Patent Application
20240278249
This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on Aug. 14, 2023, is named RICEP0022USC1.xml and is 86,220 bytes in size.
The disclosure describes novel reagents, instruments, and methods for detection and quantitation of specific nucleic acid sequences for scientific and clinical research and diagnostics applications.
Most commercial nucleic acid (NA) assays require the use of enzymes for molecular or signal amplification. Enzymes such as DNA polymerase have been optimized to be fast and specific. Reconstitution of lyophilized enzymes in resource-limited conditions reduces the need for a cold chain. Isothermal nucleic acid amplification assays such as NEAR, LAMP and NASBA enable DNA/RNA profiling without complex temperature cycling equipment. Despite these many advances, existing nucleic acid detection technologies still face challenges for rapid PoC (point of care) detection of pathogen biomarkers, because it is difficult to design/evolve enzymes that simultaneously capture all desirable properties (e.g., fast, high fidelity, and robust to chemicals/inhibitors).
A number of existing nucleic acid analysis technologies are enzyme-free, including microarrays, fluorescence in situ hybridization (FISH), branched DNA dendrimers (Panomics), and fluorescent barcoding (Nanostring). In these approaches, the DNA or RNA target molecules stoichiometrically recruit or are converted into a limited number of fluorescent groups. This is unlike PCR where even a single nucleic acid molecule is amplified endlessly to produce an arbitrarily high number of amplicon molecules. Consequently, expensive and bulky equipment is needed for these approaches to achieve the molecular sensitivity needed to detect and analyze the small amounts of DNA or RNA target present in biological samples, restricting their use for PoC applications and in the limited resources conditions.
Another group of nucleic acid identification techniques employs solution-based enzyme-free DNA amplification approaches. Here a single-stranded DNA target molecule can catalytically release DNA oligonucleotides with identical sequence to the target from the preassembled DNA detection complexes in unlimited manner. Clinically relevant limits of detection have yet to be demonstrated for this family of approaches. In these systems, there is false positive amplification due to DNA “breathing” events that result in release of amplicon molecules in the absence of the detection of a target sequence.
Thus, in accordance with the present disclosure, there is provided a device comprising a surface having a plurality oligonucleotide complexes, wherein said oligonucleotide complexes each comprise:
Each of the first DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 5 and about 80 nucleotides, or a length of between about 15 and about 80 nucleotides. Each of the fifth DNA sequences may have a length of between about 5 and about 20 nucleotides. Each of the fourth DNA sequences may have a length of between about 5 and about 80 nucleotides.
In another embodiment, there is provided a fluidic reaction chamber comprising:
Each of the first DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the fifth DNA sequences may have a length of between about 5 and about 20 nucleotides. Each of the fourth DNA sequences may have a length of between about 5 and about 80 nucleotides. The materials contacting first and second surfaces that form the inner and outer boundaries of the chamber may have thickness between 40 microns (40 μm) and 2 millimeters (2 mm).
The fluidic reaction chamber may be circular, oval, square, rectangular, triangular, hexagonal, octagonal, rhomboid or trapezoid, or annular as defined herein. The fluidic reaction chamber may not be at a uniform temperature, and the warmest region of the reaction chamber may be at least 10° C. higher than the coldest region of the reaction chamber. The coldest region of the reaction chamber may be between about 50° C. and about 75° C. The hottest region of the reaction chamber may be between about 80° C. and about 100° C. The fluidic reaction chamber may further comprise a fluid disposed within the fluidic reaction chamber, said fluid solution comprising a DNA polymerase, dNTPs, and PCR buffer.
In yet another embodiment, there is provided a method of amplifying a target nucleic acid comprising (a) providing a fluidic reaction chamber according to claim 16, wherein said fluidic reaction chamber is in operable relationship to a first and a second heat source, wherein said first and second heat sources are capable of applying differing first and a second heat levels to said annular chamber, wherein said first and second heat levels are not the same; (b) introducing into said fluidic reaction chamber a fluid comprising a target nucleic acid sequence, a DNA polymerase, dNTPs and a polymerase chain reaction (PCR) buffer; and (c) applying first and second heat levels to said fluidic reaction chamber. The method may further comprise detecting amplification of said target nucleic acid.
