EP3387431A1 - Systèmes, méthodes et compositions pour améliorer la spécificité de l'hybridation des acides nucléiques - Google Patents

Systèmes, méthodes et compositions pour améliorer la spécificité de l'hybridation des acides nucléiques

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Publication number
EP3387431A1
EP3387431A1 EP16874070.2A EP16874070A EP3387431A1 EP 3387431 A1 EP3387431 A1 EP 3387431A1 EP 16874070 A EP16874070 A EP 16874070A EP 3387431 A1 EP3387431 A1 EP 3387431A1
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EP
European Patent Office
Prior art keywords
melting
nucleic acid
microarray
dna
probe
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EP16874070.2A
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German (de)
English (en)
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EP3387431A4 (fr
Inventor
Michael Okura
Michael D. Okura
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Individual
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Individual
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Publication of EP3387431A1 publication Critical patent/EP3387431A1/fr
Publication of EP3387431A4 publication Critical patent/EP3387431A4/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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q1/6832—Enhancement of hybridisation reaction

Definitions

  • the present invention generally relates to systems, methods and compositions for improving the specificity of nucleic acid hybridization. More particularly, to specifically in improving the accuracy of microarray technology, such as microarray gene expression profiling, single nucleotide polymorphism (SNP) analysis and any assay requiring hybridization, including PCR and Next Gen DNA sequencing.
  • microarray technology such as microarray gene expression profiling, single nucleotide polymorphism (SNP) analysis and any assay requiring hybridization, including PCR and Next Gen DNA sequencing.
  • Nucleic acid hybridization methods are currently used to detect the presence of nucleic acid regions known or suspected to be associated with the natural functioning of a living organism or nucleic acid residues obtained from various sources. Nucleic acid hybridization can also be used to detect sections of nucleic acid regions known or believed to be associated with an organism's disease state, metabolic state or life stage that the organism is experiencing during its life cycle. The accuracy of hybridization typically can be revealed during melting curve analysis of hybridized nucleic acid regions. Still further, there is a need for systems, methods, and compositions of matter to improve the specificity of binding between nucleic acid regions of nucleic acid sources.
  • Microarray technology has been the dominant genomics methodology but suffered from problems with repeatability and inaccuracy.
  • NGS next generation sequencing
  • Genomics is certainly one of the fastest developing areas of the life sciences but large gaps continue to exist in the price performance of NGS in relation to other genomics techniques. While there have been dramatic pricing drops for the actual sequencing process, the price of NGS when used for gene expression profiling and SNP analysis is not competitive with microarrays that range in the hundreds of dollars per assay.
  • NGS techniques were developed for whole genome sequencing and sequence all DNA present in the sample. Analysis of specific parts of the genome or a subset of genes requires capture-enrichment assays. These consist of standard microarray chips which hybridize specific sequences but allow other unwanted sequences to be washed away. While, enrichment can be over 100 fold using this methodology, only between 30% to 60% of the captured DNA can come from the desired sections of the genome. As a result, capture enrichment assays typically are not very efficient and the depth or redundancy of sequence coverage varies with each experiment.
  • the melting curve microarray originated as a method for improving the accuracy of microarray gene expression profiling.
  • the use of its technology is envisioned to simplify and lower cost for single nucleotide polymorphism (SNP) analysis.
  • SNP single nucleotide polymorphism
  • dsDNA double stranded DNA
  • liquid phase reactions These experiments were done in tubes or liquid phase with the DNA free in solution.
  • a common limitation to liquid phase melting curves is the inability to achieve one base pair resolution of detection.
  • the application of melting curve analysis to the microarray or solid phase reaction is a relatively new and not completely understood process.
  • the present invention is directed to methods, systems, and compositions of matter are provided to enhance the inter-nucleic acid binding at the surface of a solid and to obtain melting curve patterns to optimize the matching between nucleic acid regions.
  • CESB Charged Enhanced Specificity of Binding
  • the enhanced melting curves are due to the additional attractive force the positively charged surface exerts on the DNA.
  • CESB can create hybridization conditions with maximum specificity and without any loss of sensitivity. More preferably, CESB can occur whenever a positively charged surface is present with the correct ion concentration in the buffer.
  • creating enhanced melting curves and CESB preferably requires a positively charged surface and interplay with the ion concentration of the buffer.
  • the surface charge density of the solid surface is even and consistent. If, for example, the surface charge density varied from spot to spot, the results would vary and be inconsistent.
  • the advantages of using the positively charged microarray surface not only create an enhanced melting curve that can detect the binding and melting of perfectly matched and 1 bp mismatched target, but also create conditions that separate the temperature ranges of melting leading to a temperature of hybridization with maximum levels of specificity for the detection of perfectly matched target DNA without loss of any sensitivity.
  • charge enhanced specificity of binding can be used to improve the specificity of any hybridization reaction provided the reaction can be done in a solid phase format.
  • a list of methods that would benefit from CESB may include but is not limited to southern blots, northern blots, microarray, PCR and any form of next generation DNA sequencing incorporating a hybridization step.
  • this assay comprises 12 different mutations occurring within 6 base pairs.
  • this test can be performed by melting curve analysis or by CESB during hybridization, or by CESB in a solid phase PCR format according to the methods disclosed in the present invention.
  • CESB and enhanced melting curves can be performed in the liquid phase format with special adaptations that allow a miniature solid surface with positive charge to be attached to a probe or primer. This allows liquid phase methods like PCR to benefit from CESB.
