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WO2016086004A1 - Détection d'acide nucléique - Google Patents

Détection d'acide nucléique Download PDF

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Publication number
WO2016086004A1
WO2016086004A1 PCT/US2015/062463 US2015062463W WO2016086004A1 WO 2016086004 A1 WO2016086004 A1 WO 2016086004A1 US 2015062463 W US2015062463 W US 2015062463W WO 2016086004 A1 WO2016086004 A1 WO 2016086004A1
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WIPO (PCT)
Prior art keywords
nucleic acid
restriction endonuclease
probe
target
solid support
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PCT/US2015/062463
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English (en)
Inventor
Kenneth D. Smith
Mariya Smit
Nina Yazvenko
Andrei L. Gindilis
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Cascade Biosystems, Inc.
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Publication of WO2016086004A1 publication Critical patent/WO2016086004A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers

Definitions

  • This document relates to methods and materials for detecting nucleic acid.
  • this document relates to methods and materials for using an enzymatic amplification cascade of restriction endonucleases to detect nucleic acid.
  • PCR polymerase chain reaction
  • real-time PCR can be used to amplify and detect small amounts of template DNA present in a sample.
  • PCR involves adding a nucleic acid primer pair and a heat-stable DNA polymerase, such as Taq polymerase, to a sample believed to contain a targeted nucleic acid to be amplified.
  • a thermal cycling process sets in motion a chain reaction in which the targeted DNA template located between the primers is exponentially amplified.
  • the presence of this amplified template can be detected using techniques such as gel electrophoresis or the amount of amplified template can be assessed using techniques such as those involving the use of fluorescently labeled probes.
  • This document provides methods and materials for detecting target nucleic acid that involve signal amplification rather than target amplification. For example, this document provides methods and materials for detecting the presence or absence of target nucleic acid within a sample, methods and materials for detecting the amount of target nucleic acid present within a sample, kits for detecting the presence or absence of target nucleic acid within a sample, kits for detecting the amount of target nucleic acid present within a sample, and methods for making such kits.
  • the methods and materials provided herein can include performing an enzymatic amplification cascade of restriction endonucleases as described herein (referred to as a restriction cascade exponential amplification or RCEA) to detect target nucleic acid in a manner that is rapid, inexpensive, sensitive, and specific, without requiring any complex
  • RCEA restriction cascade exponential amplification
  • the methods and materials provided herein can be used in addition to or can replace current PCR-based nucleic acid detection approaches such as quantitative PCR as the detection limit is around 10 attomolar (10 ⁇ 17 M) or approximately 200 target molecules per sample.
  • the methods described herein include two independent biorecognition steps, and are insensitive to non-specific binding and can be used to detect target sequences in the presence of heterologous DNA and possibly other contaminants.
  • the methods and materials provided herein can allow clinicians, medical professionals, laboratory personnel, and researchers to detect any type of target nucleic acid.
  • the methods and materials provided herein can be used in genotyping applications to detect nucleic acid mutations (e.g., single nucleotide polymorphisms (SNPs)), genome rearrangements, and genome epigenetic events (e.g., DNA methylation events), can be used in diagnostic or prognostic applications to detect viruses or microorganisms (e.g., bacteria, fungi, and protozoa), can be used in gene expression applications to detect mRNA levels within particular cell types, and can be used in forensic applications to compare the identity between samples or to assess a sample's origin.
  • SNPs single nucleotide polymorphisms
  • genome rearrangements e.g., DNA methylation events
  • genome epigenetic events e.g., DNA methylation events
  • viruses or microorganisms e.g., bacteria, fungi, and
  • kits provided herein for detecting the presence of bacteria can be designed for use as a home detection kit such that a user can detect the presence or absence of E. coli nucleic acid within a biological sample (e.g., blood sample, mucus sample, saliva sample, food sample, or environmental sample) obtained from the user.
  • a biological sample e.g., blood sample, mucus sample, saliva sample, food sample, or environmental sample.
  • the methods described herein can be used in a wide variety of portable and field- deployable formats, including quantitative RCEA based on real-time electrochemical detection.
  • this document features a method for assessing a sample for target nucleic acid.
  • the method includes a) contacting the sample with a probe nucleic acid comprising an amplifying restriction endonuclease and a nucleotide sequence
  • the target nucleic acid hybridizes to at least a portion of the probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site; b) contacting the double- stranded portion of nucleic acid with a recognition restriction endonuclease having the ability to cut the double-stranded portion of nucleic acid at the restriction endonuclease cut site under conditions wherein the recognition restriction endonuclease cleaves the double-stranded portion of nucleic acid at the restriction endonuclease cut site, thereby separating a portion of the probe nucleic acid comprising the amplifying restriction endonuclease from at least another portion of the probe nucleic acid; c) contacting a double stranded nucleic acid with the portion of the probe nucleic acid
  • the probe nucleic acid can be single-stranded.
  • the probe nucleic acid can be attached to a solid support (e.g., directly attached to a solid support).
  • the portion of the probe nucleic acid comprising the amplifying restriction endonuclease can be released from the solid support via the step (b).
  • Steps (a) and (b) can be performed in the same compartment.
  • Steps (a) and (b) can be performed by adding the sample to a compartment comprising the probe nucleic acid.
  • the portion of the probe nucleic acid comprising the amplifying restriction endonuclease that is separated from the at least another portion of the probe nucleic acid in step (b) comprises at least a portion of the target nucleic acid.
  • the method can include using a plurality of the probe nucleic acid in step (a).
  • the method includes using a plurality of the double stranded reporter nucleic acid in step (c).
  • the double stranded nucleic acid in step (c) can be in molar excess of the portion of the probe nucleic acid comprising the amplifying restriction endonuclease from step (b).
  • the number of molecules of the portion of the probe nucleic acid comprising the amplifying restriction endonuclease that is separated from the at least another portion of the probe nucleic acid in step (b) can be in an essentially linear relationship to the number of molecules of the target nucleic acid present in the sample.
  • One strand of the double stranded nucleic acid can be attached to a solid support (e.g., directly attached to a solid support).
  • the strand of the double stranded nucleic acid comprising the reporter molecule can be attached to the solid support.
  • the strand of the double stranded nucleic acid comprising the amplifying restriction endonuclease can be attached to the solid support.
  • the reporter molecule can be a fluorescent label, a radioactive label, an enzyme label, or a redox label.
  • the determining step can include determining the amount of the released reporter molecule.
  • the solid support can be selected from the group consisting of a microtiter plate, a bead, a slide, a microparticle, a nanoparticle, a gel, and a surface of a chamber or channel within a microfluidic device.
  • the solid support can be a ceramic based solid support, a metal based solid support, a plastic polymer-based solid support, or a biopolymer-based solid support.
  • the kit comprises or consists essentially of (a) a probe nucleic acid comprising an amplifying restriction endonuclease and a nucleotide sequence complementary to a sequence of the target nucleic acid, wherein at least a portion of the target nucleic acid is capable of hybridizing to at least a portion of the probe nucleic acid to form a double- stranded portion of nucleic acid comprising a restriction endonuclease cut site for a recognition restriction endonuclease; and (b) a double stranded nucleic acid, wherein one strand of the double stranded nucleic acid comprises the amplifying restriction endonuclease and one strand of the double stranded nucleic acid comprises a reporter molecule (e.g., fluorescent label, a radioactive label, an enzyme label, or a redox label), the double stranded nucleic acid comprising
  • a reporter molecule e.g., fluorescent label, a radio
  • the probe nucleic acid is can be single-stranded.
  • the kit can include a solid support, and the probe nucleic acid can be attached to the solid support.
  • the kit also can include a recognition restriction endonuclease.
  • the portion of the probe nucleic acid comprising the amplifying restriction endonuclease can be releasable via cleavage with a recognition restriction endonuclease having the ability to cleave at the restriction endonuclease cut site.
  • the portion of the reporter nucleic acid comprising the label is capable of being separated from at least another portion of the reporter nucleic acid via cleavage by the amplifying restriction endonuclease.
  • Figure 1 is a general schematic of an exemplary restriction endonuclease assay.
  • A Surface immobilization of HRP conjugated to an oligonucleotide probe specific for a target gene of interest.
  • B The target DNA (an oligonucleotide or a denatured PCR amplicon) is hybridized to the immobilized probe.
  • C Addition of a restriction endonuclease that recognizes (Rrec) and cleaves the target-probe double-stranded DNA hybrid, resulting in release of the HRP marker into the reaction solution.
  • D The reaction solution is transferred into a new well and mixed with an HRP substrate for colorimetric detection. For each target DNA molecule, one HRP molecule is released, resulting in a linear dependence of the signal on the target DNA concentration.
  • E Detailed schematic of the double stranded target-probe DNA duplex, with the specific restriction site shown in dark gray.
  • Figure 2 is a typical calibration curve of the restriction endonuclease assay generated with a 40-mer oligonucleotide target (AMC-40-mer) that is fully
  • the X-axis shows concentrations (nM) of the target oligonucleotide.
  • the Y-axis shows the restriction endonuclease-generated HRP signal that was quantified by blue color formation as measured by the OD 6 55.
  • the signal values were background-corrected by subtracting the signal generated by the negative control with no target oligonucleotide added.
  • the experiments were performed in triplicate to generate mean values (black circles) and standard deviations (shown with error bars).
  • Figures 3A and 3B are graphs showing the effect of point mutations introduced into the target sequence as a percent of the positive control.
  • A Single, double, and triple mutations were introduced between the target center and the 3' end corresponding to the surface-immobilized terminus of the target-probe duplex.
  • B Mutations were introduced between the target center and the 5' end corresponding to the end of the target-probe duplex that was free in solution.
  • HRP signals (bars) are expressed as the percentages of the fully cognate positive control (dark grey bar 40, for 40-mer).
  • Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a 40-mer target with one to three mutations shown with black ovals.
  • the Bglll restriction site is indicated with thick horizontal lines.
  • Targets are named with 'rs' for mutations introduced within the restriction site, otherwise the target name contains the replacement nucleotide (mostly G) and position within the sequence, starting from the 5' target end.
  • the rs 19+24 contained two mutations at the ends of the restriction site.
  • Target oligonucleotide sequences are shown in Table 1.
  • Figure 4 is a graph showing the effect of sequence length on assay.
  • the HRP signals (bars) are expressed as the percentages of the fully cognate positive control (the dark grey bar 40-mer).
  • Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length and end sequence.
  • the Bglll restriction site is indicated with thick horizontal lines.
  • Target-probe duplex designations indicate the complementary sequence length, or fraction of complementary sequence to the total target length.
  • the target oligonucleotide sequences are shown in Table 1.
  • Figure 5 is a graph showing the effects of restriction site positioning within the double stranded DNA hybrid, and non-complementary loop addition.
  • the HRP signals (bars) are expressed as percentages of the fully cognate positive control (40-mer).
  • Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length, non-complementary ends, and/or loops.
  • the Bglll restriction site is indicated with thick horizontal lines.
  • Target designations are the following: 5' (or 3'), corresponds to the 5' (or 3') ends of the full length positive control; C, control (fully cognate); L, loop (addition of 5 or 10 nucleotides); rs5' (or rs3'), the end of restriction site to which 0, 3, or 5 (+0, +3, +5) complementary nucleotides were added.
  • rs3'+0 two targets were prepared that had different non-complementary sequences flanking the 3'- end of the restriction site (rs3'+0-A, rs3'+0-G).
  • the target oligonucleotide sequences are shown in Table 1.
  • Figure 6 is a graph of calibration curves generated with either the purified 196 bp dsDNA mecA amplicon (diamonds) or the unpurified PCR mixture (containing the target amplicon) (squares). The logarithmic trend-lines were calculated in Excel, and proved to be identical for the purified and non-purified amplicons.
  • Figure 7 is a graph showing the detection of the non-purified amplicon in the presence of a large excess of heterologous (mouse) genomic DNA. Circles and diamonds show replicate experiments performed using the amplicon-containing PCR mixture, closed and open for addition of 100 or 0 ng of mouse DNA, respectively. The triangles show the negative control supplemented with 100 ng of mouse DNA, specifically, dilutions of the whole PCR mixture that were not subjected to thermocycling (no amplicon formation as verified by gel electrophoresis).
  • Figure 8 is an exemplary schematic of an oligonucleotide probe specific to the target of interest conjugated to a mutant restriction endonuclease (either the BamHI- S17C/C34S/C54S/C64S or EcoRI-K249C), and attached to a solid substrate through biotin.
  • An oligonucleotide target complementary to the probe is added, and it hybridizes to the probe.
  • the hybrid is specifically cleaved by a free restriction endonuclease Bglll, and the mutant restriction endonuclease is released into the reaction solution.
  • Figure 9 contains two graphs depicting the testing of the restriction endonuclease- oligonucleotide conjugates, BamHI-S17C/C34S/C54S/C64S-MCA-BG-Bio and EcoRI- K249C-MCA-BG-Bio.