Each of said second DNA sequences may be identical or may not be identical. The plurality of said oligonucleotide complexes may be located in spatially discrete regions on said surface. Each of said first oligonucleotides may comprise a fluorescent moiety, and each of said second oligonucleotides may comprise a fluorescence quencher moiety. Each of said spatially discrete regions may further comprise a third oligonucleotide comprising a fourth DNA sequence and a fifth DNA sequence, wherein the fourth DNA sequence is complementary to the third DNA sequence. The third oligonucleotides may each comprise a fluorescence quencher moiety. The linking moiety may comprise an alkyne group including a strained alkyne or an azide group.
The first DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the fifth DNA sequences have a length of between about 5 and about 20 nucleotides. The fluidic reaction chamber may be circular, oval, square, triangular, rectangular, hexagonal, octagonal, rhomboid or trapezoid.
The fluidic reaction chamber may not at a uniform temperature, and the warmest region of the reaction chamber may be at least 10° C. higher than the coldest region of the reaction chamber. The coldest region of the reaction chamber may be between about 50° C. and about 75° C. The hottest region of the reaction chamber may be between about 80° C. and about 100° C.
In yet a further embodiment, there is provided a device comprising a first surface region and a second surface region,
Each of the first DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the fifth DNA sequences may have a length of between about 5 and about 20 nucleotides. Each of the fourth DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the sixth DNA sequences may have a length of between 5 and 80 nucleotides.
In still a further embodiment, there is provided a fluidic reaction chamber comprising:
Each of the first DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the fifth DNA sequences may have a length of between about 5 and about 20 nucleotides. Each of the fourth DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the sixth DNA sequences may have a length of between about 5 and about 80 nucleotides.
The fluidic reaction chamber may be circular, oval, square, triangular, rectangular, hexagonal, octagonal, rhomboid or trapezoid, or annular as defined herein. The fluidic reaction chamber may not be at a uniform temperature, and the warmest region of the reaction chamber may be at least 10° C. higher than the coldest region of the reaction chamber. The coldest region of the reaction chamber may be between about 10° C. and about 50° C. The hottest region of the reaction chamber may be between about 51° C. and about 100° C. The fluidic reaction chamber may further comprise a fluid disposed within the fluidic reaction chamber, said fluid comprising one or more oligonucleotides and hybridization buffer, and the fluid may further comprise a non-specific nucleic acid staining dye.
Yet an additional embodiment comprises a method of amplifying a target nucleic acid comprising (a) providing a fluidic reaction chamber according to claim 71, wherein said fluidic reaction chamber is in operable relationship to a first and a second heat source, wherein said first and second heat sources are capable of applying differing first and a second heat levels to said annular chamber, wherein said first and second heat levels are not the same; (b) introducing into said fluidic reaction chamber a fluid comprising a target nucleic acid sequence; and (c) applying first and second heat levels to said fluidic reaction chamber.
The method may further comprise detecting amplification of said target nucleic acid. The first or second oligonucleotide may comprise a fluorescent moiety, and the first or second oligonucleotide may comprise a fluorescence quencher. Each of said second DNA sequences may be identical or may not be identical. Each of said plurality of said oligonucleotide complexes are located in spatially discrete regions on said surface. The linking moiety may comprise an alkyne group including a strained alkyne or an azide group.
Each of the first DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the third DNA sequences may have a length of between about 15 and about 80 nucleotides. Each of the fifth DNA sequences may have a length of between about 5 and about 20 nucleotides. Each of the fourth DNA sequences may have a length of between about 5 and about 80 nucleotides. Each of the sixth DNA sequences may have a length of between about 5 and about 80 nucleotides.
The fluidic reaction chamber may be circular, oval, square, rectangular, triangular, hexagonal, octagonal, rhomboid or trapezoid, or annular as defined herein. The fluidic reaction chamber may not be at a uniform temperature, and the warmest region of the reaction chamber may be at least 10° C. higher than the coldest region of the reaction chamber. The coldest region of the reaction chamber may be between about 10° C. and about 50° C. The hottest region of the reaction chamber may be between about 51° C. and about 100° C. The fluid further comprises a non-specific nucleic acid staining dye.