  • the surface of a solid is exposed with a first solution having a composition to impart a positive charge to the surface. Thereafter, a first nucleic acid source or solution is exposed to the positively charged surface. Then, after removal of any unbound first nucleic acid, a second nucleic acid source or solution is offered to the first nucleic acid bound surface at conditions to produce a hybridized nucleic acid pair. After hybridization, the hybrid nucleic acid pair is heated sufficiently to reveal a melting curve.
  • the method, system, and compositions also provide for adjusting the melting curve shape to attain a stepwise pattern by altering the composition and/or exposure of the first solution, and/or the solution containing the first nucleic acid source and/or exposure of the first nucleic acid solution, and/or the solution containing the second nucleic acid source.
  • the melting curve shape is adjusted to attain a step wise pattern by altering the composition of at least one of the first solution, the first nucleic acid solution, and the second nucleic acid solution.
  • the positively charged particle comprises a surface coating of positively charged chemicals.
  • the positively charged chemicals can be selected from the group consisting of polyethyleneimine, epoxide, amine, epoxysilane and any chemical compound with a positive charge. More preferably, the positively charged chemical is polyethyleneimine and is present in the amount from about 1% to about 10%.
  • the nucleic acid can be a segment of DNA or RNA.
  • the first nucleic acid can be a DNA or RNA fragment.
  • the first nucleic acid can be a probe.
  • the solid can be selected from the group consisting of polystyrene, microbeads, glass, metal, charcoal, colloidal gold, bentonite, polypropylene, plastics and silica. More preferably, the solid particle is glass. Even more preferably, the solid particle is a glass slide and is a micro array glass slide. The micro array glass slide comprises from about 10 to about 4.2 million probes.
  • the first nucleic acid can include a label.
  • the label can be a fluorescent dye selected from the group consisting of 2-((iodoacetyl)amino)ethyl)aminonapthylene-l -sulfonic acid) (1,5-IEDANS), fluorescein, Bodipy, FTC, Texas Red, phycoerythrin, rhodamines, carboxytetramethylrhodamine, DAPI, indopyras dyes, Cascade Blue, Oregon Green, eosins, erythrosin, pyridyloxazoles, benzoxadiazoles, aminonapthalenes, pyrenes, maleimides, coumarins, Lucifer Yellow, Propidium iodide, porhyrins, CY3, CY5, CY9, lanthanides, cryptates, and lanthanide chelates.
  • fluorescent dye selected from the group consisting of 2-(iodoacetyl)amino)eth
  • the reaction mixture can further include a buffer.
  • the present invention is also directed to methods and an apparatus for high accuracy genomic analysis platform utilizing hybridization and chemically enhanced dissociation that meets these needs.
  • the methods and apparatus according to the present invention can be used in capture/enrichment, gene expression profiling and targeted sequencing.
  • Embodiments of the present invention provide a solution to improving the accuracy and stringency of microarrays and/or other genomic analysis methods relying on nucleic acid hybridization and melting curve analysis. Methods are provided by controlling the surface chemistry of the slide and development of an improved microarray reader. In an advantageous embodiment, there is a method of producing, through initial synthesis, manufacture or through secondary applications, a positively charged surface or surface coating, on the surface of microarray slides or other types of surfaces used for similar purposes, such as micro beads, which enhances melting curve analysis to the point of allowing the detection or differentiation of small changes in sequences between nucleic acid binding partners.
  • polyethyleneimine, epoxide or a variety of other positively charged chemicals or even the use of an electrical current across the surface to generate a positive charge can be used for the enhancement of DNA microarray melting curve analysis or other hybridization based assays.
  • the present invention is directed to a method of enhanced inter-nucleic acid binding at the surface of a solid, to capture/enrichment, detecting the presence, measuring the amount or verifying the sequence of a target polynucleotide of interest in a test sample.
  • the method comprises exposing the surface with a solution sufficient to attain a positively charged surface; exposing a first nucleic acid solution to the positive charged surface to produce a first nucleic acid bound surface; wherein the first nucleic acid solution comprises a first probe.
  • the method further comprises exposing a second nucleic acid solution to the first nucleic acid bound surface to produce a hybridized nucleic acid pair; wherein the second nucleic acid solution comprises the target polynucleotide; whereby the first probe is complementary to portions of the target polynucleotide sequence.
  • the method further comprises heating the hybridized nucleic acid pair sufficiently to reveal a bi-phasic melting curve shape; whereby the positively charged surface improves stringency during hybridization of the nucleic acid pair by changing kinetics of unbinding of the target polynucleotide, correlating with temperature changes and thermal dissociation characteristics for analysis, to the point that detection, quantification or differentiation of small sequence differences between nucleic acid hybrids in the target polynucleotide and the first probe.
  • the small sequence difference can be one base pair such that a temperature range of melting of one base pair mismatched target polynucleotide and temperature range of melting of perfectly matched target polynucleotide have different temperature ranges and no longer overlap.
  • the positively charged surface of the solid changes the kinetics by narrowing the temperature range of melting between the one base pair mismatch and the perfectly matched target polynucleotide thereby producing a distinctive melting curve with a change in a slope of the curve effectively forming a biphasic melting curve consisting of two separate melting curves with a short section between the two curves where no melting occurs.
  • the present invention is directed to a method of capture/enrichment, detecting the presence, measuring the amount or verifying the sequence of a target polynucleotide of interest in a test sample.
  • the method comprises the steps of forming a reaction mixture by combining in an assay medium: (i) a first reagent comprising a first probe bound to a solid particle, and (ii) an aliquot of the test sample suspected of containing the target nucleotide sequence.
  • the first probe comprises a first single stranded nucleic acid fragment complementary to a first of two separated strands of a selected segment of the target nucleotide sequence.