  • the restriction endonuclease conjugates were tested for the presence of oligonucleotide parts and immobilization as described by the schematic in Figure 8.
  • the X-axis shows the target MCA-BG-Bio concentration in ⁇ (the negative controls at the left had no target added); and the Y-axis shows the HRP reporter signals quantified colorimetrically as OD 6 55.
  • a line graph shows the titration of free restriction endonuclease conjugates using the HRP reporter systems.
  • the X-axis shows the conjugate concentrations. Negative controls were prepared with no addition of conjugates, and used for background subtraction.
  • the Y-axis shows the background- corrected HRP signals (OD655) generated using the two mutant enzyme conjugates, BamHI-S17C/C34S/C54S/C64S and EcoRI-K249C.
  • FIG 10 is an exemplary schematic of the restriction cascade exponential amplification (RCEA) assay.
  • RCEA restriction cascade exponential amplification
  • Each step of this exponential cascade of cleavage reactions increases the amount of free Ramp in the reaction solution.
  • the linker cleavage releases HRP, which is quantified colorimetrically.
  • E Each initial target-probe hybridization event produces an exponentially amplified number of HRP molecules, with the value dependent on the assay time.
  • Figure 11 is a graph of the RCEA limit of detection evaluated using the oligonucleotide target MCA-BG-Bio.
  • the X-axis shows the target concentrations (M), and the Y-axis shows the background-corrected HRP signal values (with the background calculated as the signal generated for zero target concentrations).
  • M target concentration
  • Y-axis shows the background-corrected HRP signal values (with the background calculated as the signal generated for zero target concentrations).
  • the HRP signal values were expressed as the percentages of the maximum background-corrected OD 6 55, corresponding to each series.
  • Open circles show the data generated using the Direct Restriction Assay (DRA) with no amplification.
  • DRA Direct Restriction Assay
  • Figure 12 is a graph showing the dependence between the assay signal and the duration of the amplification stage analyzed using the BamHI-S17C/C34S/C54S/C64S system.
  • the X-axis shows the duration of the RCEA amplification stage (min).
  • the Y- axis shows the background-corrected HRP signal values (with the background calculated for each time point as the mean signal generated for zero target concentrations).
  • the HRP signal values were expressed as the percentages of the maximum background-corrected OD 6 55 obtained for 1 fM concentration after 75 minutes of incubation. Two different target concentrations 100 fM (open circles) and 1 fM (closed diamonds) were used for the assays.
  • Figure 13 is a graph of the target sequence alteration and addition of foreign DNA on performance of the RCEA assay employing the EcoRI-K249C system.
  • the X-axis shows the experimental conditions: AMC, the target type (BG-40 is fully cognate; 12/40 contains 12 cognate nucleotides centered around the restriction site and non-cognate ends); gDNA, presence or absence of foreign, mouse genomic DNA (80 ng per reaction); Wash, whether the beads carrying immobilized Ramp conjugates were washed after the target hybridization before addition of the recognition restriction endonuclease Bglll.
  • the Y-axis shows the background-corrected HRP signal values (with the background calculated as the mean signal generated for zero target concentration).
  • FIGS 14A-F are schematics of the methods and materials provided herein for detecting target nucleic acid using probe nucleic acid (101), a recognition restriction endonuclease (Rrec, 106), amplifying recognition endonuclease (Ramp, 103) and reporter nucleic acid (121).
  • Figures 15A-15C are schematics of an exemplary configuration of probe nucleic acid that can be used with the methods and materials provided herein for detecting target nucleic acid.
  • the methods and materials described herein include using a nucleic acid conjugated to an amplifying restriction endonuclease (Ramp), with signal generated based on the hybridization of a target DNA to the nucleic acid conjugated to the Ramp, and specific cleavage of the resulting nucleic acid-target hybrid by a free recognition restriction endonuclease.
  • the released Ramp is proportionate to the initial DNA target amount.
  • the released Ramp is amplified by contacting it with an excess of Ramp attached to one strand of a double stranded nucleic acid, which contains the cut site for the Ramp.
  • the Ramp conjugated to the double stranded nucleic acid is incapable of cleaving itself as well as adjacent nucleic acid.
  • the released Ramp cleaves the double stranded nucleic acid, releasing more Ramp, with each cleavage step increasing the amount of free Ramp in the reaction solution.
  • the other strand of the double stranded nucleic acid can contain a reporter molecule (e.g., horseradish peroxidase (HRP)).
  • HRP horseradish peroxidase
  • a method for detecting target nucleic acid can include contacting a sample (e.g., a sample to be tested or suspected of containing target nucleic acid) with probe nucleic acid.
  • the probe nucleic acid can be designed to have a single- stranded portion with a nucleotide sequence that is complementary to at least a portion of the target nucleic acid to be detected.
  • target nucleic acid present within the sample can hybridize with the complementary sequence of this single-stranded portion of the probe nucleic acid to form a double-stranded section with one strand being target nucleic acid and the other strand being probe nucleic acid.
  • the single- stranded portion of the probe nucleic acid having the nucleotide sequence that is complementary to at least a portion of the target nucleic acid to be detected can be designed such that hybridization with the target nucleic acid creates a restriction endonuclease cut site.
  • target nucleic acid present within the sample can hybridize with the complementary sequence of the single-stranded portion of the probe nucleic acid to form a double-stranded section that creates a cut site for a restriction endonuclease.
  • This cut site created by the hybridization of target nucleic acid to probe nucleic acid can be referred to as a recognition restriction endonuclease cut site.
  • a restriction endonuclease that cleaves nucleic acid at such a recognition restriction endonuclease cut site can be referred to as a recognition restriction endonuclease.
  • the probe nucleic acid also can be designed to include a restriction endonuclease.
  • This restriction endonuclease which can be a component of the probe nucleic acid, can be referred to as an amplifying restriction endonuclease.
  • An amplifying restriction endonuclease is typically a different restriction endonuclease than the restriction endonuclease that is used as a recognition restriction endonuclease.
  • probe nucleic acid is designed to contain an amplifying restriction endonuclease and to have a nucleotide sequence such that the target nucleic acid can hybridize to the probe nucleic acid and create a recognition restriction endonuclease cut site for a recognition restriction endonuclease.
  • the probe nucleic acid can be attached to a solid support (e.g., a well of a microtiter plate, bead, or particle).
  • a solid support e.g., a well of a microtiter plate, bead, or particle.
  • the probe nucleic acid can be attached to a solid support such that cleavage at the recognition restriction endonuclease cut site via the recognition restriction
  • the endonuclease releases a portion of the probe nucleic acid that contains the amplifying restriction endonuclease.
  • the target nucleic acid After contacting the sample that may or may not contain target nucleic acid with the probe nucleic acid that is attached to a solid support, the target nucleic acid, if present in the sample, can hybridize to the probe nucleic acid and create the recognition restriction endonuclease cut site.
  • the recognition restriction endonuclease whether added to the reaction or already present in the reaction, can cleave the probe nucleic acid at the recognition restriction endonuclease cut sites that are formed by the hybridization of target nucleic acid to the probe nucleic acid, thereby releasing the portion of the probe nucleic acid that contains the amplifying restriction endonuclease from the solid support.
  • the number of amplifying restriction endonuclease-containing portions of the probe nucleic acid that are released from the solid support can be in an essentially linear relationship (e.g., essentially a one-for-one relationship) with the number of target nucleic acid molecules that hybridize with the probe nucleic acid to form the recognition restriction endonuclease cut site.
  • the portions of the probe nucleic acid containing the amplifying restriction endonuclease that were released from the solid support can be collected and placed in contact with a double-stranded nucleic acid, where one strand of the nucleic acid contains the same amplifying restriction endonuclease and the other strand contains a reporter molecule.
  • the double-stranded nucleic acid can be referred to as a "reporter nucleic acid" herein.
  • the released portions of the probe nucleic acid if present, can be transferred from one well of a microtiter plate (e.g., a 96-well plate) that contained the probe nucleic acid to another well of a microtiter plate that contains reporter nucleic acid.
  • the reporter nucleic acid can contain a restriction endonuclease cut site for the amplifying restriction endonuclease that was present on the probe nucleic acid and/or that was present on the reporter nucleic acid itself.
  • This restriction endonuclease cut site for an amplifying restriction endonuclease can be referred to as an amplifying restriction endonuclease cut site. If portions of the probe nucleic acid containing an amplifying restriction endonuclease are present and placed in contact with the reporter nucleic acid, then the reporter nucleic acid can be cleaved at the amplifying restriction endonuclease cut site by the amplifying restriction endonuclease.
  • the number of reporter nucleic acid molecules that are cleaved can greatly exceed the number of amplifying restriction endonucleases present in the reaction.
  • the number of cleaved reporter nucleic acid molecules can greatly exceed (e.g., exponentially exceed) the number of amplifying restriction endonucleases present in the reaction and therefore can greatly exceed (e.g., exponentially exceed) the number of target nucleic acid molecules that were present in the sample contacted with the probe nucleic acid.
  • Such a greatly expanded relationship e.g., an exponential relationship
  • the presence or absence of released reporter molecules can be determined.
  • the presence of released reporter molecules can indicate that the sample contained the target nucleic acid, while the absence of released reporter molecules can indicate that the sample lacked the target nucleic acid.
  • the amount of released reporter molecules can be determined. In such cases, the amount of released reporter molecules can indicate the amount of target nucleic acid present in the sample.
  • a standard curve using known amounts of target nucleic acid can be used to aid in the determination of the amount of target nucleic acid present within a sample.
  • a reporter nucleic acid can be attached to a solid support (e.g., a well of a microtiter plate, agarose bead, or particle).
  • a reporter nucleic acid can be attached to a solid support such that cleavage at an amplifying restriction endonuclease cut site by an amplifying restriction endonuclease (e.g., amplifying restriction endonucleases of the probe nucleic acid and of the reporter nucleic acid when the same particular restriction endonuclease is used for both) releases a portion of the reporter nucleic acid that contains the label.
  • the resulting reaction mixture can be collected and assessed for the presence, absence, or amount of released portions of the reporter nucleic acid using the reporter molecule.
  • the released portions of the reporter nucleic acid can be transferred from one well of a microtiter plate (e.g., a 96- well plate) that contained the reporter nucleic acid to another well of a microtiter plate, where the transferred material can be assessed for a signal from the reporter molecule.
  • Probe nucleic acid described herein typically includes at least one single-stranded DNA section that is designed to hybridize with a desired target nucleic acid and thereby create a recognition restriction endonuclease cut site.
  • the other portions of the probe nucleic acid can include DNA, RNA, a nucleic acid analog, or other molecules.
  • probe nucleic acid can include biotin such that the probe nucleic acid can be attached to a streptavidin-coated solid support.
  • the single-stranded section of the probe nucleic acid that is designed to hybridize with a desired target nucleic acid and create a recognition restriction endonuclease cut site can be RNA or a nucleic acid analog (e.g., a peptide nucleic acid (PNA)) provided that such a single-stranded section can (i) hybridize with the desired target nucleic acid and (ii) create a recognition restriction endonuclease cut site with the complementary target nucleic acid sequence that is capable of being cleaved by the recognition restriction endonuclease.
  • PNA peptide nucleic acid
  • restriction endonucleases that can be used as recognition restriction endonucleases to cleave a recognition restriction endonuclease cut site that is created between an RNA section of the probe nucleic acid and a DNA section of the target nucleic acid include, without limitation, Hhal, Alul, Taql, Haelll, EcoRI, Hindll, Sail, and Mspl restriction endonucleases.
  • the probe nucleic acid can have various configurations.
  • the probe nucleic acid can be designed to be a single nucleic acid strand such that the entire nucleic acid component of probe nucleic acid is single-stranded prior to contact with a target nucleic acid.
  • probe nucleic acid can be designed to have a first strand and a second strand.
  • the first strand can be attached to solid support and can be designed to have a single-stranded section having a nucleotide sequence that is complementary to at least a portion of target nucleic acid.
  • the second strand can include an amplifying restriction endonuclease and can have a single-stranded section having a nucleotide sequence that can hybridize to first strand.
  • the first and second strands can be synthesized or obtained separately and then mixed together to form probe nucleic acid.
  • the first strand can be synthesized, biotinylated, and attached to a streptavidin-coated solid support.
  • the second strand can be incubated with the first strand to form nucleic acid probe.
  • a probe nucleic acid can contain more than two strands.
  • probe nucleic acid can include first, second, and third strands.
  • first strand can be attached to solid support
  • the second strand can be hybridized to first strand and can include a single-stranded section having a nucleotide sequence that is complementary to at least a portion of target nucleic acid
  • a third strand can be hybridized to the second strand and can be attached to an amplifying restriction endonuclease. Similar two, three, or more strand configurations can be used to make reporter nucleic acid.
  • Probe nucleic acid described herein can be any length provided that the single- stranded section of the probe nucleic acid that is designed to hybridize with a desired target nucleic acid is capable of hybridizing to the target nucleic acid and provided that the amplifying restriction endonuclease of the probe nucleic acid is capable of cleaving its amplifying restriction endonuclease cut site after the probe nucleic acid is cleaved by a recognition restriction endonuclease.