Another embodiment comprises a system comprising a reaction chamber, comprising (a) a first region and second region, wherein a first oligonucleotide comprising a first nucleotide sequence is functionalized to the first surface region and a second oligonucleotide comprising a second nucleotide sequence is functionalized to the second surface region, and wherein the first nucleotide sequence and the second nucleotide sequence are not identical; (b) a buffer solution amenable for DNA hybridization at a non-uniform temperature, wherein the buffer solution contacts the first surface region and the second surface region; (c) a third oligonucleotide comprising a third nucleotide sequence, wherein the third oligonucleotide is hybridized to the first oligonucleotide; and (d) a first temperature zone and a second temperature zone, wherein the first temperature zone has a temperature at least 10° C. greater than a temperature of the second temperature zone.
The first surface region may be located on a first surface and the second surface region is located on the first surface. The first surface region may be located on a first surface and the second surface region is located on a second surface, wherein the first surface and the second surface are different surfaces. The first nucleotide sequence may comprise a first nucleotide region that is not complementary to the third nucleotide sequence. The third nucleotide sequence may comprise a second nucleotide region that is not complementary to first nucleotide sequence.
The buffer solution may comprise at least 60% by mass water and a cation at a concentration of at least 1 mM. The length of the first oligonucleotide and the length of the second oligonucleotide may be between 5 nucleotides and 20,000 nucleotides. The first oligonucleotide and the length of the second nucleotide may be between 5 nucleotides and 200 nucleotides. The length of the third oligonucleotide may be between 5 nucleotides and 20,000 nucleotides. The first oligonucleotide, the second oligonucleotide and the third oligonucleotide may be identical or differently and may comprise a nucleic acid selected from the group consisting of DNA, RNA, a nucleotide analog, and any combination thereof.
The nucleotide analog may be selected from the group consisting of LNA, PNA a morpholino-oligonucleotide, and any combination thereof. At least one of the first oligonucleotide, the second oligonucleotide and the third oligonucleotide may be functionalized with a chemical moiety, wherein the chemical moiety allows detection of oligonucleotides. The chemical moiety may be selected from the group consisting of TAMRA, ROX, HEX, an organic fluorophore, a quantum dot, a nanoparticle, methylene blue, an electrochemically active molecule, and any combination thereof.
The buffer solution may comprise a detectable molecule, wherein the detectable molecule exhibits a different unit signal when non-irreversibly bound to an oligonucleotide than when free in solution, such as detectable molecule selected from the group consisting of a SybrGreen dye, a Syto dye, and a EvaGreen dye. The first surface and the second surface may be identical or differently selected from the group consisting of glass, quartz, plastic, a polymer, metal, composite material, and surface self-assembled monolayers. The polymer may be PDMS.
The first surface region may comprise a temperature that is 10° C. below a maximum temperature of the buffer solution, and wherein the second surface region may comprise a temperature that is 10° C. below the maximum temperature of the buffer solution. The system may further comprise at least one heating/cooling element in contact with the first surface region and the second surface region. The at least one heating/cooling element may be selected from the group consisting of a hot plate, a heating fan, an IR-heater, and a water bath. The hot plate may be selected from the group consisting of a thermo-resistive heater and a Peltier element. The system may further comprise an enzyme that modifies nucleic acids in a template-directed manner, such as where the enzyme facilitates template-directed extension of a nucleic acid template.
A method for enzyme-free amplification and detection of a nucleic acid target, comprising (a) contacting a sample with a composition in a reaction chamber, wherein the composition comprises:
The composition may be localized in the reaction chamber, and wherein the first surface region is located on first surface and the second surface region is located the first surface. The composition may be localized in the reaction chamber, wherein the first surface region may be located on a first surface and the second surface region may be located on a second surface, and wherein the first surface and the second surface are different surfaces. Detecting potential amplification may further comprise optical detection of fluorescence changes through a detection device selected from the group consisting of a photodiode, a photomultiplier tube, a fluorescence microscope, a CCD camera, and any other optical detection device. Detecting potential amplification may further comprise electrochemical detection through an electrochemical potentiostat/galvanostat. Detecting potential amplification may further comprise measuring the mass of the first surface region using quartz crystal microbalance technique.
