMPLE PLATE" (i.e., template nucleic acid) is brought in via another inlet channel. In fact, the present application reports that a nanoreactor is formed by coalescence of the sample & reagent aqueous fluids. Parent application as-filed, line 13 on page 4.
Only the present invention as-claimed provides that amplification reagents may be brought on chip in a first aqueous fluid and target nucleic acid may be brought on chip in distinct, second aqueous fluid. Nobile and Cho both fail to teach or suggest any on-chip merger of reagent and template. In fact, Nobile's only mention of droplet coalescence is as
a risk to be avoided
! Nobile, [0081]. Cho do not teach or suggest distinct aqueous fluids merging or coalescing on-chip.
Factual analysis of Cho and Nobile thus reveal that those references cannot reasonably be used to support a claim of obviousness of independent claim 1, as amended.
Because the only independent claim is not obvious over Cho and Nobile, 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: October 1, 2020
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 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

CITED REFERENCE SUMMARIES: Ref US20050202489A1: Claims:
A method for amplifying nucleic acids, which comprises: introducing into a reaction vessel via different inlet channels a reactant aqueous solution containing reactants for nucleic acid amplification and a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction; creating a plurality of reactant aqueous solution droplets surrounded by the fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel; and amplifying the nucleic acids in the reactant aqueous solution droplets.
introducing into a reaction vessel via different inlet channels a reactant aqueous solution containing reactants for nucleic acid amplification and a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction;
creating a plurality of reactant aqueous solution droplets surrounded by the fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel; and
amplifying the nucleic acids in the reactant aqueous solution droplets.
The method of claim 1 , wherein the fluid is at least one selected from the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, and vegetable oil.
The method of claim 1 , wherein the operation of amplifying the nucleic acids is performed by one selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and loop-mediated isothermal amplification (LAMP).
The method of claim 3 , wherein the operation of amplifying the nucleic acids is performed by PCR.
The method of claim 4 , wherein the operation of amplifying the nucleic acids is performed by PCR in which the entire surface of a substrate is repeatedly heated and cooled to a predetermined temperature.
The method of claim 4 , wherein the operation of amplifying the nucleic acids is performed by continuous-flow PCR in which a substrate is heated to have at least two different temperature areas and the reactant aqueous solution droplets alternately and repeatedly pass through the different temperature areas.
The method of claim 6 , wherein the substrate is heated to have at least two different temperature areas using a plurality of independently controllable heaters.
The method of claim 4 , wherein the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel further comprises introducing into the reaction vessel at least one aqueous solution of a negative control and a positive control for quantitative assay and a standard sample aqueous solution via respective inlet channels different from the inlet channels for the introduction of the reactant aqueous solution and the fluid, and the operation of creating the reactant aqueous solution droplets further comprises creating a plurality of control aqueous solution droplets and a plurality of standard sample aqueous solution droplets by contacting the at least one aqueous solution of the negative control and the positive control and the standard sample aqueous solution with the fluid in the reaction vessel.
the operation of creating the reactant aqueous solution droplets further comprises creating a plurality of control aqueous solution droplets and a plurality of standard sample aqueous solution droplets by contacting the at least one aqueous solution of the negative control and the positive control and the standard sample aqueous solution with the fluid in the reaction vessel.
The method of claim 8 , wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, the standard sample aqueous solution is adjusted to at least two concentrations and then introduced into the reaction vessel.
The method of claim 4 , wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, at least two reactant aqueous solutions containing at least two primers are introduced into the reaction vessel via respective inlet channels.
The method of claim 4 , wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, a nucleic acid aqueous solution, a common component containing polymerase and dNTP, and a primer are introduced into respective inlet channels and mixed before being introduced into the reaction vessel to form the reactant aqueous solution.