  • the solid particle is a positively charged solid particle.
  • the first probe is complementary to mutually exclusive portions of the target polynucleotide sequence.
  • the reaction mixture is then subjected under denaturing conditions rendering the target polynucleotide sequence in the sample to be single stranded.
  • the reaction mixture is then incubated under hybridization conditions to cause hybridization between the first probe and the first strand of the selected segment of the target polynucleotide sequence. In the presence of the target polynucleotide, substantially all of the first probe will be hybridized to the first strand of the selected segment of the target polynucleotide sequence producing bound target polynucleotide sequence.
  • the reaction mixture is then exposed to disassociation conditions.
  • the reaction mixture is then monitored. Preferably, dissociation correlates with changes in the presence of the bound target polynucleotide providing disassociation curve analysis.
  • the positively charged solid particle enhances thermal disassociation characteristics for analysis, to the point of allowing the detection, amount or differentiation of small sequence differences between nucleic acid hybrids in the target polynucleotide and the first probe.
  • the small sequence differences can be down to one base pair.
  • the target polynucleotide can be a segment of DNA or RNA.
  • the first probe can be a DNA or RNA fragment.
  • the first probe can be bound to the solid particle by a linker.
  • the solid particle can be selected from the group consisting of polystyrene, microbeads, glass, metal, charcoal, colloidal gold, bentonite, polypropylene, plastics and silica. More preferably, the solid particle is glass. Even more preferably, the solid particle is a glass slide and is a micro array glass slide. The micro array glass slide comprises from about 10 to about 4.2 million probes.
  • the positively charged particle comprises a surface coating of positively charged chemicals.
  • the positively charged chemicals can be selected from the group consisting of polyethyleneimine, epoxide, amine, epoxysilane and any chemical compound with a positive charge. More preferably, the positively charged chemical is polyethyleneimine and is present in the amount from about 1% to about 10%.
  • the positively charged particle can comprise a surface coating of positively charged chemicals generated by use of an electrical current across the surface to generate a positive charge.
  • the target polynucleotide sequence can include a label.
  • the label can be a fluorescent dye selected from the group consisting of 2- ((iodoacetyl)amino)ethyl)aminonapthylene-l -sulfonic acid) (1,5-IEDANS), fluorescein, Bodipy, FTC, Texas Red, phycoerythrin, rhodamines, carboxytetramethylrhodamine, DAPI, indopyras dyes, Cascade Blue, Oregon Green, eosins, erythrosin, pyridyloxazoles, benzoxadiazoles, aminonapthalenes, pyrenes, maleimides, coumarins, Lucifer Yellow, Propidium iodide, porhyrins, CY3, CY5, CY9, lanthanides, cryptates, and lanthanide chelates.
  • the step of exposing the reaction mixture to disassociation conditions can be carried out over a temperature range from about 0° C to about 100° C, with temperature increase increments of from about 0.01° C to about 5.0° C.
  • the steps of forming, subjecting, incubating, exposing and monitoring were carried out by an automated micro array device.
  • the first probe can include a label.
  • the label can be a fluorescent dye selected from the group consisting of 2-((iodoacetyl)amino)ethyl)aminonapthylene-l -sulfonic acid) (1,5- IEDANS), fluorescein, Bodipy, FTC, Texas Red, phycoerythrin, rhodamines, carboxytetramethylrhodamine, DAPI, indopyras dyes, Cascade Blue, Oregon Green, eosins, erythrosin, pyridyloxazoles, benzoxadiazoles, aminonapthalenes, pyrenes, maleimides, coumarins, Lucifer Yellow, Propidium iodide, porhyrins, CY3, CY5, CY9, lanthanides, cryptates, and lanthanide chelates.
  • the reaction mixture can further include a buffer.
  • the present invention is directed to a micro array apparatus for genome sequence analysis
  • a base structure comprises: a melting curve microarray reader cassette; wherein the cassette configured to hold microarray slides; a thermal control chamber comprising a heat control unit and a fluids control unit; wherein the heat control unit measures temperature data for melting curve analysis; an optical system for measuring the presence or absence, and concentration of labeled nucleic acid sample providing the concentration data for melting curve analysis; and an automatic focusing system.
  • a computerized Z-axis is added to the thermal control chamber to speed up a focusing procedure and allow automatic incremental adjustments of focus.
  • the melting curve data is sufficient to distinguish between the melting of different sequences of target DNA with one base pair sensitivity for each probe spot of a microarray, allowing for scanning of entire genome sequencing.
  • FIG. 1 illustrates an exemplary prototype melting curve microarray reader machine according to an embodiment of the present invention.
  • FIG. 2 illustrates screenshots of three custom software programs, according to embodiments of the present invention.
  • FIG. 3 illustrates a comparison of the initial results obtained with the Keck Center Microarray Chips according to the present invention. A total of 10 probe spots are displayed in each graph. All data is raw and unadjusted.
  • FIG. 4 illustrates a comparison of the melting curves between perfectly matched target and 1 base pair mismatch target binding the 25 mer mouse GAPDH probe sequence according to the present invention.
  • Graph A shows melting curve analysis from a microarray with identical probe spots containing the same 25 mer probe. Note the extremely sharp melting curve and Tm of about 57° C.
  • Graph B is a microarray with the same probe type as graph A but hybridized with target containing a 1 bp mismatch or SNP. Note that the Tm is about 48.5° C a difference of 8.5° C compared to graph A.
  • FIGS. 5A-B illustrates exemplary results after treatment of slides with 5% polyethyleneimine according to an embodiment of the present invention.