  • the single-stranded section of the probe nucleic acid that is designed to hybridize with a desired target nucleic acid can be between about 10 and about 500 or more nucleotides (e.g., between about 10 and about 400 nucleotides, between about 10 and about 300 nucleotides, between about 10 and about 200 nucleotides, between about 10 and about 100 nucleotides, between about 10 and about 50 nucleotides, between about 10 and about 25 nucleotides, between about 20 and about 500 nucleotides, between about 30 and about 500 nucleotides, between about 40 and about 500 nucleotides, between about 50 and about 500 nucleotides, between about 15 and about 50 nucleotides, between about 15 and about 25 nucleotides, between about 20 and about 50 nucleotides, or between about 18 and about 25 nucleotides) in length.
  • nucleotides e.g., between about 10 and about 400 nucleotides, between about 10 and about 300 nu
  • the recognition restriction endonuclease cut site that will be created by the hybridization of target nucleic acid to this single-stranded section of the probe nucleic acid can be located at any position alone the single-stranded section.
  • the recognition restriction endonuclease cut site to be created can be towards the 5 ' end, towards the '3 end, or near the center of the single-stranded section of the probe nucleic acid.
  • the overall length of the probe nucleic acid described herein can be between about 10 and about 2500 or more nucleotides (e.g., between about 10 and about 2000 nucleotides, between about 10 and about 1000 nucleotides, between about 10 and about 500 nucleotides, between about 10 and about 400 nucleotides, between about 10 and about 300 nucleotides, between about 10 and about 200 nucleotides, between about 10 and about 100 nucleotides, between about 10 and about 50 nucleotides, between about 10 and about 25 nucleotides, between about 20 and about 500 nucleotides, between about 30 and about 500 nucleotides, between about 40 and about 500 nucleotides, between about 50 and about 500 nucleotides, between about 75 and about 500 nucleotides, between about 100 and about 500 nucleotides, between about 150 and about 500 nucleotides, between about 15 and about 50 nucleotides, between about 15 and about 25 nucleotides, between between about
  • the recognition restriction endonuclease cut site to be created by hybridization of target nucleic acid to the probe nucleic acid can be a cut site of any type of restriction endonuclease.
  • any type of restriction endonuclease can be used as a recognition restriction endonuclease to cleave probe nucleic acid upon target nucleic acid hybridization.
  • restriction endonucleases that can be used as recognition restriction endonucleases include, without limitation, EcoRI, EcoRII, BamHI, Hindlll, Taql, Notl, Hinfl, Sau3A, PovII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl, Pstl, Sad, Sail, Seal, Sphl, Stul, Xbal, Aarl, Banll, BseGI, BspPI, CM, EcoNI, Hsp92II, NlalV, Rsal, Tail, Aasl, Bbsl, BseJI, BspTI, Clal, EcoO109I, I-Ppol, NmuCI, RsrII, Taqal, Aatll, Bbul, BseLI, BsrBI, Cpol, , Kasl, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, ,
  • nucleic acid encoding a naturally-occurring restriction endonuclease can be genetically engineered to create a modified restriction endonuclease that has the ability to recognize a particular cut site.
  • Common computer algorithms can be used to locate restriction endonuclease cut sites along the nucleotide sequence of any desired target nucleic acid.
  • sequence of the restriction endonuclease cut site along with additional flanking sequence can be used to design the complementary sequence of the probe nucleic acid that is used to hybridize to the target nucleic acid and create the recognition restriction endonuclease cut site upon target nucleic acid hybridization.
  • flanking sequence e.g., 5' flanking sequence, 3' flanking sequence, or both 5 ' and 3 ' flanking sequence
  • probe nucleic acid can be designed to have a single-stranded section that is designed to hybridize with desired target nucleic acid and to form a single recognition restriction endonuclease cut site upon target nucleic acid hybridization.
  • probe nucleic acid can be designed to have a single-stranded section that is designed to hybridize with desired target nucleic acid and to form more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) recognition restriction endonuclease cut site upon target nucleic acid hybridization.
  • the multiple recognition restriction endonuclease cut sites can be cut sites for the same restriction endonuclease or cut sites for different restriction endonucleases.
  • probe nucleic acid can be designed to have a single-stranded section that is designed to hybridize with desired target nucleic acid and to form one recognition restriction endonuclease cut site for an EcoRI recognition restriction endonuclease and one recognition restriction endonuclease cut site for an Xbal recognition restriction endonuclease upon target nucleic acid hybridization.
  • each recognition restriction endonuclease can be used individually or in combination (e.g., as a mixture) to cleave probe nucleic acid that hybridized to target nucleic acid and formed the corresponding recognition restriction endonuclease cut site via such hybridization.
  • Probe nucleic acid can be designed such that any target nucleic acid can be detected.
  • target nucleic acid examples include, without limitation, human nucleic acid, microbial nucleic acid (e.g., bacterial, fungal, or protozoan nucleic acid), viral nucleic acid, nucleic acid containing a point mutation, SNP, or gene rearrangement, mammalian nucleic acid, methylated nucleic acid, and mR A.
  • restriction endonucleases having the ability to cleave a recognition restriction endonuclease cut site that is created between a DNA section of the probe nucleic acid and the R A target nucleic acid can be used as recognition restriction endonucleases.
  • restriction endonucleases include, without limitation, Hhal, Alul, Taql, Haelll, EcoRI, Hindll, Sail, and Mspl restriction endonucleases.
  • restriction endonucleases having the ability to cleave a recognition restriction endonuclease cut site that includes a methylated nucleotide to be assessed can be used as recognition restriction endonucleases.
  • restriction endonucleases having the ability to recognize methylated nucleotides include, without limitation, Dpnl, Glal, Hpall, Mspl, Acil, Hhal, and Sssl restriction endonucleases.
  • a control can include detecting the same target nucleic acid without the methylated nucleotide.
  • a combination of methylation insensitive and methylation sensitive restriction endonucleases can be used to assess a sample for methylated target nucleic acid. For example, similar generation of cleavage products using both methylation insensitive and methylation sensitive restriction endonucleases designed for the same site can indicate that the target nucleic acid lacks methylation at that site, while an increased level of cleavage products using a methylation insensitive restriction endonuclease as compared to the level generated using a methylation sensitive restriction endonuclease designed for the same site can indicate that the target nucleic acid is methylated at that site.
  • the methods described herein are flexible and adaptable to new DNA targets by designing corresponding target-specific probes for the recognition step.
  • the main probe design feature is the presence of a restriction site for a recognition restriction
  • the RCEA amplification step can be common for many different assays as described herein.
  • the nucleotide sequence of target nucleic acid to be detected can be obtained from, for example, common nucleic acid databases such as GenBank ® .
  • a portion of target nucleic acid sequence can be selected using a computer-based program.
  • a computer-based program can be used to detect restriction endonuclease cut sites within a portion of target nucleic acid. Such information can be used to design probe nucleic acid such that the single-stranded section creates at least one recognition restriction endonuclease cut site upon hybridization of the target nucleic acid.
  • nucleic acid component of the probe nucleic acid can be synthesized using commercially available automated oligonucleotide synthesizers such as those available from Applied Biosystems (Foster City, CA).
  • probe nucleic acids can be synthesized de novo using any of a number of procedures widely available in the art. Examples of such methods of synthesis include, without limitation, the ⁇ - cyanoethyl phosphoramidite method (Beaucage et al, Tet.
  • nucleic acid synthesizers In some cases, recombinant nucleic acid techniques such as PCR and those that include using restriction enzyme digestion and ligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA) can be used to obtain the nucleic acid component of the probe nucleic acid.
  • recombinant nucleic acid techniques such as PCR and those that include using restriction enzyme digestion and ligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA) can be used to obtain the nucleic acid component of the probe nucleic acid.
  • Probe nucleic acid described herein can be attached to a solid support.
  • solid supports include, without limitation, a well of a microtiter plate (e.g., a 96-well microtiter plate or ELISA plate), beads (e.g., magnetic, glass, plastic, or gold-coated beads), slides (e.g., glass or gold-coated slides), micro- or nano-particles (e.g., carbon nanotubes), platinum solid supports, palladium solid supports, and a surface of a chamber or channel within a microfluidic device.
  • a microtiter plate e.g., a 96-well microtiter plate or ELISA plate
  • beads e.g., magnetic, glass, plastic, or gold-coated beads
  • slides e.g., glass or gold-coated slides
  • micro- or nano-particles e.g., carbon nanotubes
  • platinum solid supports palladium solid supports, and a surface of a chamber or channel within
  • a solid support can be a ceramic based solid support (e.g., a silicon oxide-based solid support), a plastic polymer-based solid support (e.g., a nylon, nitrocellulose, or polyvinylidene fluoride -based solid support), a metal based solid support, or a biopolymer-based (e.g., an agarose, cross- linked dextran, or cellulose-based solid support) solid support.
  • Probe nucleic acid can be directly or indirectly attached to a solid support.
  • biotin can be a component of the probe nucleic acid, and the probe nucleic acid containing biotin can be indirectly attached to a solid support that is coated with streptavidin via a biotin-streptavidin interaction.
  • probe nucleic acid can be attached to a solid support via a covalent or non-covalent interaction.
  • probe nucleic acid can be covalently attached to magnetic beads as described elsewhere (Albretsen et al., Anal. Biochem., 189(1):40-50 (1990)).
  • Probe nucleic acid can be designed to contain any type of restriction endonuclease as an amplifying restriction endonuclease.
  • an amplifying restriction endonuclease of the probe nucleic acid is typically a different restriction endonuclease than the restriction endonuclease that is used as a recognition restriction endonuclease.
  • a restriction endonuclease other than an EcoRI restriction endonuclease e.g., a Hindlll or BamHI restriction endonuclease
  • restriction endonucleases that can be used as amplifying restriction endonucleases include, without limitation, EcoRI, EcoRII, BamHI, Hindlll, TaqI, NotI, Hinfl, Sau3A, PovII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl, PstI, Sad, Sail, Seal, Sphl, Stul, Xbal, Aarl, Banll, BseGI, BspPI, CM, EcoNI, Hsp92II, NlalV, Rsal, Tail, Aasl, Bbsl, BseJI, BspTI, Clal, EcoO109I, I-Ppol, NmuCI, RsrII, Taqal, Aatll, Bbul, BseLI, BsrBI, Cpol, Kasl, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, ,
  • Restriction endonucleases such as Time-SaverTM Qualified or a High Fidelity (HF ® ) restriction endonuclease from New England BioLabs, Inc. (Ipswich, MA) can be be used in order to complete restriction endonuclease reactions in less time, and/or prevent cleavage activity at non-cognate sites (star activity) under a wide range of reaction conditions.
  • HF ® High Fidelity
  • an amplifying restriction endonuclease can have one or more amino acid additions, deletions, or substitutions relative to a naturally-occurring sequence and be used as an amplifying restriction endonuclease. Substitutions can be made such that catalytically activity conjugates can be produced. Amino acid substitutions typically are located at positions on the surface of the restriction endonuclease that are considered non-essential to activity. Amino acid substitutions can be conservative or non- conservative. Conservative amino acid substitutions replace an amino acid with an amino acid of the same class, whereas non-conservative amino acid substitutions replace an amino acid with an amino acid of a different class.
  • conservative substitutions include amino acid substitutions within the following groups: (1) glycine and alanine; (2) valine, isoleucine, and leucine; (3) aspartic acid and glutamic acid; (4) asparagine, glutamine, serine, and threonine; (5) lysine, histidine, and arginine; and (6) phenylalanine and tyrosine.
  • Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class.
  • Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product.
  • Non- conservative amino acid substitutions also can make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue.
  • non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid or a polar amino acid for an acidic amino acid.
  • a mutant restriction endonuclease e.g., an EcoRI restriction endonuclease described elsewhere (Dylla-Spears, et al.. Analyt. Chem., 81, 10049-10054 (2009)) having a cysteine substituted for a lysine at residue 249 can be used as an amplifying restriction endonuclease.
  • a wild-type or mutant BamHI restriction endonuclease can be used.
  • a BamHI restriction endonuclease having the amino acid set forth in GenBank® accession number P23940.1 (GI No. 135217) where a serine is substituted for the cysteines at residues 34, 54, and 64 (C34S/C54S/C64S) and cysteine is substituted for serine at residue 17 (S17C) can be used as an amplifying restriction endonuclease.
  • Such a restriction endonuclease can be designated BamHI-S17C/C34S/C54S/C64S.
  • a BamHI restriction endonuclease having the amino acid set forth in GenBank® accession number P23940.1 (GI No. 135217) where cysteine is substituted for serine at residue 17 (S17C) can be used as an amplifying restriction endonuclease.
  • Such a restriction endonuclease can be designated BamHI-S17C.
  • a single probe nucleic acid molecule can contain one, two, three, four, five, or more EcoRI amplifying restriction endonuclease molecules.