The method of claim 1 , wherein the reaction vessel comprises two primary reaction channels and a single secondary reaction channel connected to a terminal end of each of the two primary reaction channels, and the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, the operation of creating the reactant aqueous solution droplets, and the operation of amplifying the nucleic acids are performed in each of the two primary reaction channels, and wherein after the operation of amplifying the nucleic acids, the method further comprises: combining the reactant aqueous solution droplets in a starting end of the secondary reaction channel to create a plurality of combined reactant aqueous solution droplets in the secondary reaction channel; and amplifying the nucleic acids in the combined reactant aqueous solution droplets.
wherein after the operation of amplifying the nucleic acids, the method further comprises:
combining the reactant aqueous solution droplets in a starting end of the secondary reaction channel to create a plurality of combined reactant aqueous solution droplets in the secondary reaction channel; and
amplifying the nucleic acids in the combined reactant aqueous solution droplets.
The method of claim 1 , wherein the reaction vessel comprises at least one reaction chamber, and wherein after the operation of amplifying the nucleic acids in the reactant aqueous solution droplets, the method further comprises: combining the reactant aqueous solution droplets in the reaction chamber; and amplifying the nucleic acids in the combined reactant aqueous solution droplets.
wherein after the operation of amplifying the nucleic acids in the reactant aqueous solution droplets, the method further comprises:
combining the reactant aqueous solution droplets in the reaction chamber; and
The method of claim 13 , wherein the operation of combining the reactant aqueous solution droplets is carried out by centrifugation.
An apparatus for amplifying nucleic acids, which comprises: a substrate; a reaction vessel formed inside of the substrate; at least one first inlet channel formed inside the substrate, connected to an end of the reaction vessel, and allowing introduction of a reactant aqueous solution containing reactants for nucleic acid amplification into the reaction vessel; a second inlet channel formed inside the substrate, connected to the end of the reaction vessel, and allowing introduction of a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction into the reaction vessel; and a heating unit installed on the substrate and heating the substrate, the reactant aqueous solution contacting with the fluid to create a plurality of reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and the nucleic acids being amplified in the reactant aqueous solution droplets.
a reaction vessel formed inside of the substrate;
at least one first inlet channel formed inside the substrate, connected to an end of the reaction vessel, and allowing introduction of a reactant aqueous solution containing reactants for nucleic acid amplification into the reaction vessel;
a second inlet channel formed inside the substrate, connected to the end of the reaction vessel, and allowing introduction of a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction into the reaction vessel; and
a heating unit installed on the substrate and heating the substrate,
the reactant aqueous solution contacting with the fluid to create a plurality of reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and the nucleic acids being amplified in the reactant aqueous solution droplets.
The apparatus of claim 15 , wherein the fluid is at least one selected from the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, and vegetable oil.
The apparatus of claim 15 , wherein the substrate has a hydrophobic surface property.
The apparatus of claim 17 , wherein the substrate is made of one material selected from the group consisting of poly(dimethylsiloxane) (PDMS), silicon, silicon dioxide, plastic, and glass.
The apparatus of claim 15 , wherein the substrate comprises a lower substrate and an upper substrate, and the reaction vessel, the first inlet channel, and the second inlet channel are formed between the lower substrate and the upper substrate.
The apparatus of claim 15 , wherein the nucleic acids are amplified in the reactant aqueous solution droplets by one selected from the group consisting of PCR, LCR, SDA, NASBA, TMA, and LAMP.
The apparatus of claim 20 , wherein the nucleic acids are amplified by PCR in the reactant aqueous solution droplets.
The apparatus of claim 21 , wherein the reaction vessel is a serpentine reaction channel, the first inlet channel and the second inlet channel are connected to a staring end of the reaction channel, and an outlet port for releasing the fluid and an amplification product is connected to a terminal end of the reaction channel.
The apparatus of claim 22 , wherein the heating unit is installed at a lower surface of the substrate and comprises at least two heaters for heating the substrate to allow the substrate to have at least two different temperature areas.
The apparatus of claim 22 , wherein the heating unit comprises a plurality of independently controllable heaters installed in parallel at a lower surface of the substrate to heat the substrate so that the substrate has at least two temperature areas.
The apparatus of claim 23 , wherein the reaction channel alternately and repeatedly passes through the temperature areas.