In still a further embodiment, there is provided a method for enzyme-dependent amplification and detection of a nucleic acid target, comprising (a) contacting a sample with a composition in a reaction chamber, wherein the composition comprises:
The maximum temperature of a region of the chamber may be between 80° C. and 100° C. The minimum temperature of the chamber may be between 20° C. and 80° C. The surface region may not be heated to within 10° C. of the maximum temperature of a region of the chamber. Detecting potential amplification may further comprise optical detection of fluorescence changes through a detection device selected from the group consisting of a photodiode, a photomultiplier tube, a fluorescence microscope, a CCD camera, and any other optical detection device. Detecting potential amplification may further comprise electrochemical detection through an electrochemical potentiostat/galvanostat. Detecting potential amplification may further comprise measuring the mass of the first surface region using quartz crystal microbalance technique.
In one embodiment of the present disclosure, surface oligonucleotide functionalization can be performed by various chemical and physical methods including but not limited to covalent immobilization, electrostatic interaction or non-covalent immobilization such as biotin-avidin (or their analogs).
In one embodiment of the present disclosure, the detection target molecule may be single-stranded DNA, double-stranded DNA, RNA or their mixtures.
In one embodiment of the present disclosure, amplicon detection and reaction monitoring is through methods including but not limited to: fluorescence (including surface plasmon resonance, SPR), UV absorbance, electrochemical detection (including pH change and charge transfer), quartz crystal microbalance (QCM) In one embodiment of the present disclosure, the proposed approaches can be used for distinguishing nucleic acid sequence variants, including single nucleotide variants (SNVs) that may be indicative of drug resistance or disease prognosis.
In one embodiment of the present disclosure, the proposed approaches can be used for quantitation of one, several, or many target molecules in specific biological samples.
As used herein, the term “annular” may be used to reference the shape of the chamber, as discussed above, and may have its normal meaning of round, oval or discoid. However, annular may also be interpreted in this context to have other regular or irregular shapes so long as the chamber constitutes a continuous circuit with no end and no beginning.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, for the method being employed to determine the value, or that exists among the study subjects. Such an inherent variation may be a variation of ±10% of the stated value.
Throughout this application, the term “irreversible linking” is used to indicate a chemical interaction that is stable under usual circumstances of the intended application. Irreversible linking in some embodiments can refer to covalent attachment such as by azide-alkyne click chemistry, and in other embodiments can refer to biotin-avidin interactions or other non-covalent long-lived interactions.
Throughout this application, the term “PCR buffer” is used to indicate an aqueous solution with salinity and chemical composition compatible with DNA amplification by a DNA polymerase via the polymerase chain reaction (PCR). The buffer may be used in conjunction with the DNA polymerase itself, primers and/or dNTPS.
Throughput this application, the term “hybridization buffer” is used to indicate an aqueous solution with salinity and chemical composition compatible with DNA hybridization and formation of stable DNA duplexes by complementary DNA oligonucleotides. All PCR buffers can be considered hybridization buffers, but not vice versa.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Here, the inventors present devices, systems, and methods for DNA amplification assay. The disclosure employs solid-phase separation of reagents to prevent unintended molecular events resulting in false positives, and uses convection flow circulation to enable spontaneous dissociation of double-stranded amplicons. Three related prior art technologies and their limitations compared to the present invention are described below.