  • a single probe nucleic acid molecule can contain two or more (e.g., two, three, four, five, or more) different types of amplifying restriction endonucleases.
  • a single probe nucleic acid molecule can contain three EcoRI amplifying restriction endonuclease molecules and two Banll amplifying restriction endonuclease molecules.
  • an amplifying restriction endonuclease can be attached by an ionic or covalent attachment.
  • covalent bonds such as amide bonds, disulfide bonds, and thioether bonds, or bonds formed by crosslinking agents can be used.
  • a non-covalent linkage can be used.
  • the attachment can be a direct attachment or an indirect attachment.
  • a linker can be used to attach an amplifying restriction endonuclease to a nucleic acid component of the probe nucleic acid.
  • nucleic acid can include a thiol modification
  • a restriction endonuclease can be conjugated to the thiol-containing nucleic acid based on succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate (SMCC) using techniques similar to those described elsewhere (Dill et al., Biosensors and Bioelectronics, 20:736-742 (2004)).
  • SMCC succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate
  • a biotinylated nucleic acid and a streptavidin-containing restriction endonuclease can be attached to one another via a biotin-streptavidin interaction.
  • a restriction endonuclease can be conjugated with streptavidin using, for example, sulfosuccinimidyl 6-(3'-[2-pyridyldithio]- propionamido)hexanoate.
  • An amplifying restriction endonuclease can be attached at any location of a nucleic acid component of the probe nucleic acid.
  • an amplifying restriction endonuclease can be attached at an end (e.g., a 5' end or 3' end) of a nucleic acid component, in the middle of a nucleic acid component, or at any position along the length of a nucleic acid component.
  • Reporter nucleic acids described herein contain a double-stranded section of nucleic acid, where one strand of the nucleic acid contains at least one amplifying restriction endonuclease and the other strand contains at least one reporter molecule, and wherein the double-stranded section includes an amplifying restriction endonuclease cut site.
  • the reporter nucleic acid can include DNA, RNA, nucleic acid analogs, or other molecules.
  • one strand of a reporter nucleic acid can include biotin such that the reporter nucleic acid can be attached to a streptavidin-coated solid support.
  • one or both strands of the double-stranded section of the reporter nucleic acid that contains an amplifying restriction endonuclease cut site can be RNA or a nucleic acid analog (e.g., a peptide nucleic acid (PNA)) provided that such a double-stranded section is capable of being cleaved by an amplifying restriction endonuclease.
  • PNA peptide nucleic acid
  • restriction endonucleases that can be used as amplifying restriction endonucleases to cleave a DNA:RNA hybrid section of reporter nucleic acid include, without limitation, Hhal, Alul, Taql, Haelll, EcoRI, Hindll, Sail, and Mspl restriction endonucleases.
  • Reporter nucleic acid described herein can be any length provided that the double- stranded section that contains an amplifying restriction endonuclease cut site is capable of being cleaved by released amplifying restriction endonuclease of probe nucleic acid and/or of reporter nucleic acid.
  • the double-stranded section of reporter nucleic acid can be between about 10 and about 500 or more nucleotides (e.g., between about 10 and about 400 nucleotides, between about 10 and about 300 nucleotides, between about 10 and about 200 nucleotides, between about 10 and about 100
  • the double-stranded section of reporter nucleic acid can be between 5 and 50 nucleotides in length.
  • An amplifying restriction endonuclease cut site of a reporter nucleic acid can be located at any position alone the double-stranded section.
  • an amplifying restriction endonuclease cut site can be towards the 5' end, towards the '3 end, or near the center of the double- stranded section of a reporter nucleic acid.
  • the overall length of a reporter nucleic acid described herein can be between about 10 and about 2500 or more nucleotides (e.g., between about 10 and about 2000 nucleotides, between about 10 and about 1000 nucleotides, between about 10 and about 500 nucleotides, between about 10 and about 400 nucleotides, between about 10 and about 300 nucleotides, between about 10 and about 200 nucleotides, between about 10 and about 100 nucleotides, between about 10 and about 50 nucleotides, between about 10 and about 25 nucleotides, between about 20 and about 500 nucleotides, between about 30 and about 500 nucleotides, between about 40 and about 500 nucleotides, between about 50 and about 500 nucleotides, between about 75 and about 500 nucleotides, between about 100 and about 500 nucleotides, between about 150 and about 500 nucleotides, between about 15 and about 50 nucleotides, between about 15 and about 25 nucleotides,
  • reporter nucleic acid can be designed to have a double-stranded section that contains a single amplifying restriction endonuclease cut site.
  • reporter nucleic acid provided herein can be designed to have a double-stranded section that contains more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) amplifying restriction endonuclease cut site.
  • the multiple amplifying restriction endonuclease cut sites can be cut sites for the same restriction endonuclease or cut sites for different restriction endonucleases.
  • reporter nucleic acid can be designed to have a double-stranded section that contains one amplifying restriction endonuclease cut site for an EcoRI amplifying restriction endonuclease and one amplifying restriction
  • nucleic acid component of a reporter nucleic acid Any appropriate method can be used to obtain the nucleic acid component of a reporter nucleic acid.
  • common molecular cloning and chemical nucleic acid synthesis techniques can be used to obtain nucleic acid components of a reporter nucleic acid.
  • nucleic acid components of a reporter nucleic acid can be synthesized using commercially available automated oligonucleotide synthesizers such as those available from Applied Biosystems (Foster City, CA).
  • reporter nucleic acid can be synthesized de novo using any of a number of procedures widely available in the art.
  • Examples of such methods of synthesis include, without limitation, the ⁇ -cyanoethyl phosphoramidite method (Beaucage et al, Tet. Let., 22: 1859-1862 (1981)) and the nucleoside H-phosphonate method (Garegg et al., Tet. Let., 27:4051-4054 (1986); Froehler et al, Nucl. Acid Res., 14:5399-5407 (1986); Garegg et al, Tet. Let., 27:4055-4058 (1986); and Gaffney et al, Tet. Let., 29:2619-2622 (1988)). These methods can be performed by a variety of commercially-available automated
  • oligonucleotide synthesizers In some cases, recombinant nucleic acid techniques such as PCR and those that include using restriction enzyme digestion and ligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA) can be used to obtain the nucleic acid component of reporter nucleic acid.
  • recombinant nucleic acid techniques such as PCR and those that include using restriction enzyme digestion and ligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA) can be used to obtain the nucleic acid component of reporter nucleic acid.
  • Reporter nucleic acid described herein can be attached to a solid support.
  • solid supports include, without limitation, a well of a microtiter plate (e.g., a 96-well microtiter plate or ELISA plate), beads (e.g., magnetic, glass, plastic, or gold- coated beads), slides (e.g., glass or gold-coated slides), micro- or nano-particles (e.g., carbon nanotubes), platinum solid supports, palladium solid supports, and a surface of a chamber or channel within a microfluidic device.
  • a microtiter plate e.g., a 96-well microtiter plate or ELISA plate
  • beads e.g., magnetic, glass, plastic, or gold- coated beads
  • slides e.g., glass or gold-coated slides
  • micro- or nano-particles e.g., carbon nanotubes
  • platinum solid supports palladium solid supports, and a surface of a chamber or channel within a microfluidic device.
  • a solid support can be a silicon oxide-based solid support, a plastic polymer-based solid support (e.g., a nylon, nitrocellulose, or polyvinylidene fluoride-based solid support) or a biopolymer-based (e.g., agarose, a cross-linked dextran or cellulose-based solid support) solid support.
  • a plastic polymer-based solid support e.g., a nylon, nitrocellulose, or polyvinylidene fluoride-based solid support
  • a biopolymer-based e.g., agarose, a cross-linked dextran or cellulose-based solid support
  • Reporter nucleic acid can be directly or indirectly attached to a solid support via either strand of the reporter nucleic acid.
  • the strand of the reporter nucleic acid that includes the reporter molecule can be directly or indirectly attached to the solid support, and the strand of the reporter nucleic acid that includes the amplifying restriction endonuclease can be attached to the solid support via its association with the other strand.
  • the strand of the reporter nucleic acid that includes the amplifying restriction endonuclease can be directly or indirectly attached to the solid support and the strand of the reporter nucleic acid that includes the reporter molecule can be attached to the solid support via its association with the other strand.
  • biotin can be a component of one strand of a reporter nucleic acid, which can be indirectly attached to a solid support that is coated with streptavidin via a biotin-streptavidin interaction.
  • one strand of a reporter nucleic acid can be attached to a solid support via a covalent or non-covalent interaction.
  • reporter nucleic acid can be covalently attached to magnetic beads as described elsewhere (Albretsen et al., Anal. Biochem., 189(l):40-50 (1990)).
  • Probe nucleic acid 101 and reporter nucleic acid 121 can have various
  • probe nucleic acid 101 can be designed to have a single nucleic acid strand such that the entire nucleic acid component of probe nucleic acid 101 is single-stranded, prior to contact with target nucleic acid 104.
  • Probe nucleic acid 101 can be, for example, in a reaction chamber 100 (e.g., a microtiter plate well) and attached to solid support 102 such that amplifying restriction endonuclease 103 is released from solid support 102 upon cleavage of a nucleic acid component of probe nucleic acid 101 by a recognition restriction
  • the reaction product from first reaction chamber 100 containing released portion 107 (containing the amplifying restriction endonuclease 103), if target nucleic acid 104 was present, can be transferred (e.g., manually or automatically) to second reaction chamber 120.
  • Second reaction chamber 120 can contain reporter nucleic acid 121, wherein one strand of reporter nucleic acid 121 includes a reporter molecule (e.g., a label) 123 (RM) and one strand includes amplifying restriction endonuclease 103.
  • the double stranded portion of reporter nucleic acid 121 contains at least one amplifying restriction endonuclease cut site 124.
  • Reporter nucleic acid 121 can be attached (e.g., immobilized) to solid support 122 via either strand of the reporter nucleic acid.
  • Reporter molecule 123 and amplifying restriction endonuclease 103 are released from solid support 122 upon cleavage at the restriction endonuclease cut site of the amplifying restriction endonuclease of reporter nucleic acid 121.
  • probe nucleic acid 101 can be designed to have first strand 128 and second strand 108.
  • First strand 128 can be attached to solid support 102 and can be designed to have a single-stranded section having a nucleotide sequence that is complementary to at least a portion of target nucleic acid 104.
  • Second strand 108 can include amplifying restriction endonuclease 103 and can have a single-stranded section having a nucleotide sequence that can hybridize to first strand 128.
  • first strand 128 and second strand 108 can be synthesized or obtained separately and then mixed together to form probe nucleic acid 101.
  • first strand 128 can be synthesized, biotinylated, and attached to a streptavidin- coated solid support. After synthesizing the nucleic acid component of second strand 108 and attaching amplifying restriction endonuclease 103 to the synthesized nucleic acid component, second strand 108 can be incubated with first strand 128 to form nucleic acid probe 101.
  • probe nucleic acid 101 can contain more than two strands.
  • probe nucleic acid can include first strand 150, second strand 152, and third strand 154.
  • first strand 150 can be attached to solid support 102
  • second strand 152 can be hybridized to first strand 150 and can include a single-stranded section having a nucleotide sequence that is complementary to at least a portion of target nucleic acid 104
  • third strand 154 can be hybridized to second strand 152 and can be attached to amplifying restriction endonuclease 103.
  • Similar strand configurations can be used to make reporter nucleic acid containing two or more strands.
  • a buffer can be used to suppress restriction endonuclease activity during the process of making and/or assembling probe nucleic acids or reporter nucleic acids that include a restriction endonuclease.
  • a buffer having salt e.g., at least about 100 mM NaCl, 125 mM NaCl, 150 mM NaCl, 175 mM NaCl, or 200 mM NaCl
  • no magnesium can be used when attaching a reporter nucleic acid to a solid substrate to reduce or limit self-cleavage of the reporter nucleic acid.
  • the solid substrate can be washed to remove any unattached material, and the buffer/inhibitor can be removed or replaced with a buffer that supports enzymatic activity of the restriction endonuclease (e.g., a buffer with 10 mM MgCk and 50 mM NaCl).
  • Reporter nucleic acid containing a restriction endonuclease and a cleavage site for that restriction endonuclease can exhibit minimal or no self-cleavage once the reporter nucleic acid is attached to a solid surface.
  • reporter molecules that can be used to detect cleaved reporter nucleic acid.
  • reporter molecules that can be a component of reporter nucleic acid include, without limitation, fluorescent labels (with or without the use of quenchers), dyes, antibodies, radioactive material, enzymes (e.g., horse radish peroxidase, alkaline phosphatese, laccase, galactosidase, or luciferase), redox labels (e.g., ferrocene redox labels), metallic particles (e.g., gold nanoparticles), or green fluorescent protein-based labels.