The apparatus of claim 22 , wherein the second inlet channel linearly extends from the starting end of the reaction channel and the first inlet channel is formed perpendicularly to the second inlet channel.
The apparatus of claim 22 , further comprising: a third inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of at least one selected from a negative control aqueous solution and a positive control aqueous solution for quantitative assay into the reaction channel; and a fourth inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of a standard sample aqueous solution for quantitative assay into the reaction channel.
a third inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of at least one selected from a negative control aqueous solution and a positive control aqueous solution for quantitative assay into the reaction channel; and
a fourth inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of a standard sample aqueous solution for quantitative assay into the reaction channel.
The apparatus of claim 27 , wherein the fourth inlet channel is connected to at least two standard sample inlet channels for introduction of standard samples and a distilled water inlet channel for diluting the standard samples to a predetermined concentration.
The apparatus of claim 28 , wherein the fourth inlet channel has a serpentine shape.
The apparatus of claim 22 , wherein the apparatus comprises at least two first inlet channels and the first inlet channels are correspondingly connected to at least two primer inlet channels for introduction of at least two different primers into the first inlet channels.
The apparatus of claim 30 , wherein each of the first inlet channels has a serpentine shape.
The apparatus of claim 22 , wherein the first inlet channel is connected to a primer inlet channel for introduction of a primer into the first inlet channel and a common component inlet channel for introduction of a common component comprising polymerase and dNTP into the first inlet channel.
The apparatus of claim 32 , wherein a portion of a starting end side of the reaction channel has a serpentine shape.
The apparatus of claim 15 , wherein the reaction vessel comprises two serpentine primary reaction channels and a single serpentine secondary reaction channel connected to a terminal end of each of the two primary reaction channels, and the first inlet channel and the second inlet channel are connected to starting ends of the two primary reaction channels.
The apparatus of claim 34 , wherein the heating unit comprises a plurality of independently controllable heaters installed at a lower surface of the substrate to provide two temperature areas for each of the two primary reaction channels and the single secondary reaction channel.
The apparatus of claim 15 , wherein the reaction vessel is a reaction chamber, the first inlet channel and the second inlet channel are connected to an end of the reaction chamber, and an outlet channel for releasing the fluid and an amplification product is connected to the other end of the reaction chamber.
The apparatus of claim 36 , wherein the apparatus comprises a plurality of reaction chambers, first inlet channels, second inlet channels, and outlet channels.
The apparatus of claim 36 , wherein the heating unit comprises a single heater installed at a lower surface of the substrate and heating the entire surface of the substrate to a predetermined temperature.
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 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.
The apparatus for amplifying genetic material according to claim 10 , wherein downstream of the orifice, a length of the first conduit from a starting position to an ending position is arranged relative to at least the first and second temperature zones such that the length of the first conduit is exposed to alternating processes of heating and cooling at a temperature and time sufficient to amplify any of the nucleic acid templates by polymerase chain reaction.
The apparatus according to claim 10 , wherein the orifice is sized between 50 Î¼m to 300 Î¼m.
The apparatus according to claim 10 , wherein the orifice is sized to approximately 150 Î¼tm.
The apparatus according to claim 10 , wherein an intersection area of the orifice and the first conduit includes a diameter of between greater than 50 Î¼m to about 800 Î¼m.
The apparatus according to claim 10 , wherein an intersection area of the orifice and the first conduit includes a diameter of about 400 Î¼m.
The apparatus according to claim 10 , further comprising collecting means for collecting one or more of the amplified nucleic acid templates from the first conduit downstream of the heating and cooling sources.
The apparatus according to claim 16 , wherein the collecting means comprises a filter.
The apparatus according to claim 10 , wherein the first and second temperature zones are circumferentially arranged on opposed sides of a curved surface, wherein the length of the first conduit is helically wound around the curved surface to provide alternating portions of the length of first conduit adjacent either the first temperature zone or the second temperature zone.
The apparatus according to claim 18 , wherein the curved surface comprises a mandrel.
The apparatus according to claim 10 , wherein the temperature zones are arranged along opposed heating and cooling linear surfaces, respectively, wherein the length of firs
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