SURFACE-BASED DETECTION OF NUCLEIC ACID IN A CONVECTION FLOW FLUIDIC DEVICE

Abstract

The present disclosure provides methods, composition and devices for performing convection-based PCR and non-enzymatic amplification of nucleic acid sequences. Techniques and reagents employed in these methods include toehold probes, strand displacement reactions, Rayleigh-Benard convection, temperature gradients, multiplexed amplification, multiplexed detection, and DNA functionalization, in open and closed systems, for use in nucleic tests and assays.

Claims

1-110. (canceled)

111. A device comprising a surface having a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete regions of the said surface and each comprise: a first oligonucleotide comprising a first DNA sequence and a linking moiety for irreversibly linking the first oligonucleotide to the surface, and a second oligonucleotide comprising a second DNA sequence and a third DNA sequence, wherein the second DNA sequence is complementary to the first DNA sequence and is hybridized thereto, wherein said second DNA oligonucleotide does not comprise a fluorescent moiety and not irreversibly linked to the surface.

112. The device of claim 111, wherein each of said second DNA sequences are not identical.

113. The device of claim 111, wherein each of said second DNA sequences not identical.

114. The device of claim 111, wherein each of said first oligonucleotides comprise a fluorescent moiety.

115. The device of claim 111, wherein said each of said second oligonucleotides comprise a fluorescence quencher.

116. The device of claim 112, wherein each of said spatially discrete regions further comprises 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.

117. The device of claim 116, wherein the third oligonucleotides each comprise a fluorescence quencher moiety.

118. The device of claim 111, wherein each of the said oligonucleotides have a length of between about 5 and about 120 nucleotides.

119. A fluidic reaction chamber comprising: a first surface, a second surface that does not contact the first surface, wherein said first and second surfaces face each other, a material contacting the first surface and the second surface and that forms an outer boundary of said reaction chamber, and a material contacting the first surface and the second surface and that forms and inner boundary of said reaction chamber, wherein the first surface comprises a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete regions of the first surface and each comprise: a first oligonucleotide comprising a first DNA sequence and a linking moiety for irreversibly linking the first oligonucleotide to the surface, and a second oligonucleotide comprising a second DNA sequence and a third DNA sequence, wherein the second DNA sequence is complementary to the first DNA sequence and is hybridized thereto, wherein said second DNA oligonucleotide is not irreversibly linked to the first surface, and optionally does not comprise a fluorescent moiety.

120. The fluidic reaction chamber of claim 119, wherein each of said second DNA sequences are not identical.

121. The fluidic reaction chamber of claim 119, wherein each of said second DNA sequences not identical.

122. The fluidic reaction chamber of claim 119, wherein each of said first oligonucleotides comprise a fluorescent moiety.

123. The fluidic reaction chamber of claim 119, wherein said each of said second oligonucleotides comprise a fluorescence quencher moiety.

124. The fluidic reaction chamber of claim 120, wherein each of said spatially discrete regions further comprises 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.

125. The fluidic reaction chamber of claim 124, wherein the third oligonucleotides each comprise a fluorescence quencher moiety.

126. The fluidic reaction chamber of claim 119, wherein each of the said oligonucleotides have a length of between about 5 and about 120 nucleotides.

127. The fluidic reaction chamber of claim 119, wherein the materials contacting first and second surfaces and forming the inner and outer boundaries of the chamber have shape of circle, oval, square, rectangle, triangle, hexagon, octagon, rhombus or trapeze, and provide distance between said first and second surfaces of between about 40 microns (40 m) and about 2 millimeters (2 mm).

128. The fluidic reaction chamber of claim 119, wherein the fluidic reaction chamber is not at a uniform temperature, and wherein the warmest region of the reaction chamber is between about 80 C. and about 100 C., and the coldest region of the reaction chamber is between about 45 C. and about 75 C.

129. The fluidic reaction chamber of claim 119, further comprising a fluid disposed within the fluidic reaction chamber, said fluid solution comprising a DNA polymerase, dNTPs, and PCR buffer.

130. A method of amplifying a target nucleic acid comprising (a) providing a fluidic reaction chamber according to claim 119, 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.