  • FIGS. 6A-B illustrates exemplary results after treatment of slides with 10% polyethyleneimine according to an embodiment of the present invention.
  • FIG. 7 illustrates an exemplary example of DNA melting curves in liquid phase solutions and solid phase according to an embodiment of the present invention.
  • FIGS. 8A illustrate an exemplary prototype microarray to perform DNA melting curve analysis according to an embodiment of the present invention.
  • FIG. 8B illustrates the thermal control chamber and heating block of the modified microarray scanner, according to the embodiments of the present invention.
  • FIG. 9A illustrates the redesigned array cassette of the modified microarray scanner of FIG. 9B according an embodiment of the present invention.
  • FIG. 9B illustrates the microarray installed in the modified microarray scanner of FIG. 8B according to an embodiment of the present invention.
  • FIG 10 illustrates an exemplary example of application of DNA to an amine coated array according to an embodiment of the present invention
  • FIG. 11A illustrates an exemplary comparison of microarray images obtained during a melting experiment between the temperatures of 45° and 65° C according to the embodiments of the present invention.
  • FIG. 11B illustrates exemplary results of array hybridized with human cDNA stained with Cy3 dye according to the embodiments of the present invention.
  • FIG. 12 illustrates the effects of 10% 2-Mercaptoethanol on DNA melting curves according to an embodiment of the present invention
  • FIG. 13 illustrates an exemplary example of epoxysilane surface attachment and blocking or deactivation of the surface by ethanolamine according to an embodiment of the present invention
  • FIGS. 14A-B illustrate epoxy silane attachment to a glass microarray according to an embodiment of the present invention
  • FIGS. 15A-E illustrate DNA melting curves on unblocked epoxy coated microarrays with glass cover slips according to the present invention
  • FIGS. 16A-D illustrate stability comparisons of unblocked epoxy slides before and after one month according to an embodiment of the present invention
  • FIGS. 17A-E illustrate melting curves on unblocked epoxy coated slides with plastic cover slips according to the present invention
  • FIGS. 18A-B illustrates an exemplary comparison of the type of melting curves obtained using standard and optimized surface chemistry of Microarray Inc. arrays according to the present invention.
  • Graph A is a melting curve on an array with optimized surface chemistry which is able to detect that approximately 18% of the target bound on this spot is one base pair mismatch (Tm 59° C) and about 82% perfect match (Tm 67° C).
  • Graph B is a melting curve on an array with standard surface chemistry and is unable to detect two distinct types of target and instead shows one large melting curve with a Tm of about 55° C. Note that the slope of graph A is steeper and that melting is occurring at a higher temperature.
  • FIG. 19 illustrates an exemplary diagram of how positively charged microarray surface can attract negatively charged nucleic acids directly above the charged surface, enhancing nucleic acid melting according to an embodiment of the present invention.
  • FIGS. 20A-C illustrates an exemplary overview of the KRAS Mutation Assay according to an embodiment of the present invention; and an exemplary example of the preferred embodiment of the invention and the potential for diagnostic applications.
  • FIGS. 21A-C illustrate the exemplary results of the KRAS Mutation Assay according to the present invention.
  • FIGS. 22A-E illustrate the exemplary results of the KRAS Mutation Assay by Codon according to the present invention
  • FIGS. 23A-B illustrate the exemplary results DNA Melting Kinetics on Standard Microarray surface chemistry
  • FIGS. 25A-C illustrate exemplary results of DNA melting curves obtained from optimized charge enhanced specificity surface chemistry according to a preferred embodiment of the present invention
  • FIGS. 26A-B illustrate exemplary binding mechanisms of liquid phase and solid phase PCR according to an embodiment of the present invention
  • FIGS. 27 A-C illustrate an exemplary example of a custom synthesized oligo bound to a nano particle according to an embodiment of the present invention
  • FIG. 28 illustrates an exemplary schematic diagram of hybrid liquid-solid phase PCR according to a preferred embodiment of the present invention.
  • a method for improving the accuracy and stringency of microarrays and/or other genomic analysis methods relying on nucleic acid hybridization and melting curve analysis by controlling the surface chemistry of the slide comprises producing a positively charged surface or surface coating, on the surface of microarray slides or other types of surfaces used for similar purposes, such as nano particles and micro beads, which enhances melting curve analysis to the point of allowing the detection or differentiation of small changes in sequences between nucleic acid binding partners.
  • an improved microarray reader machine to collect melting curve data on microarray slides containing 1000 probe spots or more.
  • the accuracy or resolution of melting curve analysis was to be sufficient to distinguish between the melting of perfect matched dsDNA and dsDNA with the smallest possible change in sequence, a one base pair mismatch.
  • the methods and apparatus according to the present invention can be used in capture/enrichment, gene expression profiling and targeted sequencing. Particularly, in an embodiment of the present invention, there is a method of capture/enrichment of a target polynucleotide of interest in a test sample. In another embodiment of the present invention, there is a method of detecting the presence of a target polynucleotide of interest in a test sample. In yet another embodiment of the invention, there is a method of measuring the amount of a target polynucleotide of interest in a test sample. In another and more preferable embodiment of the present invention there is a method of verifying the sequence of a target polynucleotide of interest in a test sample.
  • FIG. 1 illustrates an exemplary device according to the present invention.
  • a cost saving measure the first melting curve microarray reader was a modified Axon 4000a (Molecular Devises, Sunnyvale, CA) machine in which heat control and fluidics were combined with existing scanning capability.