  • fluorescent labels with or without the use of quenchers
  • dyes include, without limitation, fluorescent labels (with or without the use of quenchers), dyes, antibodies, radioactive material, enzymes (e.g., horse radish peroxidase, alkaline phosphatese, laccase, galactosidase, or luciferase), redox labels (e.g., ferrocen
  • the detector can be an electrode for amperometric assay of redox molecules.
  • the electrode at high electrode potential can provide an oxidation of the reduced form of ferrocene, thereby converting it to an oxidized form of ferrocene.
  • the generated current can be proportional to the concentration of ferrocene label in the solution.
  • reporter nucleic acid can contain a fluorescent label and a quencher such that cleaved reporter nucleic acid provides a fluorescent signal and uncleaved reporter nucleic acid does not provide a fluorescent signal.
  • the reporter nucleic acid can contain a reporter molecule (e.g., a fluorescent label or an enzyme such as horse radish peroxidase) and can be attached to a solid support (e.g., a well of a microtiter plate).
  • the reporter nucleic acid can be attached to a solid support such that cleavage at an amplifying restriction endonuclease cut site by an amplifying restriction endonuclease releases a portion of the reporter nucleic acid that contains the label.
  • the resulting reaction mixture can be collected and assessed for the presence, absence, or amount of released portions of the reporter nucleic acid using the reporter molecule.
  • the released portions of the reporter nucleic acid if present, can be transferred from one well of a microtiter plate (e.g., a 96-well plate) that contained the reporter nucleic acid to another well of a microtiter plate, where the transferred material can be assessed for a signal from the reporter molecule.
  • a microtiter plate e.g., a 96-well plate
  • Any number of molecules of a label can be attached to one reporter nucleic acid molecule.
  • a reporter nucleic acid molecule can contain one, two, three, four, five, or more fluorescent molecules.
  • a label can be attached by an ionic or covalent attachment.
  • covalent bonds such as amide bonds, disulfide bonds, and thioether bonds, or bonds formed by crosslinking agents can be used.
  • a non-covalent linkage can be used.
  • the attachment can be a direct attachment or an indirect attachment.
  • a linker can be used to attach a label to a nucleic acid component of reporter nucleic acid.
  • nucleic acid can include a thiol modification
  • a label can be conjugated to the thiol-containing nucleic acid based on succinimidyl 4-[N-maleimidomethyl]cyclo-hexane-l-carboxylate (SMCC) using techniques similar to those described elsewhere (Dill et ah, 2004, supra).
  • SMCC succinimidyl 4-[N-maleimidomethyl]cyclo-hexane-l-carboxylate
  • a biotinylated nucleic acid and a streptavidin-containing label can be attached to one another via a biotin-streptavidin interaction.
  • a label can be conjugated with streptavidin using, for example, sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate.
  • a label can be attached at any location of a nucleic acid component of reporter nucleic acid.
  • a label can be attached at an end (e.g., a 5' end or 3' end) of a nucleic acid component, in the middle of a nucleic acid component, or at any position along the length of a nucleic acid component of reporter nucleic acid.
  • Reporter nucleic acid can be designed to contain any type of restriction endonuclease as an amplifying restriction endonuclease.
  • an amplifying restriction endonuclease of a report nucleic acid is typically a different restriction endonuclease than the restriction endonuclease that is used as a recognition restriction endonuclease and is typically has the same cleavage site as the amplifying restriction endonuclease of the probe nucleic acid.
  • a restriction endonuclease other than an EcoRI restriction endonuclease e.g., a Hindlll restriction endonuclease
  • an amplifying restriction endonuclease for both the probe nucleic acid and the reporter nucleic acid.
  • restriction endonucleases that can be used as amplifying restriction endonucleases include, without limitation, EcoRI, EcoRII, BamHI, Hindlll, Taql, Notl, Hinfl, Sau3A, PovII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl, Pstl, Sad, Sail, Seal, Sphl, Stul, Xbal, Aarl, Banll, BseGI, BspPI, CM, EcoNI, Hsp92II, NlalV, Rsal, Tail, Aasl, Bbsl, BseJI, BspTI, Clal, EcoO109I, I-Ppol, NmuCI, RsrII, Taqal, Aatll, Bbul, BseLI, BsrBI, Cpol, Kasl, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, ,
  • a single reporter nucleic acid molecule can contain one, two, three, four, five, or more EcoRI amplifying restriction endonuclease molecules.
  • a single reporter nucleic acid molecule can contain two or more (e.g., two, three, four, five, or more) different types of amplifying restriction endonucleases.
  • a single reporter nucleic acid molecule can contain three EcoRI amplifying restriction endonuclease molecules and two Banll amplifying restriction endonuclease molecules.
  • an amplifying restriction endonuclease can be attached by an ionic or covalent attachment.
  • covalent bonds such as amide bonds, disulfide bonds, and thioether bonds, or bonds formed by crosslinking agents can be used.
  • a non-covalent linkage can be used.
  • the attachment can be a direct attachment or an indirect attachment.
  • a linker can be used to attach an amplifying restriction endonuclease to a nucleic acid component of a reporter nucleic acid.
  • nucleic acid can include a thiol modification
  • a restriction endonuclease can be conjugated to the thiol-containing nucleic acid based on succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate (SMCC) using techniques similar to those described elsewhere (Dill et al., Biosensors and Bioelectronics, 20:736-742 (2004)).
  • SMCC succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate
  • a biotinylated nucleic acid and a streptavidin-containing restriction endonuclease can be attached to one another via a biotin-streptavidin interaction.
  • a restriction endonuclease can be conjugated with streptavidin using, for example, sulfosuccinimidyl 6-(3'-[2-pyridyldithio]- propionamido)hexanoate.
  • An amplifying restriction endonuclease can be attached at any location of a nucleic acid component of a reporter nucleic acid.
  • an amplifying restriction endonuclease can be attached at an end (e.g., a 5' end or 3' end) of a nucleic acid component, in the middle of a nucleic acid component, or at any position along the length of a nucleic acid component.
  • the methods and materials provided herein can be used to detect target nucleic acid in any type of sample.
  • blood samples, serum samples, saliva samples, nasal swab samples, stool samples, urine samples, tissue samples (e.g., tissue biopsy samples), environmental samples (e.g., water samples, soil samples, and air samples), food samples (e.g., meat samples, produce samples, or drink samples), and industrial samples (e.g., air filter samples and samples collected from work stations) can be collected and assessed for target nucleic acid.
  • tissue samples e.g., tissue biopsy samples
  • environmental samples e.g., water samples, soil samples, and air samples
  • food samples e.g., meat samples, produce samples, or drink samples
  • industrial samples e.g., air filter samples and samples collected from work stations
  • a sample to be assessed can be processed to obtain nucleic acid.
  • a nucleic acid extraction can be performed on a tissue sample to obtain a sample that is enriched for nucleic acid.
  • a sample can be
  • a sample to be assessed can be contacted with a probe nucleic acid as described herein.
  • This contacting step can be carried out for any period of time and at any temperature that allows target nucleic acid to hybridize with probe nucleic acid.
  • this step can be performed between 10 seconds and 24 hours (e.g., between 30 seconds and 12 hours, between 30 seconds and 8 hours, between 30 seconds and 4 hours, between 30 seconds and 2 hours, between 30 seconds and 1 hour, between 1 minute and 24 hours, between 1 minute and 12 hours, between 1 minute and 8 hours, between 1 minute and 4 hours, between 1 minute and 2 hours, between 1 minute and 1 hour, between 5 minutes and 1 hour, between 10 minutes and 1 hour, between 15 minutes and 1 hour, or between 30 minutes and 1 hour).
  • 10 seconds and 24 hours e.g., between 30 seconds and 12 hours, between 30 seconds and 8 hours, between 30 seconds and 4 hours, between 30 seconds and 2 hours, between 30 seconds and 1 hour, between 1 minute and 24 hours, between 1 minute and 12 hours, between 1 minute and 8 hours, between
  • the initial temperature can be between 15°C and 100°C (e.g., between 23°C and 98°C, between 23°C and 90°C, between 23°C and 85°C, between 23°C and 75°C, between 23°C and 65°C, between 23°C and 55°C, between 23°C and 45°C, between 23°C and 35°C, between 30°C and 95°C, between 30°C and 85°C, between 30°C and 75°C, between 30°C and 65°C, between 30°C and 55°C, between 30°C and 45°C, between 20°C and 40°C, between 20°C and 30°C, and between 25°C and 35°C).
  • the temperature during this contacting step can remain constant or can be increased or decreased.
  • the initial temperature can be between about 20°C and about 85°C, and then the temperature can be allowed to decrease to room temperature over a period of about 30 seconds to about 30 minutes (e.g., between about 30 seconds and about 15 minutes, between about 30 seconds and about 10 minutes, between about 1 minute and about 30 minutes, between about 1 minute and about 15 minutes, or between about 1 minute and about 5 minutes).
  • the temperature can be allowed to decrease to room temperature over a period of about 30 seconds to about 30 minutes (e.g., between about 30 seconds and about 15 minutes, between about 30 seconds and about 10 minutes, between about 1 minute and about 30 minutes, between about 1 minute and about 15 minutes, or between about 1 minute and about 5 minutes).
  • probe nucleic acid can occur in the presence of the recognition restriction endonucleases, or a separate step of adding the recognition restriction endonucleases to the reaction can be performed.
  • the recognition restriction e.g., a sample to be tested or suspected to contain target nucleic acid
  • endonuclease step can be carried out for any period of time and at any temperature that allows the recognition restriction endonuclease to cleave recognition restriction endonuclease cut sites formed by the hybridization of target nucleic acid to the probe nucleic acid.
  • this step can be performed between one second and 24 hours (e.g., between one second and 30 minutes, between one second and one hour, between five seconds and one hour, between 30 seconds and 24 hours, between 30 seconds and 12 hours, between 30 seconds and 8 hours, between 30 seconds and 4 hours, between 30 seconds and 2 hours, between 30 seconds and 1 hour, between 1 minute and 24 hours, between 1 minute and 12 hours, between 1 minute and 8 hours, between 1 minute and 4 hours, between 1 minute and 2 hours, between 1 minute and 1 hour, between 5 minutes and 1 hour, between 10 minutes and 1 hour, between 15 minutes and 1 hour, or between 30 minutes and 1 hour).
  • one second and 24 hours e.g., between one second and 30 minutes, between one second and one hour, between five seconds and one hour, between 30 seconds and 24 hours, between 30 seconds and 12 hours, between 30 seconds and 8 hours, between 30 seconds and 4 hours, between 30 seconds and 2 hours, between 30 seconds and 1 hour, between 1 minute and 24 hours, between 1 minute and 12 hours, between 1 minute and 8 hours, between 1 minute and 4 hours
  • the temperature can be between 15°C and 75°C (e.g., between 15°C and 75°C, between 15°C and 65°C, between 15°C and 55°C, between 15°C and 45°C, between 15°C and 35°C, between 15°C and 30°C, between 23°C and 55°C, between 23°C and 45°C, between 30°C and 65°C, between 30°C and 55°C, between 30°C and 45°C, between 30°C and 40°C, between 35°C and 40°C, and between 36°C and 38°C). Any appropriate concentration of recognition restriction endonuclease can be used.
  • Any appropriate concentration of recognition restriction endonuclease can be used.
  • restriction endonuclease between about 0.001 units and 1000 units (e.g., between about 0.001 units and 750 units, between about 0.001 units and 500 units, between about 0.001 units and 250 units, between about 0.001 units and 200 units, between about 0.001 units and 150 units, between about 0.001 units and 100 units, between about 0.001 units and 50 units, between about 0.001 units and 25 units, between about 0.001 units and 10 units, between about 0.001 units and 1 unit, between about 0.001 units and 0.1 units, between about 0.01 units and 1000 units, between about 0.1 units and 1000 units, between about 1 unit and 1000 units, between about 10 units and 1000 units, between about 50 units and 1000 units, between about 0.5 units and 100 units, or between about 1 unit and 100 units) of restriction endonuclease can be used.
  • Other restriction endonuclease reaction conditions such as salt conditions can be used according to manufacturer's instructions.
  • the resulting reaction product containing cleaved nucleic acid can be used in the next step.
  • cleaved nucleic acid of a reaction product can be removed from uncleaved nucleic acid and used in the next step of the method.
  • probe nucleic acid is attached to a solid support, the released portions of probe nucleic acid that contain an amplifying restriction endonuclease can be collected and placed in contact with reporter nucleic acid as described herein.
  • the resulting reaction products of a particular step can be manually or automatically (e.g., robotically) transferred to a location containing nucleic acid for the next step (e.g., reporter nucleic acid), which nucleic acid can be attached or not attached to a solid support.
  • a location containing nucleic acid for the next step e.g., reporter nucleic acid
  • one reaction of a method described herein can be carried out at one location (e.g., a chamber) of a microfluidic device or blister package device, and the reaction products that are generated can be moved to another location (e.g., another chamber) that contains nucleic acid for the next step (e.g., reporter nucleic acid) via a channel.
  • cleaved nucleic acid of a reaction product can be used in the next step of the method by removing the uncleaved nucleic acid from the reaction product.