131. The method of claim 130, further comprising detecting amplification of said target nucleic acid.

132. A device comprising a first surface region and a second surface region, the first surface region comprising a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the first surface region and each comprise: a first oligonucleotide comprising a first DNA sequence and a linking moiety for irreversibly linking the first oligonucleotide to the first surface region, and a second oligonucleotide comprising a second DNA sequence and a third DNA sequence, wherein the second sequence is complementary to the first sequence, and the second surface region comprising a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the second surface region and each comprise: a third oligonucleotide comprising a fourth DNA sequence and a linking moiety for irreversibly linking the third oligonucleotide to the second surface region, and wherein the fourth sequence is complementary to the second sequence, the third sequence, or a combination of at least six continuous nucleotides of the second sequence and six continuous nucleotides of the third sequence.

133. A device comprising a first surface region and a second surface region, the first surface region comprising a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the first surface region and each comprise: a first oligonucleotide comprising a first DNA sequence and a linking moiety for irreversibly linking the first oligonucleotide to the first surface region, and a second oligonucleotide comprising a second DNA sequence and a third DNA sequence, wherein the second sequence is complementary to the first sequence, and the second surface region comprising a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the second surface region and each comprise: a third oligonucleotide comprising a fourth DNA sequence and a linking moiety for irreversibly linking the third oligonucleotide to the second surface region, and a fourth oligonucleotide comprising a fifth DNA sequence and a sixth DNA sequence, wherein the fifth sequence is complementary to the fourth sequence, and wherein the second sequence is complementary to the fifth sequence or is complementary to the sixth sequence.

134. The device of claim 132, wherein the first or second oligonucleotide comprises a fluorescent moiety.

135. The device of claim 132, wherein the first or second oligonucleotide comprises a fluorescence quencher.

136. The device of claim 132, wherein each of said second DNA sequences are identical.

137. The device of claim 132, wherein each of said second DNA sequences are not identical.

138. The device of claim 132, wherein each of said oligonucleotides have a length of between about 5 and about 120 nucleotides.

139. A fluidic reaction chamber comprising: a first surface, a second surface that does not contact the first surface, wherein said first and second surfaces face each other, a material contacting the first surface and the second surface and that forms an outer boundary of said reaction chamber, and a material contacting the first surface and the second surface and that forms and inner boundary of said reaction chamber, wherein (a) the first surface comprises a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the first surface region and each comprise: a first oligonucleotide comprising a first DNA sequence and a linking moiety for irreversibly linking the first oligonucleotide to the first surface region, and a second oligonucleotide comprising a second DNA sequence and a third DNA sequence, wherein the second sequence is complementary to the first sequence, and the second surface region comprises a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the second surface region and each comprise: a third oligonucleotide comprising a fourth DNA sequence and a linking moiety for irreversibly linking the third oligonucleotide to the second surface region, and wherein the fourth sequence is complementary to the second sequence, the third sequence, or a combination of at least six continuous nucleotides of the second sequence and six continuous nucleotides of the third sequence; or (b) the first surface region comprises a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the first surface region and each comprise: a first oligonucleotide comprising a first DNA sequence and a linking moiety for irreversibly linking the first oligonucleotide to the first surface region, and a second oligonucleotide comprising a second DNA sequence and a third DNA sequence, wherein the second sequence is complementary to the first sequence, and the second surface region comprises a plurality oligonucleotide complexes, wherein said oligonucleotide complexes are located in spatially discrete locations of the second surface region and each comprise: a third oligonucleotide comprising a fourth DNA sequence and a linking moiety for irreversibly linking the third oligonucleotide to the second surface region, and a fourth oligonucleotide comprising a fifth DNA sequence and a sixth DNA sequence, wherein the fifth sequence is complementary to the fourth sequence, and wherein the second sequence is complementary to the fifth sequence or is complementary to the sixth sequence.

140. The fluidic reaction chamber of claim 139, wherein the first or second oligonucleotide comprises a fluorescent moiety.

141. The fluidic reaction chamber of claim 139, wherein the first or second oligonucleotide comprises a fluorescence quencher.

142. The fluidic reaction chamber of claim 139, wherein each of said second DNA sequences are identical.

143. The fluidic reaction chamber of claim 139, wherein each of said second DNA sequences are not identical.

144. The fluidic reaction chamber of claim 139, wherein each of the said oligonucleotides have a length of between about 5 and about 120 nucleotides.