  • Initial experiments utilized a commercial microarray chip "Check It Chips" with large 300 ⁇ probe spots and 70 mer probe sequences for the human genome printed in blocks of 100 spots for a total of 2 blocks or 200 probe spots per array (commercially available from Arrayit Corporation, Sunnyvale, CA).
  • these slides typically have an amine coated surface and probes attached via UV cross linking.
  • Human cDNA stained with Cy3 dye (Arrayit Corp.) was used as target DNA for hybridization.
  • melting experiments can be carried out in a temperature range from about 0° C to about 100° C.
  • melting experiments were carried out over a temperature range from about 40° C to about 70°C, preferably with temperature increase increments of 1°C and fluidics buffer flush of 600 ⁇ of 2.5x SSC.
  • Embodiments of the present invention provide a solution to improving the accuracy and stringency of microarrays and/or other genomic analysis methods relying on nucleic acid hybridization and melting curve analysis. Methods are provided by controlling the surface chemistry of, for example, a microarray slide and development of an improved microarray reader. In an advantageous embodiment, there is a method of producing, through initial synthesis, manufacture or through secondary applications, a positively charged surface or surface coating, on the surface of microarray slides or other types of surfaces used for similar purposes, such as micro beads, which enhances melting curve analysis to the point of allowing the detection or differentiation of small changes in sequences between nucleic acid binding partners.
  • polyethyleneimine, epoxide or a variety of other positively charged chemicals or even the use of an electrical current across the surface to generate a positive charge can be used for the enhancement of DNA microarray melting curve analysis or other hybridization based assays.
  • the target polynucleotide when energy in the form of heat is applied to the microarray, 2 different types of chemical bonds must be broken to allow the target polynucleotide to melt away or dissociate, the hydrogen bonds between the complementary probe and target polynucleotide, and the attractive force with the positively charged surface. Because there are 2 different types of bonds being broken at the same time, this causes a change in the way the target polynucleotide dissociates. Rather than melting apart over a range of temperature, for example, a 5° C range, the target polynucleotide can melt away over a much shorter temperature range of, for example, 1° C or even less. Note that in order to exert this effect, the positively charged surface bonds with the target polynucleotide and produces a beneficial effect.
  • the present invention is directed to a method of capture/enrichment, detecting the presence, measuring the amount or verifying the sequence of a target polynucleotide of interest in a test sample.
  • the method comprises the steps of forming a reaction mixture by combining in an assay medium: (i) a first reagent comprising a first probe bound to a solid particle, and (ii) an aliquot of the test sample suspected of containing the target nucleotide sequence.
  • the first probe comprises a first single stranded nucleic acid fragment complementary to a first of two separated strands of a selected segment of the target nucleotide sequence.
  • the solid particle is a positively charged solid particle.
  • the first probe is complementary to mutually exclusive portions of the target polynucleotide sequence.
  • the reaction mixture is then subjected under denaturing conditions rendering the target polynucleotide sequence in the sample to be single stranded.
  • the reaction mixture is then incubated under hybridization conditions to cause hybridization between the first probe and the first strand of the selected segment of the target polynucleotide sequence.
  • substantially all of the first probe will be hybridized to the first strand of the selected segment of the target polynucleotide sequence producing bound target polynucleotide sequence.
  • the reaction mixture is then exposed to disassociation conditions.
  • nucleic acid hybridization as an analytical tool is based on the double stranded duplex structure of DNA.
  • the hydrogen bonds between the purine and pyrimidine bases of the respective strands in double stranded DNA can be reversibly broken.
  • the two complementary strands of DNA resulting from this melting or denaturation of DNA will associate (also referred to as reannealing or hybridization) to reform the duplexed structure.
  • Contact of a first single stranded nucleic acid either DNA or RNA, which comprises a base sequence sufficiently complementary to a second stranded nucleic acid under appropriate conditions, will result in the formation of nucleic acid hybrids, as the case may be.
  • the reaction mixture is then monitored.
  • dissociation correlates with changes in the presence of the bound target polynucleotide providing disassociation curve analysis.
  • the positively charged solid particle enhances thermal disassociation characteristics for analysis, to the point of allowing the detection, amount or differentiation of small sequence differences between nucleic acid hybrids in the target polynucleotide and the first probe.
  • the small sequence differences can be down to one base pair.
  • the target polynucleotide can be a segment of deoxyribonucleic acid (DNA) sequence or ribonucleic acid (RNA) sequence.
  • the target polynucleotide sequence of interest can be any polynucleotide sequence present naturally in a sample. It can be in a material in or derived from a cellular system.
  • the polynucleotide sequence can be any gene or polynucleotide sequence of interest (DNA or RNA).
  • the first probe can be a nucleic acid fragment, preferably, a DNA or RNA fragment. More preferably, the first probe comprises a first single stranded nucleic acid fragment complementary to a first of two separated strands of a selected segment of the target polynucleotide sequence.
  • the nucleic acid fragments can be produced or obtained by any method known to those of ordinary skill in the art, e.g., synthetic production methods or enzymatic production methods, both in vitro and in vivo.
  • DNA and RNA probes preferably are single stranded nucleic acid molecules generally synthesized by gene machines or made using recombinant DNA methods known to those skilled in the art.
  • the first probe will exhibit detectable hybridization at one or more points with the target polynucleotide sequence of interest. More preferably, the nucleic acid probe fragment attached to the solid particle can be of almost any length, provided that the fragment is long enough to form a stable nucleic acid hybrid with the selected segment of the target polynucleotide sequence.
  • the first probe nucleic acid fragment will typically have a minimum 4-base sequence, one case greater than an amino acid codon.
  • the first probe nucleic acid fragment is from about 4 to about 80 nucleotides in length. The more nucleotides, the greater the specificity.