  • a magnetic force can be used to remove the magnetic beads and any attached uncleaved nucleic acid from the reaction product.
  • Any appropriate method can be used to detect cleaved reporter nucleic acid to determine the presence, absence, or amount of target nucleic acid in a sample.
  • size separation techniques can be used to assess reaction products for cleaved reporter nucleic acid. Examples of such size separation techniques include, without limitation, gel electrophoresis and capillary electrophoresis techniques.
  • a melt curve analysis can be performed to assess reaction products for cleaved reporter nucleic acid.
  • a label can be used to aid in the detection of cleaved nucleic acid (e.g., reporter nucleic acid).
  • labels include, without limitation, fluorescent labels (with or without the use of quenchers), dyes, antibodies, radioactive material, enzymes (e.g., horse radish peroxidase, alkaline phosphatese, laccase, galactosidase, or luciferase), redox labels (e.g., ferrocene redox labels), metallic particles (e.g., gold nanoparticles), and green fluorescent protein based labels.
  • the release of fluorescently labeled portions of reporter nucleic acid from a solid support can be assessed using common fluorescent label detectors.
  • cleaved reporter nucleic acid can be detected electrochemically.
  • the reporter nucleic acid can include a ferrocene redox label.
  • Reporter nucleic acid containing ferrocene can be obtained by coupling ferrocene carboxylic acid with an amino-modified oligonucleotide using the carbodiimide reaction in the presence of an excess of ferrocene carboxylic acid.
  • the detector can be an electrode for amperometric assay of redox molecules.
  • the electrode at high electrode potential can provide an oxidation of the reduced form of ferrocene, thereby converting it to an oxidized form of ferrocene.
  • the generated current can be proportional to the concentration of ferrocene label in the solution.
  • the methods and materials provided herein can be used to assess one or more samples for target nucleic acid in real-time.
  • a fluorescent label/quencher system or an electrochemical redox label system can be used to detect cleavage of reporter nucleic acid in real time.
  • the methods and materials provided herein can be used to assess one or more samples (e.g., two, three, four, five, six, seven, eight, nine, ten, 20, 50, 100, 500, 1000, or more) for a single type of target nucleic acid.
  • the methods and materials provided herein can be used in a multiplex manner to assess one or more samples for more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, 20, 50, 100, 500, 1000, or more) type of target nucleic acid.
  • target nucleic acid for ten different sequences e.g., ten different sequences from a single bacterial species or strain, or a different sequence from ten different bacterial species or strains
  • a different label can be used to correspond to each probe nucleic acid such that the detected signals can indicate which of the ten target nucleic acids are being detected.
  • kits for performing the methods described herein.
  • this document provides kits that clinicians, medical professionals, laboratory personnel, and researchers can use to detect any type of target nucleic acid.
  • a kit provided herein can include probe nucleic acid with or without being attached to a solid support and/or reporter nucleic acid with or without being attached to a solid support.
  • such a kit can include a recognition restriction
  • kits can be configured into a microfluidic device that allows for the movement of probe nucleic acid, reporter nucleic acid, or recognition restriction endonucleases (or any combination thereof) as well as a cleaved portion of any such nucleic acid in a manner that allows a detection method provided herein to be carried out with or without the nucleic acid being attached to a solid support.
  • a kit provided herein can be a microfluidic device capable of receiving a sample and contacting that sample with probe nucleic acid.
  • the probe nucleic acid can be designed to include a length of nucleotides followed by the sequence complementary to the target nucleic acid, which can create a recognition restriction endonuclease cut site, followed by an amplifying restriction endonuclease.
  • the distance from the recognition restriction endonuclease cut site to the amplifying restriction endonuclease can be relatively short (e.g., 100, 50, 25, 10, or less nucleotides), while the distance from the recognition restriction endonuclease cut site to the beginning of the length of nucleotides can be relatively long (e.g., 50, 100, 150, 200, 500, 1000, 2000, or more).
  • cleavage of the probe nucleic acid at the recognition restriction endonuclease cut site can result in a relatively small portion that contains the amplifying restriction endonuclease and is capable of travelling faster than the larger uncleaved probe nucleic acid. This difference can allow the cleaved portion containing the amplifying restriction
  • a valve can be used to prevent the larger uncleaved probe nucleic acid from entering.
  • a filter can be used to limit the ability of larger uncleaved probe nucleic acid from proceeding to the next reaction location. Similar approaches can be used during other steps of a method provided herein to separate cleaved nucleic acid from uncleaved nucleic acid.
  • a kit provided herein can be a portable or self-contained device, packet, vessel, or container that can be used, for example, in field applications.
  • a kit can be configured to allow a user to insert a sample for analysis.
  • the sample can be heated (e.g., heated to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, or more °C) and/or cooled by a heating or cooling mechanism located within the kit.
  • a heating or cooling mechanism located within the kit.
  • an exothermic or endothermic chemical reaction can be initiated within the kit to increase, decrease, or maintain the temperature.
  • exothermic or endothermic chemical reactions can be carried out within the kit without being in fluid communication with the reactions of the target nucleic acid detection method.
  • An iron oxidation reaction is an example of an exothermic chemical reaction that can be used to heat a kit provided herein.
  • An endothermic chemical reaction that can be used to cool a kit provided herein can be a reaction that includes the use of ammonium chloride and water, potassium chloride and water, or sodium carbonate and ethanoic acid.
  • the kit when detecting DNA target nucleic acid, the kit can be designed to generate, if needed, enough heat to denature double stranded DNA present within the sample.
  • kits provided herein can include a temperature indicator (e.g., color indicator or thermometer) to allows a user to assess temperature.
  • a temperature indicator e.g., color indicator or thermometer
  • kits can be designed to provide a user with a "yes" or "no" indication about the presence of target nucleic acid within a tested sample.
  • a label having the ability to generate a change in pH can be used, and a visual indicator (e.g., a pH-based color indicator) can be used to inform the user of the presence of target nucleic acid based on a change in pH.
  • the mecA gene sequences from members of the Staphylococcus genus were collected using the Integrated Microbial Genomes (IMG) web site of DOE Joint Genome Institute (http://img.jgi.doe.gov/).
  • IMG Integrated Microbial Genomes
  • the sequence alignment in Clustal W showed very high conservation (nearly 100% identity over 2 kb length) of this gene among the MRSA strains.
  • a mecA gene fragment commonly used for qPCR (McDonald, et al. (2005) J Clin
  • Microbiol 43: 6147-6149 was selected.
  • the mecA amplicon (196 bp) was generated as described elsewhere (McDonald, et al., 2005, supra) using the primers: MCA-For, 5 '-GGC AATATTACCGCACCTC A-3 ' (starting at position 1644 of mecA gene alignment, SEQ ID NO: 1), and MCA-Rev, 5'- GTCTGCCACTTTCTCCTTGT-3 ' (starting at position 1820, SEQ ID NO: 2).
  • the PCR reaction mixture was prepared from 25 of iQ Supermix (Bio-Rad Laboratories, Inc., Hercules, CA), 1 ⁇ , of both forward and reverse primers (20 ⁇ ), 0.01 to 10 ng of template strain TCH1516 genomic DNA, and nuclease-free water to a total reaction volume of 50 ⁇ .
  • PCR was performed using one cycle of denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 90 sec, with the final extension step at 72°C for 7 min.
  • amplicons were analyzed by agarose gel electrophoresis for the presence of a single band of 196 bp.
  • the amplicon was purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA), and DNA concentrations were measured with a NanoDrop 3300 Fluorospectrometer with the PicoGreen reagent (Thermo Scientific, Wilmington, DE).
  • a detailed restriction map of the amplicon (minus the primers) of 175 bp in length was built.
  • 6 Class II restriction enzymes variant isoschizomers recognizing the same sequence were considered as one enzyme
  • BgUl had a relatively long, 6 bp palindromic restriction site AGATCT.
  • the amplicon sequences flanking the BgUl site from both sides had relatively high sequence complexity and did not contain repeat sequences or form stable secondary structures (that may impede the target-probe hybridization) as assessed using DINAMelt software (Markham and Zuke (2005) Nucl Acids Res 33: W577-W581).
  • a 40-mer probe designated MCA-BG (5'- CAATTAAGTTTGCATAAGATCTATAAAT- ATCTTCTTT ATG-3 ' (SEQ ID NO: 3), was designed from the amplicon sequence with the BgUl site in the center (underlined in SEQ ID NO:3).
  • the probe was modified with biotin at the 5' end for surface attachment, and a thiol group was added to the 3' end for conjugation to the molecular marker HRP.
  • the reaction mixture was purified from the excess of DTT by applying twice to P-6 columns (Bio- Rad). Finally, equal volumes (90 ⁇ _, each) of the purified SH-modified oligonucleotide solution and activated HRP were mixed together, and the reaction was incubated at 4°C overnight.
  • the resultant HRP-oligonucleotide conjugate, MCA-BG-HRP contained a 5 ⁇ oligonucleotide concentration with an excess of unbound HRP.
  • the preparation was used directly for surface immobilization.
  • target hybridization to the surface-immobilized HRP -probes, working solutions of target oligonucleotides (0 - 100 nM) were prepared in PBS.
  • the positive control was the fully complementary target 40-mer, and negative control was PBS without oligonucleotides added.
  • Test and control target solutions were added to the wells (20 ⁇ per well) coated with the HRP -probes. Hybridization was performed at 37°C for 30-60 min with gentle shaking (100 rpm), and unbound targets were removed by washing 6X with PBST and 2X with PBS.
  • the general scheme of the proposed restriction enzyme assay is shown in Figure 1.
  • the oligonucleotide probe was conjugated to HRP to generate the MCA-BG-HRP conjugate.
  • the conjugate was attached to the streptavidin coating of ELISA plate wells via the 5' biotin ( Figure 1 A).
  • a single-stranded (ss) target DNA (an oligonucleotide or a denatured PCR am licon) was hybridized to the immobilized probes ( Figure IB).
  • a restriction reaction was carried out using the BgUl enzyme, which was specific for the target-probe ds DNA hybrid ( Figure 1C).
  • the restriction enzyme cleaved its cognate site which was formed by the DNA hybrid, releasing the HRP marker into the reaction solution.
  • the reaction solution was transferred into a new well and mixed with an HRP substrate for colorimetric detection ( Figure ID).
  • oligonucleotide target Table 1. Oligonucleotide probe and targets. Capital letters show sequences that are cognate between a target oligonucleotide and the robe, with the restriction site shown in bold.
  • Target length 30-mer GAAGATATTTATAGATCTTATGCAAACTTA 29 30 30 65.8
  • Target length 22-mer ATATTTATAGATCTTATGCAAA 30 22 22 58.6
  • Target length 20-mer TATTTATAGATCTTATGCAA 31 20 20 57.3
  • Target length 18-mer ATTTATAGATCTTATGCA 33 18 18 55.4
  • Tm was calculated for a target-probe hybrid in PBS (150 mM Na + ).
  • the assay generated relatively high HRP signals. This was true even if the duplex had additional non-complementary sequences.
  • Example 3 Sensitivity of the restriction enzyme assay for amplicon detection is similar to that of fluorogenic-based qPCR.
  • the restriction enzyme assay was used for detection of the 196 bp mecA amplicon (described in Example 1) as follows.
  • the ds amplicon was generated by PCR using the purified MRS A genomic DNA (strain TCH1516) as a template. Purified amplicons were diluted in PBS as described above for oligonucleotide targets (Example 1). To use unpurified dsDNA amplicons, the PCR reaction mixture was collected either at the end- point or at an intermediate cycle during PCR. Serial dilutions were made using the pre- cycling PCR mixture (containing the primers and template, but no amplicon). When required, heterologous mouse genomic DNA was added as the last step to each dilution, at 100 ng per well.
  • test samples containing dsDNA targets were denatured to make the target strand available for hybridization to the immobilized probes by heating at 95°C for 5 minutes followed by incubation on ice for 2 minutes, and then immediately added to the wells coated with the HRP conjugate for target-probe hybridization. Hybridization was performed at 37°C for 30 minutes with gentle shaking (100 rpm), and unbound targets were removed by washing as described herein for oligonucleotide targets (Example 1).
  • the restriction enzyme cleavage of the hybridized target-probe dsDNA was done using 20 of the reaction mixture per well.
  • the mixture contained 1 : 10 dilution of 10X NEBuffer 3 in nuclease-free water and 0.5 U/ (1 :20 dilution of the stock) of BgUl restriction enzyme (New England Biolabs, Ipswich, MA).
  • BgUl restriction enzyme New England Biolabs, Ipswich, MA.
  • the restriction protocol recommended by the manufacturer was used with the omission of bovine serum albumin (BSA) from the reaction mixture (since BSA presence is known to increase the HRP substrate oxidation background).