145. The fluidic reaction chamber of claim 139, wherein the materials contacting first and second surfaces and forming the inner and outer boundaries of the chamber have shape of circle, oval, square, rectangle, triangle, hexagon, octagon, rhombus or trapeze, and provide distance between said first and second surfaces of between about 40 microns (40 m) and about 2 millimeters (2 mm).

146. The fluidic reaction chamber of claim 139, wherein the fluidic reaction chamber is not at a uniform temperature, and wherein the warmest region of the reaction chamber is between about 51 C. and about 100 C., and the coldest region of the reaction chamber is between about 10 C. and about 50 C.

147. The fluidic reaction chamber of claim 139, further comprising a fluid disposed within the fluidic reaction chamber, said fluid comprising one or more oligonucleotides, hybridization buffer, and optionally does not comprise non-specific nucleic acid staining dye.

148. A method of amplifying a target nucleic acid comprising: (a) providing a fluidic reaction chamber according to claim 139, 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.

149. The method of claim 148, detecting amplification of said target nucleic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0104] 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.

[0105] FIG. 1 shows a schematic of an embodiment of the fluidic chip vertically mounted on heaters at differential temperatures. The fluidic chip comprises a reaction chamber with a surface for functionalizing DNA oligonucleotides. In some embodiments, this chip is used for performing multiplex PCR-based detection of nucleic acids.

[0106] FIG. 2 shows an embodiment of the system, in which the fluidic chip is mounted vertically on the heaters at differential temperatures. A horizontally mounted light source excites fluorescent moieties on DNA functionalized to the chip; fluorescence signal is quantitated via the shown detector.

[0107] FIG. 3 shows camera pictures of the fluidic chip (left), the chip mounted on heaters (middle) and the chip with rectangular-shaped chambers mounted on the heaters (right).

[0108] FIG. 4 shows an example of the chemical approach for functionalization of the glass slide with DNA oligonucleotide to form part of the detection probe. The process is a two-step reaction of the PDITC (p-phenylene-diisothiocyanate) activated glass with an azido-PEG-amine (11-azido-3,6,9-trioxaundecan-1-amine) followed by a conjugation with an alkyne-functionalized oligonucleotide via copper-catalyzed alkyne-azide cyclo-addition that results in an oligonucleotide attachment through a hydrophilic PEG linker. Hydrophilicity of the functionalized surface prevents non-specific absorption of the reaction components such as target DNA, primers and enzymes. Copper-free reaction of the azide-functionalized surface with oligonucleotides modified with strained cycloalkynes also results in stable and highly specific covalent attachment of the oligonucleotides to the surface.

[0109] FIG. 5 shows an example of the functionalized probe structure. The oligonucleotide functionalized to the surface is labeled with a fluorophore and the oligonucleotide hybridized to the surface-functionalized probe is labeled with a quencher. When a detection target displaces the quencher-labeled oligonucleotide from the surface, the fluorescence intensity of the surface increases. Bottom panels show fluorescence microscopy images before and after target introduction; the spot diameter here is 1 mm Oligonucleotides were as follows:

TABLE-US-00001 Alk-TMR-1 (SEQIDNO:1) /5Hexynyl/tttt/i6-TAMN/tggatgctgaatacttgtgataa taca 32-RQ (SEQIDNO:2) ccgtagaggtgtattatcacaagtattcagcatcca/3IAbRQSp/ 54 (SEQIDNO:3) tggatgctgaatacttgtgataatacacctctacgg

[0110] FIG. 6 shows a schematic for polymerase chain reaction (PCR) amplification of a genomic DNA (gDNA) template within the fluidic chip when convection flow is applied; sequence (10) indicates forward primer and sequence (11) indicates reverse primer. The double-stranded amplicon, comprising domains 10-15-16-17 in the forward strand and domains 11-12-13-14 in the reverse strand is denatured in the hot 95 C. zone, and then is carried by convection to the 60 C. zone, where it can displace a quencher-labeled oligonucleotide to generate increased fluorescence in the corresponding spot.