  • the first probe can be bound to the solid particle by a spacer linker.
  • the solid particle can be any insoluble particle that is capable of attaching DNA or RNA.
  • the DNA or RNA can be attached to the solid particle by any known methods known to those of ordinary skill of the art including but not limited to chemical bonds, including covalent bonds, ionic bonds and electrostatic attractions.
  • the solid particle can be selected from the group consisting of polystyrene, microbeads, glass, metal, charcoal, colloidal gold, bentonite, polypropylene, plastics and silica. More preferably, the solid particle is glass. Even more preferably, the solid particle is a glass slide and is a micro array glass slide.
  • any number of probes can be possible and can be tailored accordingly.
  • the micro array glass slide comprises from about 10 to about 4.2 million probes.
  • probes comprise a single stranded nucleic acid fragment complementary to a first of two separated strands of a selected segment of a target polynucleotide sequence.
  • the nucleic acid fragments can be fragments from deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences.
  • the nucleic acid fragment is single stranded.
  • the nucleic acid fragments can be produced or obtained by any method known to those of ordinary skilled in the art, e.g., synthetic production methods or enzymatic production methods, both in vitro and in vivo.
  • DNA and RNA probes are single- stranded nucleic acid molecules generally synthesized by so called gene machines or made using recombinant DNA methods.
  • the first probe strands are not attached directly to the solid particle, such as a microarray surface, but preferably attached by using a linker, which can elevate the DNA off the surface.
  • the linkers can be made primarily to allow the DNA to be at a greater distance off the surface of the slide, but can also have additional chemical properties, for example, if the linker were to have a positive charge, it may be able to replicate the results achieved with just a positively charged surface.
  • hybridization conditions means those conditions which enable the hybridization between the first probe attached to the solid particle to a first strand of the selected segment of target polynucleotide sequence.
  • hybridization techniques and melting curve analysis are typically known to those skilled of ordinary skill in the art and can be used in the present invention.
  • the choice of solid particle can be governed by the effect of rate of hybridization and binding of the probe to the target DNA.
  • the solid particle preferably should provide sufficient sensitivity in order to detect the amount of target nucleotide sequence available for hybridization. Other consideration will be the ease of synthesis of the probe, the availability of instrumentation, the ability to automate and convenience.
  • the solid particle by controlling the surface chemistry of the solid particle. More preferably, the solid particle will have a positively charged surface or surface coating, which enhances melting curve analysis to the point of allowing the detection or differentiation of small changes in sequences between nucleic acid binding partners, the detection or differentiation of small changes in sequences can be up to one base pair.
  • any cationic or other positively charged chemicals can be used to coat the solid particle surface.
  • the positively charged particle comprises a surface coating of positively charged chemicals.
  • the positively charged chemicals can be selected from the group consisting of polyethyleneimine, epoxide, amine and including but not limiting any chemical compound known to those with ordinary skill in the art with a positive charge. More preferably, the positively charged chemical is polyethyleneimine and is present in the amount from about 1 % to about 10%.
  • the positively charged particle can comprise a surface coating of positively charged chemicals generated by use of an electrical current across the surface to generate a positive charge that can be used for the enhancement of DNA micro array melting curve analysis or other hybridization based assays.
  • the target polynucleotide sequence can include a label.
  • the label can be any label or tag known to those skilled in the art.
  • the label can include dyes, radioactive labels, gold, silver, beads, antibody or any other label known to those skilled in the art to label or tag a polynucleotide sequence.
  • the label can be a fluorescent dye selected from the group consisting of 2-((iodoacetyl)amino)ethyl)aminonapthylene-l -sulfonic acid) (1,5-IEDANS), fluorescein, Bodipy, FTC, Texas Red, phycoerythrin, rhodamines, carboxytetramethylrhodamine, DAPI, indopyras dyes, Cascade Blue, Oregon Green, eosins, erythrosin, pyridyloxazoles, benzoxadiazoles, aminonapthalenes, pyrenes, maleimides, coumarins, Lucifer Yellow, Propidium iodide, porhyrins, CY3, CY5, CY9, lanthanides, cryptates, and lanthanide chelates. More preferably, CY3 is used as the dye.
  • the step of exposing the reaction mixture to disassociation conditions means exposing the reaction mixture to any melting temperature or melting conditions known to those skilled in the art.
  • Disassociation and melting can be used interchangeably from here throughout the specification.
  • Disassociation or melting conditions can be any conditions known but not limited to those with skilled in the art, including heat, chemicals, electrical current or other types of fluid or sound waves. More preferably, disassociation means melting conditions.
  • the melting temperature can be calculated using the probe sequence and buffer composition.
  • the melting temperature typically is the lowest temperature that will allow all of the target to be release from the probe. If the melting temperature is too low the target will not be released. Using a high melting temperature can have no negative effect on the results other than consuming more time and energy.
  • the determining the range of temperature for the melting (dissociation) reaction to take place it usually starts at or below the hybridization temperature and ends a little above the melting temperature.
  • the step of exposing the reaction mixture to disassociation conditions can be carried out over a temperature range from about 0° C to about 100° C.
  • the step of exposing the reaction mixture to disassociation conditions can be carried out over a temperature range from about 40° C to about 70° C, with temperature increase increments of from about 0.01 ° C to about 5.0 ° C.
  • the temperature increase increments can be carried out from about 0.01 ° C to about 5.0 ° C.
  • the temperature range can vary depending on the probe sequence and probe length, and can be adjusted according to those skilled in the art.
