The technology of polymerase chain reaction has been a common and often indispensable technique in medical and biological studies and applications. Digital PCR (dPCR) allows quantification of DNA in a sample. dPCR is advantageous for reasons of accuracy (absolute titer quantification), sensitivity (single molecule detection), dynamic range, and robustness against inhibition. A mobile dPCR allows immediate quantification of samples, but the samples typically need to be purified before a dPCR can be conducted. In addition, a vast amount of samples may need to be tested, compared to typical lab settings.
Systems and methods of a portable continuous flow dPCR device that automates the entire analysis on a continuous flow of samples from a fluid are described herein.
The present disclosure provides systems and methods that perform digital droplet PCR analysis on a continuous fluid stream. The instrument draws in a sample of molecules, such as DNA in aqueous suspension, mixes and dilutes that sample with PCR mastermix, a diluent such as water, and one or more suitable PCR probes without disrupting flow of the fluid stream significantly. The resultant sample liquid is then broken into droplets that stochastically contain the target molecules. The droplets are then thermocycled to amplify their nucleic acid contents by PCR. In the end, the individual droplets are counted to determine the original starting concentration in the sample.
In accordance with one aspect of the disclosure, a system for continuous flow polymerase chain reaction (PCR) is provided. The system comprises an injector, a mixer a droplet generator, a detector, a digital PCR system, and a controller. The injector takes in a sample from a sample inlet and aliquots the sample into a volume necessary for a PCR reaction, dispenses waste, and hands off the sample aliquots separated by an immiscible oil phase to a mixer one aliquot at a time. The mixer takes in the sample aliquot, mixes it with the PCR master mix and diluting water, dispenses waste, and hands off the sample mixture to a coalescer in aliquots separated by an immiscible oil phase. The coalescer takes in the sample mixture, coalesces it with primers that are dispensed from the cassette, dispenses waste, and hands off the reaction mixture separated by an immiscible oil phase to the droplet generator. The droplet generator converts the sample mixture into an emulsion where aqueous droplets of the reaction mixture are maintained inside of an immiscible oil phase. The aqueous reaction droplets are then passed to the digital PCR system to enable amplification of target molecule (e.g., DNA) molecules in the droplets. Post amplification, a detector determines whether or not target molecule (e.g., DNA) amplification occurred for each of the droplets. The controller processes data outputted from the detector and controls the system so that the system runs automatically and continuously.
In another aspect of this disclosure, a method for continuous flow PCR is provided. First a sample of a fluid stream is taken in at a sample inlet and passed through an injector to produce sample aliquots, with each aliquot being separated by an immiscible oil phase. Each sample aliquot is mixed, e.g., using a mixer, with reagents such as PCR master mix, primers, probes, and diluting water to produce a sample mixture. The primers and/or probes may be PCR primers modified with fluorophores that bind to a target molecule, such as DNA. The reagents may come from a cassette or from reagent storage.
The foregoing and other advantages of the invention will appear from the following description. In the description, reference is made to the accompanying drawings, which form a part hereof, and in which there is shown by way of illustration a preferred embodiment of the invention. Such embodiment does not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and herein for interpreting the scope of the invention.
“Polymerase chain reaction” or “PCR” refers to a technology widely used in molecular biology to amplify a single copy or a few copies of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
The PCR technology uses reaction mixture that comprises DNA templates containing DNA to be amplified, primers, enzyme such as Taq polymerase, deoxynucleoside triphosphates (dNTPs)—the building-blocks from which the DNA polymerase synthesizes a new DNA strand, buffer that provides a suitable chemical environment for the amplifying process, and other chemicals. PCR master mix comprises those components except primers. Primers are short DNA fragments containing sequences complementary to the target region along with a DNA polymerase are used to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.
The PCR methods comprises placing the reaction mixture in a thermocycler and, in the thermocycler, undergoing a series of 20-40 repeated temperature changes—called cycles—with each cycle commonly consisting of 2-3 discrete temperature steps. The cycling is often preceded by a single temperature step at a high temperature (>90° C.)—also called hot start, and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time in each cycle depend on parameters, such as the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature of the primers.