[0111] FIG. 7 shows results of PCR amplification within the convection chip. The left panel shows an agarose gel electrophoresis of amplification products. Lane 1 shows the amplification product of 10 ng of NA18562 gDNA template amplified for 30 minutes in the convection chip. Primer concentrations are 600 nM for the forward primer and 200 nM reverse primers. Lane 2 shows a negative control with primers, polymerase, dNTPs, and probe spot, but no gDNA input. The ladder is a 50 bp ladder (New England Biolabs) as a reference. The right panel shows fluorescence time course of the spot intensity through an amplification reaction. The probes and primers for this experiment were designed for target rs7517833 (see FIG. 20).

[0112] FIG. 8 demonstrates convection flow in the fluidic chip. The top left panel shows a fluorescence image of fluorescent tracking beads in the absence of a temperature gradient across the chip (60 C. for both heaters). The top right panel shows a time-lapse (2 second) fluorescence image of the fluorescence tracking beads when a temperature gradient is applied (95 C. for left heater and 60 C. for right heater). The bottom panel summarizes observed mean convection flow velocity based on chamber thickness.

[0113] FIG. 9 shows a schematic for an array-based readout of multiple amplicons within the fluidic chip. The right panel shows a fluorescence image of the chip with 24 printed spots. Spots marked M are positive control spots lacking quencher-labeled oligonucleotides. Other spots each are specific to a particular amplicon sequence.

[0114] FIG. 10 shows fluorescence images of the probe array area of the fluidic chip before and after convection PCR. Primers that generate amplicons corresponding to the probes at spots 2, 3, and 4 were introduced (600 nM each forward primer, 200 nM each reverse primer), along with 10 ng of gDNA template. High fluorescence of spot 14 is unintentional and may have resulted from poorly functionalized or hybridized DNA probe molecules.

[0115] FIG. 11 shows time-course fluorescence for 9-plex PCR amplification in the convection fluidic chip. Each spot's fluorescence intensity was individually quantitated and normalized based on background fluorescence and the fluorescent intensity of the marker spots M. Each forward primer concentration is 200 nM and each reverse primer concentration is 100 nM, and gDNA input is 10 ng.

[0116] FIG. 12 shows 3-plex PCR amplification in the convection fluidic chip corresponding to primers for human, mouse, and rat DNA. Here, all spots in a row have the same sequence identity and report on the same amplicon. The top row are positive control probes. In the reaction chamber was 600 nM each forward primer, 200 nM each reverse primer, and 10 ng of gDNA template. Sequences used in the experiment were as follows:

[0117] Primers

TABLE-US-00002 h_ppia_fp (SEQIDNO:4) gttaacagattggaggtagtagcatttt h_ppia_rp (SEQIDNO:5) tctatcaccaccccccaact r_b2m_fp (SEQIDNO:6) caggtattttggggtatgattatggtt r_b2m_rp (SEQIDNO:7) ccaacagaatttaccaggaaacaca m_gadph_fp (SEQIDNO:8) caatacggccaaatctgaaagacaa m_gadph_rp (SEQIDNO:9) ctgcaggttctccacacctat

[0118] Arms

TABLE-US-00003 h_ppia_arm (SEQIDNO:10) agcagtgcttgctgttccttagaattttgccttgtgcgatgctgaata cttgtgataatacacctctacgggtcagg r_b2m_arm (SEQIDNO:11) ctggttcttactgcagggcgtgggaggagcgcgatgctgaatacttgt gataatacacctctacgggtcagg m_gadph_arm (SEQIDNO:12) gatagcctggggctcactacagacccatgagggcgatgctgaatactt gtgataatacacctctacgggtcagg

[0119] Quenchers

TABLE-US-00004 h_ppia_q (SEQIDNO:13) /5IAbRQ/acaaggcaaaattctaaggaacagcaagcactgctgcacg atcaggggt r_b2m_q (SEQIDNO:14) /gctcctcccacgccctgcagtaagaaccagaccccagcctttacac m_gadph_q (SEQIDNO:15) /5IAbRQ/cctcatgggtctgtagtgagccccaggctatctcatgttc ttcagagtgga

[0120] Anchor

TABLE-US-00005 (SEQIDNO:16) /DBCO/tttttcctgacccgtagaggtgtattatcacaagtattcagc atcgc/ATTO-550/

[0121] FIG. 13A shows a schematic of the reaction chamber with two surface regions, each functionalized with different DNA oligonucleotide reagents. Unlike in FIG. 1, the oligonucleotide reagents are not independent in sequence, but are rather rationally designed for enzyme-free amplification. FIG. 13B shows two possible embodiments of the two surface regions: either they are on different surfaces, or on the same surface but distally located to prevent direct interaction.