Liquid, when held at a non-uniform temperature and confined in a volume, will circulate via a process known as Rayleigh-Benard convection flow [1]. Rayleigh-Benard convection has been used for molecular diagnostics to generate low-cost devices for providing the necessary temperature cycling for PCR (convection flow PCR, cf-PCR) [2, U.S. Pat. No. 6,586,233 B2, U.S. Pat. No. 8,735,103 B2, U.S. Pat. No. 8,187,813 B2]. cf-PCR requires only a static temperature gradient maintained with a high of around 95° C. and a low of around 60° C. (annealing/extension temperature), eliminating the need for high energy consumption thermal cycling instruments.
cf-PCR has been demonstrated for both single-plex [3-5, U.S. Pat. No. 8,187,813 B2] and multiplex detection of specific DNA sequences [6]; the multiplex approach utilized end-point electrophoretic results examination. Because cf-PCR lacks the temperature uniformity of traditional qPCR assays, cf-PCR struggles in applications requiring high sequence selectivity, such as applications for detection or profiling of single nucleotide variants (SNV), therefore no SNV specific cf-PCR has yet been shown. Real-time detection of the cf-PCR has been shown solely in solution phase employing unspecific fluorescent dye (SYBR Green I) detection method [7]. This approach restricts the cf-PCR from being used in multiplex settings. Likewise, application of sequence specific real-time detection methods such as 5′-nuclease assay chemistry or hybridization probes would allow detecting not more than 5-6 targets simultaneously because of fluorophore spectral overlap. The present disclosure is differentiated from cf-PCR in that the present disclosure offers spatially resolved multiplexed readout without requiring an open-tube step for subsequent analysis. Additionally, in the enzyme-free embodiment of the disclosure, no enzyme is required for amplification.
Microarray technology is one of the main techniques for multiplexed screening of biological samples. Multiple probe sequences are functionalized to a surface, and the fluorescent signal of a particular spot is taken as the quantitative readout of the corresponding sequence. The technology has been successfully demonstrated for detecting of various types of biological analytes such as DNA, RNA, proteins, carbohydrates and cells [8-12]. Application of the microarray technology has found the most extensive use in the field of nucleic acid testing. Microarray technology has shown application of NA microarrays for whole genome hybridization, de novo sequencing, re-sequencing, comparative genomics, transcriptome hybridization or identification of single nucleotide variations [13-15]. All aforementioned NA applications require large amounts of NA targets for hybridization, consequently microarrays are typically used as a final readout on PCR amplification products. Microarray readouts are typically slow, requiring overnight hybridization, and also risks amplicon contamination due to the open-tube process.
Toehold-mediated strand displacement reaction [19-21] is a process of competitive hybridization that occurs in the absence of enzymes, and is relevant to the present disclosure. Using toehold-mediated strand displacement, enzyme-free amplification of DNA and RNA analyte sequences in homogeneous solutions has been demonstrated [22-27] (U.S. Pat. No. 8,043,810 B2, U.S. Pat. No. 8,110,353 B2). The enzyme-free amplification embodiment of the disclosure is different in that thermal convection flow is used to spontaneously dissociate double-stranded amplicons, and surface-functionalization is used to sequester reactive reagents from one another to reduce false positives. Toehold-mediated strand displacement has been applied to surface functionalized DNA oligonucleotides (U.S. Pat. No. 8,630,809 B2) for stoichiometric conversion of target analyte sequences to other sequences. The present disclosure differs in providing amplification of the detection target.
This disclosure describes reagents and devices for amplification and detection of specific nucleic acid target sequences. The disclosure utilizes solid-phase functionalization and sequestering of oligonucleotide reagents, in order to prevent unintended molecular events that result in false positives, and application of Rayleigh-Benard thermal convection flow for target regeneration and facilitating DNA surface hybridization kinetics (more efficient mixing of the reaction mixture). The Rayleigh-Benard convection flow regime can be realized by placing a reaction chamber, which consists of two 1 mm thick white-water glass microscope slides separated by double-sided sticky tape as a spacer with thickness of 250 μm, between two differentially-controlled hot plates (
The hot plates are set to maintain two different temperatures (cold heater and hot heater, respectively), which cause a temperature gradient across the reaction chamber filled with a liquid reaction mixture. Liquid residing near the hot part of the chamber has a higher temperature and, therefore, is less dense than the liquid residing in the part of the chamber with lower temperature. Such distribution of liquid densities in confined volume results in a difference between buoyancy and gravity forces (near the hot and cold heaters, respectively) that in turn results in organization of circular steady-state convective flow.