Each cycle usually comprises three steps, melting (or denaturation), annealing, and extension (or elongation). In the melting step, the reaction mixture is heated to 94-98° C. for 20-30 seconds, causing melting of the DNA template to single-stranded DNA molecules by disrupting the hydrogen bonds between complementary bases.
In the annealing step, the reaction temperature is lowered to 50-65° C. for 20-40 seconds allowing annealing—combining—of the primers to the single-stranded DNA template. This temperature is low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should only bind to a perfectly complementary part of the template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation.
In the extension step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template.
Digital PCR follows the same principle and process as those of traditional PCR, except that, in digital PCR, a sample is partitioned into many small partitions such that individual nucleic acid templates of interest can be localized in individual partitions.
Referring to
Referring to
Referring to
Referring to
Referring to
Referring to
Specifically for real-time PCR, the thermocycler must have the ability to maintain a consistent temperature, as PCR amplification efficiency is dependent upon the temperature. Referring to
Referring to
The injector 302 can have multiple ports of different specific volumes (as shown in
Referring to
The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention. The appended document describes additional features of the present invention and is incorporated herein in its entirety by reference.
The present application is a U.S. National Phase Application under 35 U.S.C. § 371 of International Application No. PCT/US2016/040172 filed Jun. 29, 2016, which claims the benefit of U.S. Prov. Pat. App. Ser. No. 62/186,321, having the same title and filed Jun. 29, 2015, and which are incorporated fully herein by reference.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US2016/040172 | 6/29/2016 | WO | 00 |
Publishing Document | Publishing Date | Country | Kind |
---|---|---|---|
WO2017/004250 | 1/5/2017 | WO | A |
Number | Name | Date | Kind |
---|---|---|---|
6143496 | Brown et al. | Nov 2000 | A |
6767706 | Quake et al. | Jul 2004 | B2 |
6960437 | Enzelberger et al. | Nov 2005 | B2 |
6977145 | Fouillet et al. | Dec 2005 | B2 |
7129091 | Ismagilov et al. | Oct 2006 | B2 |
7268167 | Higuchi et al. | Sep 2007 | B2 |
7294503 | Quake et al. | Nov 2007 | B2 |
7622081 | Chou et al. | Nov 2009 | B2 |
7772287 | Higuchi et al. | Aug 2010 | B2 |
RE41780 | Anderson et al. | Sep 2010 | E |
RE43365 | Anderson et al. | May 2012 | E |
8278071 | Brown et al. | Oct 2012 | B2 |
8304193 | Ismagilov et al. | Nov 2012 | B2 |
8329407 | Ismagilov et al. | Dec 2012 | B2 |
8399198 | Hiddessen et al. | Mar 2013 | B2 |
8633015 | Ness et al. | Jan 2014 | B2 |
8709762 | Hindson | Apr 2014 | B2 |
8730479 | Ness et al. | May 2014 | B2 |
8771747 | O'Hagan et al. | Jul 2014 | B2 |
8822148 | Ismagliov et al. | Sep 2014 | B2 |
8841093 | Takahashi et al. | Sep 2014 | B2 |
8871444 | Griffiths et al. | Oct 2014 | B2 |
8889083 | Ismagilov et al. | Nov 2014 | B2 |
8951732 | Pollack et al. | Feb 2015 | B2 |
9012390 | Holtze et al. | Apr 2015 | B2 |