  • the experiment can be carried out over a temperature range from about 0° C to about 100° C.
  • the experiment can be carried out over a temperature range from about 40° C to about 70 ° C.
  • the range of temperature over which the experiment can be conducted is determined by the hybridization temperature and the melting temperature of the target sequence and probes. Before the melting reaction is conducted, hybridization can be performed and is accomplished at one specific temperature.
  • the hybridization temperature can be a temperature suggested by the manufacturing company, for example, of the micro array and a common hybridization temperature typically can be 45° C.
  • the range of temperature can be determined by calculating or estimating the temperature in which the target polynucleotide sequence is most likely to bind its complementary first probe. This estimated temperature can vary according to the sequence of nucleic acids and type of buffer used during hybridization.
  • the temperature increase increments in the step of exposing the reaction mixture to disassociation or melting conditions, can be from about 0.01° C to about 5.0° C. Preferably, the temperature increase increments can be from about 0.01° C to about 3.0° C.
  • the temperature increase increment can be varied according to those skilled in the art, to how much resolution is needed in the melting curve graph analysis. For example, typically, for the following experiments conducted, a 1° C temperature increase worked well, however, a temperature increment increase of less than 1° C could add more data points to the graph generated for analysis, thereby increasing the resolution of the melting curve however, would have consumed more time.
  • the temperature increase increment is about 1° C.
  • the steps of forming, subjecting, incubating, exposing and monitoring preferably are carried out by an automated microarray device.
  • the first probe can include a label.
  • the label can be any label or tag known to those skilled in the art.
  • the label can include dyes, radioactive labels, gold, silver, beads, antibody or any other label known to those skilled in the art to label or tag a polynucleotide sequence.
  • the label can be a fluorescent dye selected from the group consisting of 2- ((iodoacetyl)amino)ethyl)aminonapthylene-l -sulfonic acid) (1,5-IEDANS), fluorescein, Bodipy, FTC, Texas Red, phycoerythrin, rhodamines, carboxytetramethylrhodamine, DAPI, indopyras dyes, Cascade Blue, Oregon Green, eosins, erythrosin, pyridyloxazoles, benzoxadiazoles, aminonapthalenes, pyrenes, maleimides, coumarins, Lucifer Yellow, Propidium iodide, porhyrins, CY3, CY5, CY9, lanthanides, cryptates, and lanthanide chelates.
  • CY3 is used as the dye.
  • the fluorescent polynucleotide probes are especially useful in automatic or semiautomatic recording of the results combined with continuous flow systems and instruments.
  • the reaction mixture can further include a buffer.
  • a buffer Any buffer known to those of ordinary skill in the art can be used. More preferably, the buffer is selected based on the buffer ionic strength, which can affect the reaction.
  • the present invention is directed to a microarray apparatus for genome sequence analysis
  • a base structure comprises: a melting curve microarray reader cassette; wherein the cassette configured to hold microarray slides; a thermal control chamber comprising a heat control unit and a fluids control unit; wherein the heat control unit measures temperature data for melting curve analysis; an optical system for measuring the presence or absence, and concentration of labeled nucleic acid sample providing the concentration data for melting curve analysis; and an automatic focusing system.
  • a computerized Z-axis is added to the thermal control chamber to speed up a focusing procedure and allow automatic incremental adjustments of focus.
  • the melting curve data is sufficient to distinguish between the melting of different sequences of target DNA with one base pair sensitivity for each probe spot of a microarray, allowing for scanning of entire genome sequencing.
  • the optical system preferably provides fluorescence intensity data, whereas the heat control unit typically provides the reaction mixture temperature data.
  • FIG. 11A illustrates comparison of microarray images obtained during a melting experiment between the temperatures of 45° and 65° C.
  • FIG. 11 A depicts, repeated cycles of temperature increase, buffer flush, and scans melted away most of the bound target DNA on the slide away by 65° C confirming release of target DNA.
  • the temperature range can vary depending on the probe sequence and probe length, and can be adjusted according to those skilled in the art.
  • the experiment can be carried out over a temperature range from about 0° C to about 100° C, preferably from about 40° C to about 70° C.
  • the temperature increase increments in the step of exposing the reaction mixture to melting conditions, can be from about 0.01° C to about 5.0° C. Preferably, the temperature increase increments can be from about 0.01° C to about 3.0° C.
  • the temperature increase increment can be varied according to those skilled in the art, to how much resolution is needed in the melting curve graph analysis. For example, typically, for the following experiments conducted, a 1° C temperature increase worked well, however, a temperature increment increase of less than 1° C could add more data points to the graph generated for analysis, thereby increasing the resolution of the melting curve however, would have consumed more time.
  • the temperature increase increment is about 1 ° C.
  • FIG. 11B depicts results of array hybridized with human cDNA stained with Cy3 dye. Melting analysis was performed over the temperature range of 40° - 64° C with readings at 2° C intervals.
  • Graph A (Cadherin 1 probe) depicts a melting curve showing one large melting point (arrow) at about 63° C indicating the presence of one major hybridization product.
  • Graph B (Beta Actin probe) depicts at least two major melting points (arrows) at 46° and 62° C. This result indicates the presence of multiple hybridization products. Conventional microarray analysis is not capable of making this distinction. The relative abundance of each hybridization product can be inferred from the graph.
  • FIG. 11B shows when actual melting curves were plotted by compiling the fluorescence intensity data at each temperature of scanning for each probe spot, well-formed curves were obtained. Remarkably, these curves exhibited a sharp slope or drop at which DNA melted away from the array which allowed easy discernment of the temperature of melting (Tm) as shown by the arrows (FIG. 1 IB) and the ability to detect more than one type of target attached to the individual probe spot.