All molecules dissolved in the liquid are involved in circulation between temperature zones by being dragged by the convection flow. Traveling along temperature zones the molecules experience periodic temperature variations. For example, a double stranded DNA molecule being placed in the circular convection flow experience multiple cycles of heating and cooling. If the temperature of solution in the hot zone is sufficient to melt the DNA duplex and the temperature of the cold zone is favorable to maintain given nucleic acids in a double-stranded form then the circulation of nucleic acids in this convection flow results in repeatable denaturation and annealing cycles. Observation of the multiple cycles of ds-DNA denaturation and annealing can be performed by various methods, for example using fluorescent microscopy by registering the intensity of the non-specific DNA staining dyes placed in the reaction mixture along with the DNA sample.
A prototype heating instrument consists of two resistive Kapton foil heaters glued to the aluminum plates, and can be simultaneously used to provide differential heating for up to five fluidic chips. Two low-wattage power supplies power the heaters. The proposed amplification system is tolerant to heating element temperature inaccuracies in range of ±2° C., and does not require precise computer controlled hardware.
The present disclosure represents an enzyme-free amplification of target nucleic acid, in which amplicon concentration increases linearly with time (
The linear amplification scheme demonstrates the benefits of simultaneously using solid-phase sequestering of oligonucleotide reactants and the temperature-driven convection flow. Immobilization of the oligonucleotide reagents on the different surface regions allows avoiding false positive signal molecule release (spurious amplification) in the absence of the target sequence, while the thermal convection flow, beside spontaneous transport and improved mass transfer, induces target regeneration via melting of the target-signal complex (T-S). In contrast, changing the temperature of the entire solution is undesirable because it would lead to spontaneous dissociation of all oligonucleotides from the surface.
Labeling of the signal strand S with a fluorescent dye represent one approach for real-time monitoring of the linear amplification process. A decrease of the intensity of fluorescence registered form the of the surface region 1, as well as an increase of the intensity of fluorescence registered from the surface region 2 can effectively reflects how the reaction amplification reaction proceeds.
To demonstrate that the enzyme-free amplification method exhibits multiple turnover, the inventors constructed a corresponding stoichiometric detection system using the convection device (
Real-time observation of the stoichiometric detection system can also be performed via simple labeling of the signal strand Ss with a fluorescent dye and registering the change surface region 2 fluorescence intensity.
Convection flow carries the duplex to the 85° C. zone where the duplex melts. Now two single-stranded oligonucleotides, the initial target T and released strand S1 flows back to the 35° C. zone where each of them triggers new release of oligonucleotide species from the surface. In particular, target oligonucleotide T catalytically releases second strand S1 from the surface region 1, while the initially released strand S1 triggers the release of the strand S2 from the second surface region. Thus, at the end of each convection flow cycle the amount of oligonucleotide species present in solution doubles, resulting in exponential accumulation of the amplicon species in solution phase.
Application of the FRET-based detection technique is illustrated on the example of exponential amplification (
Another application the composition claimed in the present disclosure is that the composition can be used as an efficient mean for surface-based real-time monitoring of an enzymatic nucleic acid amplification process proceeding in the solution. There are no reported examples of real-time monitoring of convection-based PCR using surface functionalized probes.
The proposed compositions and methods in this disclosure allow for simultaneous monitoring of the amplification (either enzyme-free or enzyme-based) of multiple nucleic acid target sequences. Spatial patterning of different oligonucleotide probes at different surface regions allows an array- or camera-based readout to provide independent information on the amplicon concentrations of each target amplification reaction.
The following oligonucleotide sequences are provided by way of example, but not limitation:
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application is a continuation of U.S. application Ser. No. 16/089,496, filed Sep. 28, 2018, which is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2017/024530, filed Mar. 28, 2017, which claims benefit of priority to U.S. Provisional Application No. 62/314,909, filed Mar. 29, 2016, the entire contents of each of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62314909 | Mar 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16089496 | Sep 2018 | US |
| Child | 18449422 | US |