[0122] FIG. 14A shows the mechanism for linear amplification of a target nucleic acid sequence bearing a sixth sequence (6) and a seventh sequence (7). The target nucleic acid sequence catalytically transfers multiple oligonucleotides bearing the second sequence (2) and the third sequence (3) from surface region 1 to surface region 2. Spontaneous dissociation of the double-stranded DNA molecule (23:67) in the hot zone is critical to allow rapid turnover. FIG. 14B shows the net reaction of the process described in FIG. 14A, as well as a fluorescent labeling strategy to allow real-time readout. FIG. 14C shows an alternative implementation with flipped 5/3 orientation (the half arrow-head denotes 3 end, as custom in literature).

[0123] FIG. 15 shows the mechanism for a control experiment in which the target nucleic acid sequence induces a stoichiometric rearrangement of surface-bound oligonucleotides.

[0124] FIG. 16 shows time-course fluorescence of surface region 1 when various concentrations of target nucleic acid are introduced. The fluorescence in the linear amplification chip decreases more quickly than in the stoichiometric conversion chip, supporting the mechanism of enzyme-free DNA amplification.

[0125] FIG. 17 shows the mechanism for exponential amplification of a target nucleic acid sequence.

[0126] FIG. 18 shows a visual representation of reporting enzyme-free amplification through the use of an intercalating dye, such as SybrGreen or EvaGreen. The fluorescence intensity of surface region 1 will decrease through the course of the reaction, and the fluorescence intensity of surface region 2 will increase.

[0127] FIG. 19 shows a visual representation of reporting enzyme-free amplification through the use of a fluorophore-functionalized oligonucleotides. The fluorescence intensity of surface region 1 will increase through the course of the reaction, and the fluorescence intensity of surface region 2 will remain dark through the course of the reaction.

[0128] FIG. 20 shows the list of primers and probes (anchor+arm+quencher) used for PCR amplification/detection.

DETAILED DESCRIPTION

[0129] 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.

1. CONVECTION FLOW PCR

[0130] Liquid, when held at a non-uniform temperature and confined in a volume, will circulate via a process known as Rayleigh-Benard convection flow [.sup.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) [.sup.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.

[0131] cf-PCR has been demonstrated for both single-plex [.sup.3-5, U.S. Pat. No. 8,187,813 B2] and multiplex detection of specific DNA sequences [.sup.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 [.sup.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.

2. MICROARRAYS

[0132] 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 [.sup.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 [.sup.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.

3. TOEHOLD PROBES AND ENZYME-FREE AMPLIFICATION

[0133] Toehold mediated strand displacement reaction [.sup.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 [.sup.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.

4. APPLICANTS' TECHNOLOGY

[0134] 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 m, between two differentially-controlled hot plates (FIGS. 1-3) tilted at the defined angle. The shape of the spacer determines the shape of the reaction chamber, and can be modified to alter convection flow speed and trajectory. Glass is selected as the chip material because glass facilitates maintenance of a uniform temperature gradient across the chamber and allows surface functionalization with synthetic oligonucleotides.

[0135] 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.

[0136] 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.

[0137] 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.