  • BSA bovine serum albumin
  • the restriction reaction was incubated at 37°C for 1 hour with gentle shaking (100 rpm). Finally, to quantify HRP released due to the restriction cleavage, each reaction mixture was transferred to a new ELISA plate well containing 100 ⁇ of the BioFX TMB One Component HRP
  • the HRP-generated signal was quantified by the blue color formation measured colorimetrically at the wavelength of 655 nM, using an iMark Microplate Reader (Bio-Rad).
  • OD 6 55 measurements were subjected to background subtraction using the corresponding negative control values.
  • the negative controls were prepared from PBS with no targets added.
  • the negative control was the unpurified PCR mixture (complete with the primers and template), stored on ice for the duration of the experiment without cycling.
  • Replicates of negative control values (at least four replicates per experiment) were used to calculate the mean background values, which were then used for subtraction. Experiments were performed in duplicate or triplicate, with the replicate values used for calculation of mean and standard deviation for each target. For calibration curves, the background-subtracted mean OD 6 55 values were plotted against the target concentrations.
  • the data were additionally normalized to construct calibration curves for the direct comparison of purified versus non-purified amplicons.
  • the maximum background-corrected signal (generated with the highest 100 nM concentration of target oligonucleotide) was specified as 100%, and all other values in the series were expressed as the percentages of the maximum.
  • the mecA amplicon was evaluated in the presence of a large excess of non-cognate, heterologous DNA.
  • Serial dilutions of the PCR mixture after cycling were supplemented with either 0 or 100 ng of mouse genomic DNA (open and closed circles/diamonds in Figure 7, respectively).
  • Negative control dilutions were prepared using the same PCR mixture prior to cycling (no amplicon) with 100 ng of mouse genomic DNA added, and they produced near zero HRP signal values (Figure 7, triangles).
  • the results obtained for the PCR mixture containing the amplicon showed almost no difference between the restriction enzyme assays performed in the presence or absence of the mouse DNA.
  • the calibration curves were similar in terms of absolute signal values, the limit of detection, and logarithmic nature of the signal dependence on target concentrations (Figure 7).
  • restriction enzyme assays with unpurified PCR mixtures were used for near real-time detection of amplicon formation.
  • qPCR was performed using 0.1 and 1 ng of MRS A genomic DNA as template, and collected aliquots of the PCR mixture every four cycles starting from the 8 th and ending with the 28 th cycle. The aliquots of the initial PCR mixture prior to cycling and at the 35 th cycle were used as the negative and positive controls, respectively.
  • the restriction enzyme assay detected the presence of amplicon starting from the 20 th and 24 th cycle for 1 and 0.1 ng of template, compared to detection by real-time PCR in prior experiments at 16.54 ⁇ 0.17 and 19.92 ⁇ 0.12 cycle, respectively.
  • the sensitivity of the restriction enzyme assay for amplicon detection was similar to that of the fluorogenic-based qPCR.
  • Example 4 Methods and Materials for restriction endonuclease Conjugating, HRP conjugate preparation, and manipulations with streptavidin beads restriction endonuclease conjugation to oligonucleotide probes and linkers to prepare for immobilization
  • the oligonucleotides for conjugation to restriction endonucleases carried amino groups at the 3 '-end. The groups were activated by reaction with succinimide of the Sulfo-SMCC reagent.
  • mutant enzymes with "non-essential" surface amino acid residues substituted with a cysteine were used: BamHI-S17C/C34S/C54S/C64S obtained from New England Biolabs and EcoRI-K249C provided by Dr.
  • the BamHI-S17C/C34S/C54S/C64S mutant was prepared as a 40 ⁇ stock solution in the buffer containing 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1 mM EDTA, 50% w/v glycerol.
  • 20 of the stock was added to 80 of the activated oligonucleotide solution for the final oligonucleotide concentration of 8 ⁇ .
  • EcoRI- K249C was provided as a 4 ⁇ stock solution in a storage buffer containing 20 mM sodium phosphate buffer, pH 7.3, 600 mM NaCl, 1 mM EDTA, 1 mM NaNs, 5% v/v DMSO, 10% w/v glycerol.
  • 100 ⁇ of the EcoRI-K249C stock was added to 40 ⁇ ⁇ of the activated oligonucleotide solution (for the final concentration of 2.9 ⁇ ). Both mixtures of mutant restriction endonucleases and activated
  • oligonucleotides were incubated for 2 hours at room temperature, and then stored at 4°C and -20°C for short-term and long-term storage, respectively.
  • endonuclease conjugate concentration values were estimated based on their oligonucleotide parts, since only full conjugates containing oligonucleotides were retained on the surface after immobilization.
  • HRP-oligonucleotide conjugates for immobilization were prepared using the oligonucleotides shown in Table 2. The conjugates were immobilized through biotin- streptavidin interactions on the surface of either streptavidin-precoated 96-microwell plates, or a streptavidin agarose bead suspension (both from Thermo Fisher Scientific). HRP was released from the surface attachment as the result of restriction endonuclease cleavage of probes and linkers. Reaction mixtures containing released HRP were transferred to wells of a 96-well ELISA microplate (Thermo Fisher Scientific).
  • an aliquot typically, 60 of the streptavidin agarose bead suspension was placed into a spin column (Pierce Spin Columns, screw cap with Luer lock, Thermo Fisher Scientific) and centrifuged at 1,500 g for 15 seconds to remove the manufacturer's buffer.
  • the agarose beads were re-suspended in 150 of 0.5 % bovine serum albumin (BSA, Thermo Fisher Scientific) solution in the RE-store buffer ('RE-store-BSA').
  • BSA bovine serum albumin
  • RE-store-BSA' RE-store buffer
  • the column was incubated for 30 minutes at room temperature in a Labquake Shaker Rotisserie (Thermo Fisher Scientific) set for 8 rpm. After incubation, the columns were centrifuged at 1,500 g for 15 seconds to remove the flow-through.
  • the prepared bead suspensions were used for conjugate immobilization in the RE-store-BSA buffer.
  • the immobilization reaction mixtures were incubated for 1 hour at room temperature in the rotisserie. Unless specified otherwise, the same washing procedure was performed after completion of immobilization.
  • the columns were centrifuged at 1,500 g for 15 seconds to remove the flow-through, and washed sequentially with: (i) RE-store-BSA buffer, 6 times, 200 each, (ii) RE-store buffer, 10 times, 200 ⁇ each, and (iii) IX NEBuffer 3 (New England Biolabs) (100 mM NaCl, 10 mM Tris-HCl, 10 mM MgCk, 1 mM DTT), 3 times, 200 ⁇ each. Each washing was done by re-suspending the agarose suspension in the buffer, and centrifugation at 1,500 g for 15 seconds to remove the flow-through.
  • the prepared bead suspensions with immobilized enzyme conjugates were stored on ice, and aliquoted into a micro-spin columns for subsequent assays (Pierce Micro-Spin Columns, Thermo Fisher Scientific).
  • reporter systems of the immobilized HRP conjugates by adding either (i) 5 ⁇ of 5 ⁇ solution of HRP-Ll-RB-Bio plus 5 ⁇ , of 10 ⁇ solution of ASL1-RB, or (ii) 5 ⁇ of 5 ⁇ solution of HRP-L2-RE-Bio plus 5 ⁇ of 10 ⁇ solution of ASL2-RE (Table 2), for testing of the BamHI- S 17C/C34S/C54S/C64S and EcoRI-K249C, respectively.
  • the agarose suspensions with immobilized restriction endonucleases were washed as described above, re-suspended in 158 ⁇ of IX NEBuffer 3, and divided into 2 aliquots of 77 ⁇ ⁇ each.
  • the bead suspensions with the immobilized HRP conjugates were washed and re-suspended in 60 ⁇ , ⁇ ⁇ NEBuffer 3, then divided into 6 aliquots of 10 ⁇ each.
  • the procedure started with the addition of 4 ⁇ of the restriction endonuclease Bglll (10 units ⁇ L) to each aliquot containing the beads with immobilized restriction endonuclease conjugates.
  • the restriction endonuclease Bglll 10 units ⁇ L
  • one aliquot was used to add 9 ⁇ of 10 ⁇ solution of the MCA-BG-Bio target oligonucleotide (for the final concentration of 1 ⁇ )
  • another, as a negative control by adding 9 ⁇ of IX NEBuffer 3 with no targets.
  • the resultant reactions were incubated at 37°C for 1 hour.
  • An HRP reporter system was prepared by treating streptavidin pre-coated wells with 50 per well of PBS solution containing HRP conjugates and the corresponding 2 nd strand oligonucleotides. Two reporters were prepared: (i) 50 nM solution of HRP-L1- RB-Bio plus 100 nM solution of ASL1-RB, or (ii) 50 nM solution of HRP-L2-RE-Bio plus 100 nM solution of ASL2-RE (see Table 2 for sequences), for testing of the BamHI- S17C/C34S/C54S/C64S and EcoRI-K249C, respectively.
  • the plate was incubated for 2 hours at room temperature, followed by washing four times with PBS supplemented with 0.5% (v/v) Tween-20 (PBST). Next, the wells were incubated with 1 : 100 dilution of saturated biotin solution in PBS for 15 minutes at room temperature to block the free streptavidin. Then the plate was washed three times with PBST and four times with PBS.
  • PBST 0.5% Tween-20
  • the mutant restriction endonuclease conjugates with the oligonucleotide probe MCA-BG-Bio (Table 1), BamHI-S17C/C34S/C54S/C64S-MCA-BG-Bio and EcoRI- K249C-MCA-BG-Bio, were used to prepare sample series in IX NEBuffer 3. Each series started with a 320 ⁇ / ⁇ conjugate solution, then it was diluted by a factor of 2 for each consecutive sample, with the last sample of 5 ⁇ / ⁇ . Negative controls were prepared from IX NEBuffer 3 with no conjugate added.
  • the dilution samples were added to the corresponding wells with the immobilized HRP conjugates (HRP-L1-RB- Bio or HRP-L2-RE-Bio, for BamHI-S17C/C34S/C54S/C64S -MCA-BG-Bio or EcoRI- K249C-MCA-BG-Bio, respectively).
  • the microwell plate was incubated at 37°C for 1 hour. Then, the reaction solutions were collected and applied for HRP detection.
  • Example 7 - RCEA assay protocol the recognition stage The agarose bead suspension was prepared and re-suspended in the RE-store-BSA buffer, 200 ⁇ , for the initial bead aliquot of 60 ⁇ ,. Then, the bead suspension was supplemented with 6 ⁇ ⁇ of either 400 nM solution of the BamHI- S17C/C34S/C54S/C64S-MCA-BG-Bio (2.4 pmol/initial aliquot) or 4 nM solution of the EcoRI-K249C-MCA-BG-Bio (24 fmol/initial aliquot).
  • the agarose suspensions were incubated for 1.5 hours at room temperature, then the reaction solution was removed by centrifugation, and the beads were washed. Finally, the beads with immobilized restriction endonuc leases were re-suspended in 216 ⁇ of IX NEBuffer 3, divided into 8 aliquots (27 each) and placed on ice prior to the recognition reaction.
  • the recognition reaction was set up using a 27 aliquot of the agarose bead suspension with the immobilized BamHI-MCA-BG-Bio.
  • the reaction solution was supplemented with 0.75 ⁇ / ⁇ of the recognition restriction endonuclease (Rrec) specific for the DNA target, Bglll (New England Biolabs), and 3 ⁇ of a target (i.e., AMC-BG-40) diluted in IX NEBuffer 3 at a desired concentration.
  • Rec recognition restriction endonuclease
  • AMC-BG-40 a target i.e., AMC-BG-40
  • negative controls were prepared using 3 ⁇ , IX NEBuffer 3 with no target added.
  • the recognition reaction was then incubated at 37°C for 1.5 hours with rotation (8 rpm), and the flow-through reaction solutions were collected by centrifugation.
  • the recognition reaction was set up using a 27 ⁇ aliquot of the agarose bead suspension with the immobilized EcoRI-MCA-BG-Bio. The reaction was performed in two steps, first a desired target analyte dilution in IX NEBuffer 3 (or a negative control) was added for hybridization to the immobilized probe conjugates. The recognition reaction was incubated at 37°C for 1 hour with rotation (8 rpm). Second, the restriction endonuclease cleavage was performed by adding the Rrec Bglll to the same reaction to achieve a 0.75 ⁇ / ⁇ concentration. The reaction mixture was incubated at 37°C for 20 minutes. Then, the flow-through reaction solutions were collected by centrifugation.
  • the recognition reaction protocol was separated into two steps for several experiments with the Ramp EcoRI-K249C.
  • the target dilutions were prepared in the RE- store buffer with addition of mouse genomic DNA to achieve 2.7 ng/ ⁇ concentration (80 ng/reaction).
  • the target mixture was hybridized to the probes without the addition of Rrec.
  • the reaction solution was then removed, and the beads were washed with (i) RE-store buffer (3 times, 200 ⁇ , each), and (ii) IX NEBuffer 3 (1 time, 200 ⁇ ).