9029083 | Griffiths et al. | May 2015 | B2 |
9056289 | Weitz | Jun 2015 | B2 |
9074242 | Larson et al. | Jul 2015 | B2 |
9127310 | Larson et al. | Sep 2015 | B2 |
9132394 | Makarewicz, Jr. et al. | Sep 2015 | B2 |
9156010 | Colston et al. | Oct 2015 | B2 |
9181375 | Tian et al. | Nov 2015 | B2 |
9216392 | Hindson et al. | Dec 2015 | B2 |
9222115 | Marble et al. | Dec 2015 | B2 |
9243288 | Ness et al. | Jan 2016 | B2 |
9248417 | Hindson et al. | Feb 2016 | B2 |
9273308 | Link et al. | Mar 2016 | B2 |
9366632 | Link et al. | Jun 2016 | B2 |
9441266 | Larson et al. | Sep 2016 | B2 |
9492797 | Makarewicz et al. | Nov 2016 | B2 |
9498761 | Holtze et al. | Nov 2016 | B2 |
RE46322 | Anderson et al. | Feb 2017 | E |
9562837 | Link | Feb 2017 | B2 |
9597026 | Meldrum et al. | Mar 2017 | B2 |
9752141 | Link et al. | Sep 2017 | B2 |
9968933 | Ismagilov et al. | May 2018 | B2 |
20050227264 | Nobile | Oct 2005 | A1 |
20060257893 | Takahashi | Nov 2006 | A1 |
20080014589 | Link et al. | Jan 2008 | A1 |
20120115738 | Zhou et al. | May 2012 | A1 |
20120231533 | Holl et al. | Sep 2012 | A1 |
20120301913 | Youngbull et al. | Nov 2012 | A1 |
20120302448 | Hutchison et al. | Nov 2012 | A1 |
20140045712 | Link et al. | Feb 2014 | A1 |
20140193800 | Aguanno et al. | Jul 2014 | A1 |
20140199731 | Agresti et al. | Jul 2014 | A1 |
20140202546 | Ismagilov et al. | Jul 2014 | A1 |
20140208832 | Hansen et al. | Jul 2014 | A1 |
20150018236 | Green et al. | Jan 2015 | A1 |
20160177375 | Abate et al. | Jun 2016 | A1 |
Number | Date | Country |
---|---|---|
1574586 | Nov 2012 | EP |
1735458 | Jul 2013 | EP |
WO-2010022391 | Feb 2010 | WO |
WO-2010036352 | Apr 2010 | WO |
WO-2010062654 | Jun 2010 | WO |
WO-2012112440 | Aug 2012 | WO |
WO-2013165748 | Nov 2013 | WO |
WO-2014008381 | Jan 2014 | WO |
WO-2014210207 | Dec 2014 | WO |
WO-2017004250 | Jan 2017 | WO |
WO-2018098438 | May 2018 | WO |
Entry |
---|
Chabert et al, “Droplet fusion by alternating current (AC) field electrocoalescence in microchannels”, 2005, Electrophoresis, pp. 3706-3715. (Year: 2005). |
Hatch, Andrew et al. Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection. Lab on a Chip, 14(3):562-568 (Nov. 19, 2013). |
International Application No. PCT/US2017/063293 International Search Report and Written Opinion dated Jan. 18, 2018. |
Tathagata, Ray et al. Low Power, High Throughput Continuous Flow PCR Instruments for Environmental Applications. (Retrieved from the Internet: Jan. 10, 2018) Dec. 1, 2013, pp. 1-181. |
Eow, et al. Electrostatic Enhancement of Coalescence of Water Droplets in Oil: a Review of the Current Understanding. Chemical engineering Journal 84 (3):173-192 (Dec. 15, 2001). |
International Application No. PCT/US16/40172 International Preliminary Report on Patentability dated Jan. 2, 2018. |
International Application No. PCT/US2016/040172 International Search Report and Written Opinion dated Oct. 20, 2016. |
Mazutis, et al. Single-Cell Analysis and Sorting Using Droplet-Based Microfluidics. Nat Protoc. 8(5): 870-891 (May 2013). |
U.S. Appl. No. 16/413,416 Non-Final Office Action dated Dec. 22, 2020. |
Number | Date | Country | |
---|---|---|---|
20190210027 A1 | Jul 2019 | US |
Number | Date | Country | |
---|---|---|---|
62186321 | Jun 2015 | US |