5. ENZYME-FREE LINEAR AMPLIFICATION EMBODIMENT

[0138] The present disclosure represents an enzyme-free amplification of target nucleic acid, in which amplicon concentration increases linearly with time (FIG. 14A). Two surface regions are irreversibly functionalized with two DNA oligonucleotides, donor, D (comprising domain 1) and acceptor, A (comprising domains 4-5). The donor oligonucleotide functionalized to the surface region 1 is initially hybridized with a signal strand S comprising domains 2-3, which are complementary to domains 4-5, in such a way that the single-stranded domain 2 is exposed to solution and plays a role of a toehold sequence. The acceptor strand is irreversibly functionalized to surface region 2 and initially represents a single-stranded oligonucleotide. The surface regions 1 and 2 are localized in the temperature zone held at 35 C. (35 C. zone), at which the D-S duplex is designed to be highly stable over the time scale of a detection assay. Target molecule T (comprising domains 6-7) is introduced in the reaction solution and will be transferred by convection flow to the 35 C. zone of the reaction chamber. Target T binds to the signal S via domain 7 (the toehold domain) and displaces domain 3 from surface to the solution via toehold-mediated strand displacement mechanism. Then, Rayleigh-Bernard convection flow carries the duplex to a hot zone of the chamber (held at 85 C.), where the duplex dissociates. The two single-stranded molecules, the target strand T and the signal strand S are then transferred back into the chamber's 35 C. zone where strand S binds to the acceptor oligonucleotide A functionalized to the surface region 2. At the same time allowing the single-stranded target T catalytically displaces another signal S molecule from surface region 1, completing the catalytic cycle. This trigger should proceed continuously until the signal molecules S are completely transferred from surface region 1 to surface region 2.

[0139] 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.

[0140] 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.

6. STOICHIOMETRIC DETECTION EMBODIMENT

[0141] To demonstrate that the enzyme-free amplification method exhibits multiple turnover, the inventors constructed a corresponding stoichiometric detection system using the convection device (FIG. 15). The stoichiometric system also includes two surface regions functionalized with a donor, Ds strand comprising domains 11-12 and an acceptor As strand comprising domain 16. The Ds strand is hybridized with a signal complex consisting of a bridge oligonucleotide Bs comprising domains 14-15 and a signal oligonucleotide Ss comprising domain 13. Domain 14 of the Bs strand and the As strand possess identical sequence. Target molecule T comprising domains 17-18 introduced in the reaction binds to the toehold domain 11 of the donor Ds and then displaces the signal complex into the solution. During this process target T binds the donor Ds and is unable to be regenerated in the chamber's hot zone in order to trigger the release of another signal complex from the surfaces region 1. The displaced signal complex is transferred by the convection flow into the 85 C. zone where it dissociates. After the dissociation the signal molecule Ss is transferred back to the 35 C. zone and is captured by the acceptor oligonucleotide As functionalized to the surface region 2. Thus, the amount of the captured signal strand Ss equals the amount of target T introduced into the system.

[0142] 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. FIG. 16 shows the time-based decrease of fluorescence on surface region 1 for the linear amplification and stoichiometric detection systems, given identical initial quantities of detection target. The decrease of fluorescent signal is faster in the linear amplification system than in the linear amplification assay that supports the designed mechanism in which each target molecule is catalytically transferring multiple signal molecules from surface 1 to surface 2.

7. ENZYME-FREE EXPONENTIAL AMPLIFICATION EMBODIMENT

[0143] FIG. 17 shows an embodiment of the present disclosure in which the detection target triggers the release of an amplicon product whose concentration exponentially increases with time, until reagents are consumed (FIG. 17). In this system, the two surface regions are functionalized with partially double-stranded oligonucleotide complexes: surface region 1 has a complex consisting of a single domain oligonucleotide 1 irreversibly attached to the surface and hybridized with an oligonucleotide S1 comprising domains 3-2 in such a way that the domain 3 remains single-stranded and acts as a toehold sequence. The surface region 2 mirrors surface region 1 and has a similar architecture of an oligonucleotide reagent functionalized to the surface: an oligonucleotide comprising domain 4 is irreversibly functionalized to the surface, and hybridized with an oligonucleotide S2 comprising domains 6-5 wherein domain 6 represents a single-stranded toehold. Injection of the target strand T, comprising domains 7 and 8 (identical in sequence to domains 6 and 5, respectively), into the reaction mixture results in the release of the oligonucleotide S1 from the surface region 1 in the form of double-strand amplicon.

[0144] 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.