  • the beads were re-suspended in 27 ⁇ , of IX
  • Example 8 - RCEA assay protocol the amplification stage The agarose bead suspension was re-suspended in the RE-store-BSA buffer, 200 ⁇ ⁇ for the initial bead aliquot of 60 ⁇ . To immobilize the restriction endonuclease- and HRP-linker conjugates, the bead solution was supplemented with either (i) 6 of 8 ⁇ solution of the BamHI-S17C/C34S/C54S/C64S-Ll-RB-Bio (48 pmol/initial aliquot) plus 12 ⁇ .
  • Both recognition and amplification stages were designed to use the same immobilized restriction endonuclease (i.e., the recognition reaction employing the BamHI-MCA-BG-Bio was applied for the amplification stage with the BamHI-Ll-RB- Bio/HRP-ASLl-RB conjugates).
  • the flow-through solution collected at the completion of the recognition stage (approximately 30 ⁇ per reaction) was mixed with a 10 ⁇ aliquot of the corresponding immobilized restriction endonuclease/HRP-dsDNA linker conjugate.
  • the resultant suspension was then incubated at 37°C for 75 minutes with rotation (8 rpm).
  • the incubation times varied from 0 to 75 minutes with 15 minute increments. After incubation, the reaction was centrifuged to collect the flow- through solution for HRP detection.
  • the first step of conjugate testing was to ensure that the oligonucleotides were indeed attached to the enzymes, and that the immobilized restriction endonucleases stayed on the surface under the applied assay conditions (Figure 8).
  • the mutant restriction endonuclease conjugates with the oligonucleotide probes were immobilized onto streptavidin-agarose beads through biotins of the probes. Extensive washing was used to remove all non-conjugated enzyme, then positive controls containing the target AMC-BG-40 at 1 ⁇ concentration, or negative controls containing no target were added together with the free Bglll restriction endonuclease in the restriction buffer (Figure 8A, B). After 1 hour incubation, the reaction solutions were removed from the beads and applied to the corresponding HRP reporter systems to quantify enzymatic activity of the released mutant restriction endonucleases ( Figure 8C, D).
  • the general scheme of the RCEA assay is shown in Figure 10.
  • the oligonucleotide probe MCA-BG-Bio was conjugated to Ramp, either BamHI-S17C/C34S/C54S/C64S or EcoRI-K249C.
  • the recognition chamber contained the Ramp-probe conjugate attached to agarose beads through its
  • oligonucleotide part a sample containing the target AMC-BG-40 was hybridized to the immobilized probes (Figure 10A).
  • the resultant dsDNA probe-target hybrids contained the specific recognition site of the Rrec Bglll (AGATCT).
  • Addition of this enzyme resulted in cleavage and release of Ramp into the reaction solution at a rate of approximately one free Ramp per target DNA molecule ( Figure 10B).
  • the reaction solution was next transferred to the amplification step, to a chamber containing an excess of another bead-immobilized conjugate of the same Ramp with a dsDNA oligonucleotide linker ( Figure IOC).
  • the first strand of the linker was attached to the surface through the 5'end, and to the Ramp through the 3'end.
  • the complementary second strand was conjugated to HRP through the 5'end ( Figure IOC).
  • the Ramp/HRP dsDNA linker conjugates contained the sequence of the corresponding 'self recognition site (GGATCC or GAATTC, for the BamHI-S17C/C34S/C54S/C64S or EcoRI-K249C, respectively).
  • the restriction reactions started upon addition of the free Ramp released during the recognition stage.
  • the released Ramp triggered the dsDNA linker cleavage, releasing additional Ramp, which in turn cleaved more new linkers.
  • the amplification step setup was tested to ensure that the immobilized restriction endonucleases were incapable of cleavage of their own and neighbor's linkers in the absence of free restriction endonuclease.
  • the high salt content 200 mM NaCl
  • the absence of magnesium of the applied RE-store buffer prevented restriction endonuclease cleavage.
  • agarose beads were incubated in the restriction buffer (IX NEBuffer 3) for 75 minutes. Then, the reaction solution was separated from the beads and applied for the colorimetric HRP assay.
  • the resultant HRP signals were identical to the negative control, an aliquot of the restriction buffer with no beads added.
  • the RCEA assay scheme was tested using serial dilutions of the target AMC-BG- 40, with the negative control of no target added. The mean negative control value was used to generate background-subtracted signal values for the test dilutions.
  • the RCEA assays were performed using both Ramp, and then the assay signals were expressed as the percentages of the maximum ones that were measured at 1 nM or lpM target concentrations for the BamHI-S17C/C34S/C54S/C64S or EcoRI-K249C, respectively.
  • To generate calibration curves the background-subtracted and normalized signals were plotted versus the target concentration (Figure 11).
  • the RCEA calibration curves were analyzed in parallel with the one generated using the direct restriction assay (DRA).
  • the DRA was performed using the AMC-BG- 40 target dilutions as described in Example 6 with a small modification of using streptavidin beads instead of ELISA plate wells.
  • the DRA calibration curve analysis showed the detection limit of 1 nM ( Figure 1 1).
  • the RCEA calibration curves for both Ramp showed logarithmic signal dependence on the target concentration in the range from 10 aM to 1 nM ( Figure 1 1).
  • the RCEA assay signal was above the background even at the lowest target concentration of 1 aM (10 ⁇ 18 M). The value was approximately 15% of the maximum. However, the error bars overlapped with those of the negative controls ( Figure 1 1).
  • the RCEA detection limit was evaluated as 10 "17 M, or 10 aM concentration of the oligonucleotide target. At this concentration, the assay signals were 60 to 100% of the maximum, well above the background ( Figure 1 1). Thus, the RCEA assay gained approximately 8 orders of magnitude improvement of the detection limit over the DRA.
  • the RCEA assay produced an exponentially amplified number of HRP molecules for each added DNA target.
  • the degree of signal amplification depended on (i) the restriction endonuclease enzyme turnover rate, (ii) the amplification stage duration, and (iii) the efficiency of mass transfer in the system ( Figure 1 IE).
  • the RCEA assay employed a double-phase system, thus, the reciprocal mass transfer between the solid and liquid phases was determined by multiple factors including the surface to volume ratios, mixing speeds, and chemical gradients. This test of the non- optimized RCEA system resulted in an extremely low detection limit of approximately 200 target molecules per sample, thus, approaching the detection limit of qPCR (5-100 molecules per sample).
  • Example 1 1 - Development of a real-time RCEA format Two concentrations of the oligonucleotide target AMC-BG-40, 1 and 100 fM were used to analyze the dependence between the assay signal and the duration of the amplification stage for the Ramp BamHI-S17C/C34S/C54S/C64S (Figure 12). For each time point, the signals were background-subtracted using the mean background value calculated from the corresponding negative control with no target added. Then, the data were expressed as the percentages of the maximum signal achieved for the 100 fM target concentration using the standard assay time of 75 minutes. For the high 100 fM concentration, the RCEA signal reached 50% of the maximum very quickly, at the first time point of 15 minutes ( Figure 12). In contrast, for the low concentration of 1 fJVI, the increase of signal above background was observed only after 60 minutes of incubation ( Figure 12). Thus, the time required for the RCEA assay to generate a signal exceeding the background was dependent on the initial target concentration.
  • each initial target concentration produces a separate amplification curve, and lower concentrations correspond to larger numbers of cycles (and longer times) required for the signal to exceed the background.
  • two different target concentrations applied to the RCEA assay produced two distinct curves when the signal values were plotted versus the amplification stage duration ( Figure 12).
  • the RCEA assay can be developed in a real-time format (qRCEA) for precise quantification of the initial target
  • Such format can include periodic sampling of the amplification reaction mixture and measuring of the HRP signal to plot the signal dependence over time. In some cases, it may be based on continuous electrochemical monitoring of HRP content in the reaction solution. This approach can help to evaluate the efficiency of RCEA amplification for various targets and can be used as a valuable tool for the RCEA assay optimization.
  • a partially complementary target AMC- 12/40 composed of (i) a fully cognate 12- mer portion containing the 6-mer Bglll restriction site and 3 adjacent nucleotides from each end, and (ii) non-cognate ends of 14 nucleotides each (Table 2) was applied to the RCEA assay in parallel with the fully cognate target AMC-BG-40. As described in Example 6, such targets produced zero signals, since a minimum of 16-bp target-probe duplex length was needed for significant Bglll cleavage.
  • the fully and partially cognate targets were assayed at 1 pM concentrations using the Ramp EcoRI-K249C system ( Figure 13).
  • the setup of the first recognition step was slightly changed to separate the target hybridization from the Rrec (Bglll) cleavage (see Example 9).
  • the assay signals were background-subtracted using the negative control of no oligonucleotides added, and normalized as the percentages of the AMC-BG-40 signal.
  • the partially cognate target did not produce a positive signal. In addition, it was substantially, almost 40%, below the background ( Figure 13).
  • the negative controls produced low variable signal values, probably due to some accidental Ramp release during the recognition step.
  • the partially cognate target (i) had a sequence too short for restriction endonuclease cleavage, and (ii) could still hybridize to the probes. Apparently, this target could somehow stabilize the probe-Ramp conjugates and reduce the accidental restriction endonuclease release in comparison with the negative controls with no targets added.
  • both DRA and RCEA assay showed similar performance due to the common properties of restriction endonuclease enzymes.
  • the DRA study described herein concluded that (i) point mutations in the restriction site, or (ii) probe length of 12-mer and below reduced the restriction endonuclease-generated signal to zero. The minimum probe length sufficient to generate a robust DRA signal was evaluated as 16-mer. Double, and especially triple, point mutations of the probe outside of the restriction site usually reduced the signal to 50% of the one observed for the fully cognate target. The same findings can be used for the design of RCEA assays.
  • thermophilic DNA-modifying enzymes at high temperatures, as shown for the nicking enzyme- mediated amplification (Tanner and Evans, Current Protocols in Molecular Biology, 15.14. 1 (2014)).
  • thermophilic restriction endonucleases have been used in conjunction with PCR to block the synthesis of wild-type amplicons in favor of amplifying mutant PCR products (Wei et ah, Nucleic acids research, 30, el 10 (2002)).
  • thermophilic restriction endonucleases with optimum temperatures of 50-65°C may further improve the RCEA assay stringency and thus, facilitate detection of mutations and allelic variation in target sequences outside of the restriction endonuclease recognition sites.
  • Example 13 RCEA sensitivity to foreign DNA addition
  • Mouse genomic DNA 80 ng per reaction
  • AMC-BG-40 target dilution of 1 pM was added to the AMC-BG-40 target dilution of 1 pM, and the mixture was hybridized to the immobilized Ramp conjugates without adding Rrec.
  • the reaction solution was removed, and the restriction buffer with the Rrec Bglll was added to the beads either without or after washing. If no washing was performed, the assay signal stayed at the background level ( Figure 13).
  • the excess of foreign DNA (undoubtedly, containing multiple restriction sites) interacted with both restriction endonucleases, the free Rrec, and released Ramp, efficiently removing them from the assay signal generation process.
  • the beads carrying target-probe hybrids were washed four times prior to the Rrec Bglll addition, the assay signal was restored to approximately 50% of the one observed with no foreign DNA added ( Figure 13).
  • the RCEA assays designed for analysis of complex nucleic acid mixtures can include multiple washings between the target-probe
  • the separation of target hybridization and cleavage reactions during the recognition step may also help in designing optimum hybridization stringency (i.e., salt concentrations) to distinguish fully from partially cognate DNA targets without disrupting the enzymatic reaction.

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Abstract

Cette invention concerne des procédés et des matériaux qui permettent de détecter un acide nucléique cible. Par exemple, des procédés et des matériaux pour détecter la présence ou l'absence d'un acide nucléique cible, des procédés et des matériaux pour détecter la quantité d'acide nucléique cible présente dans un échantillon, des kits pour détecter la présence ou l'absence d'un acide nucléique cible, des kits permettant de détecter la quantité d'acide nucléique cible présente à l'intérieur d'un échantillon, et des procédés de fabrication de ces kits.
PCT/US2015/062463 2014-11-26 2015-11-24 Détection d'acide nucléique WO2016086004A1 (fr)

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US11939629B2 (en) * 2017-08-01 2024-03-26 Andrei Gindilis Methods and systems that detect nucleic-acid targets
EP4156910A4 (fr) * 2020-05-29 2024-07-03 Mammoth Biosciences, Inc. Dispositif de diagnostic à nucléase programmable

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US6114117A (en) * 1996-07-25 2000-09-05 Medical Analysis Systems, Inc. Homogeneous diagnostic assay method utilizing simultaneous target and signal amplification
US6326145B1 (en) * 1998-06-13 2001-12-04 Zeneca Limited Methods for detecting target nucleic acid sequences
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11939629B2 (en) * 2017-08-01 2024-03-26 Andrei Gindilis Methods and systems that detect nucleic-acid targets
EP4156910A4 (fr) * 2020-05-29 2024-07-03 Mammoth Biosciences, Inc. Dispositif de diagnostic à nucléase programmable

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