8. ALTERNATIVE DETECTION METHODS

[0145] FIG. 14B presents one possible detection mechanism for assaying reaction progress. Alternative readout approaches also utilizing fluorescent microscopy are possible. Non-sequence-specific intercalating nucleic acid staining dyes, such as SybrGreen and Syto dyes, can be used to indicate the total amount of accumulated double-stranded product (FIG. 18). Because the acceptor strand A (domains 4-5) immobilized on the surface region 2 is in single-stranded form at the beginning of the reaction, the intercalating dye would have a low affinity to the strand A. In the course of the reaction more and more strands S (domains 2-3) will hybridize with the surface functionalized strand A (domains 4-5). The newly formed complexes A-S now would be efficiently stained with the dye that would cause an increase of the surface region 2 fluorescence. The opposite situation can be potentially observed on the surface region 1.

[0146] Application of the FRET-based detection technique is illustrated on the example of exponential amplification (FIG. 19). For example, irreversibly labeled with a fluorescent dye surface immobilized strands (domain 1 is shown labeled) can be efficiently quenched with a quencher functionalized signal strands (the strand with domains 2-3 is shown with a fluorescent quencher) before the target is added. An addition of the target will result in exponential release of the signal strands forms the surface and lighting up of the surface immobilized strands.

9. REAL-TIME DETECTION OF CONVECTION FLOW PCR

[0147] 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.

[0148] FIG. 6 shows an approach utilizing the claimed composition for real-time monitoring of the PCR reaction performed in the temperature-driven convection flow. Initially a surface region 1 residing in 60 C. zone is functionalized with an Anchor oligonucleotide comprising domain 1 and a fluorescent dye. An Arm oligonucleotide comprising domains 3-2 is hybridized to the Anchor oligonucleotide through its 2 domain. The Anchor-Arm oligonucleotide complex is in turn hybridized with a Quencher oligonucleotide comprising domains 4 and 5 and a fluorescent quencher through its domain 4; domain 5 is single-stranded. The reaction solution comprises reagents for enzymatic extension of oligonucleotide primers (domains 10 and 11), such as a DNA polymerase, mixture of deoxynucleotide triphosphates, divalent ions (Mg.sup.2+). After injection of a target molecule (gDNA), it spontaneously melts in the 95 C. Then the melted gDNA is transferred into the 60 C. zone by the convection flow where primers anneal to their specific target sequences and extend by DNA polymerase that leads to formation of the double stranded amplicon molecules comprising domains 10-15-16-17 in forward strand and 11-12-13-14 in reverse strand. The amplicon molecules melt in the 95 C. and flow back to the 60 C. zone where forward strand 10-15-16-17 displaces the Quencher oligonucleotide form the surface functionalized complex Anchor-Arm resulting in increasing of the fluorescent signal registered form the surface region 1.

10. SIMULTANEOUS MONITORING OF MULTIPLE TARGET SEQUENCES

[0149] 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.

11. EXEMPLARY OLIGONUCLEOTIDES

[0150] The following oligonucleotide sequences are provided by way of example, but not limitation:

Linear Amplification System Oligonucleotides

[0151]

TABLE-US-00006 1 (SEQIDNO:17) /5Hexynyl/TTTTTCCGTAGAGGTGTATTATCACAAGTATT 2-3 (SEQIDNO:18) /56-TAMN/TGGATGCTG-AATACTTGTGATAATACACCTCTACGG 4-5 (SEQIDNO:19) /5Hexynyl/TTTTTCCGTAGAGGTGTATTATCACAAGTATT- CAGCATCCA 6-7 (SEQIDNO:20) CCGTAGAGGTGTATTATCACAAGTATT-CAGCATCCA

Stoichiometric Detection System Oligonucleotides

[0152]

TABLE-US-00007 11-12 (SEQIDNO:21) /5Hexynyl/TTTTTTGTCAACC-ATCATCGTTCGTACCACAGTGTTC AG 13 (SEQIDNO:22) /56-TAMN/TGGATGCTGAATACTTGTGATAATACACCTCTACGG 14-15 (SEQIDNO:23) CCGTAGAGGTGTATTATCACAAGTATTCAGCATCCACTGAACACTGTG GTACGAACGATGA 16 (SEQIDNO:24) /5Hexynyl/TTTTTCCGTAGAGGTGTATTATCACAAGTATTCAGCAT CCA 17-18 (SEQIDNO:25) CTGAACACTGTGGTACGAACGATGAT-GGTTGACA

12. REFERENCES

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