  • Tm temperature of melting
  • FIG. 1 IB The melting curve for the Beta Actin probe spot depicts two distinct melting curves indicating that at least two different types of target DNA were bound.
  • the objectives of the present invention include improving a melting curve microarray reader machine, both instrumentation and software and to demonstrate the ability of the machine to collect melting curve data on microarray slides containing 1000 probe spots or more.
  • the accuracy or resolution of melting curve analysis was to be sufficient to distinguish between the melting of perfect matched dsDNA and dsDNA with the smallest possible change in sequence, a one base pair mismatch.
  • systems, methods, and compositions to optimize high-accuracy hybridization between nucleic acid regions of separate DNA or RNA molecules are also provided.
  • Systems include modification of a binding membrane with positive charge to enhance the sticking of nucleic acids contained within a sample to be analyzed with a detection nucleic acid probe. Methods for using the positive charge modified membrane and related compositions are described. Compositions used to enhance the binding and subsequent de-binding in melting curve analysis are also described.
  • Other embodiments according to the present invention include the teachings of systems, methods, and compositions of matter concerning the enhancement of nucleic acid hybridization specificity and controlling the shapes of melting curves revealed by nucleic acid hybrid pairs to optimize nucleic acid analysis.
  • the use of solid phase nucleic acid melting analysis in the presence of a positively charged solid surface preferably are used to enhance the melting curves generated by double stranded nucleic acids.
  • this enhancement involves narrowing the temperature ranges of melting of perfect match and one base pair mismatch such there is no overlap of each melting range.
  • the temperature ranges of melting are separated for the two species of nucleic acids, they become easily detectable after binding the same probe spot using melting curve analysis which is evidenced by a change in the slope of the graph.
  • this type of graph is a 2 stepped curve or enhanced melting curve which could distinguish the presence of both perfect math and one base pair mismatch binding.
  • this detection is not possible using standard microarray surfaces which are normally chemically blocked and neutral in charge.
  • any chemical coating which produces a positively charged surface can be used to coat the solid support or particle.
  • the positively charged surface comprises an active surface coating of chemicals forming a positive charge on the surface of the particle.
  • the solid support is a slide.
  • the chemical coating includes but is not limited to amines, polyethyleneimine (PEI), epoxysilane and any chemical compound with a positive charge formed on the surface of the particle. It is known to those skilled in the art, that the chemical epoxysilane is neutral in charge but produces a layer of positive charge by forming a dipole during the attachment to the glass microarray surface.
  • the presence of the positively charged solid surface can introduce an attractive force that the negatively charged target strand of nucleic acids must overcome during the melting process in addition to the hydrogen bonds already present between the probe and target strands of DNA. It is believed that this additional attractive force produced by the positively charged surface is responsible for enhancement of the melting curves both narrowing the ranges of melting and further separating the temperatures of melting between the perfect match and 1 base pair mismatch.
  • the solid support is selected from the group consisting of polystyrene, microbeads, glass, metal charcoal, colloidal gold, bentonite, polypropylene, plastics and silica.
  • the solid support or particle is glass. More preferably, the solid support is a glass slide.
  • the positively charged support or particle comprises an active surface coating of chemicals forming a positive charge on the surface of the support or particle, and the chemical is polyethyleneimine
  • the polyethyleneimine is present in the amount from about 1% to about 10%. It is reasonable to assume that the level of positive charge on PEI coated arrays of 1%, 5%, and 10% were different, with the higher concentrations having higher levels of positive charge. While positive charge is need to produce the enhanced 2 stepped melting curves, the amount of positive charge needed appears to not be limited to just one specific level of positive charge but can vary to some degree. This is evidenced by the ability of both 1% and 10% PEI coatings being able to produce 2 stepped melting curves but with different characteristics.
  • the positive charge needs to be in an appropriate range in order to work - neither too strong nor too weak.
  • No defined amount of chemical charge has been determined but it was clear from experimentation that enhancement of the melting curves was optimal at certain surface chemical concentrations.
  • the chemical composition of the buffer surrounding the nucleic acids can play a role in helping to regulate the effect of the positive charge on the DNA.
  • a buffer with a high concentration of ions was thought to shield the nucleic acids somewhat from the attractive force of the positively charged surface reducing the effective attractive force. More preferably, altering the concentration of ions in solution is another way to fine tune the amount of surface attraction the DNA experiences towards the positively charged surface.
  • the solid support i.e. micro array surface
  • the probe DNA is attached to the solid support by binding positively charged amine surface coating via its negatively charged phosphate backbone.
  • This preliminary bonding is known to those skilled in the art as electrostatic bonding and can be made into a stronger covalent bond by use of heat or uv light (FIG. 10).
  • the probe molecule is not free to move up and down and cannot contribute to in-homogenous bonding and widening of the melting curve.
  • the advantages of using the positively charged microarray surface not only create an enhanced melting curve that can detect the binding and melting of perfectly matched and 1 bp mismatched target, but also create conditions that separate the temperature ranges of melting leading to a temperature of hybridization with maximum levels of specificity for the detection of perfectly matched target DNA without loss of any sensitivity. From hereinafter, this is termed “Charged Enhanced Specificity of Binding” (CESB).
  • CESB Charged Enhanced Specificity of Binding
  • charge enhanced specificity of binding can be used to improve the specificity of any hybridization reaction provided the reaction can be done in a solid phase format.
  • a list of methods that would benefit from CESB may include but is not limited to southern blots, northern blots, microarray, PCR and any form of next generation DNA sequencing incorporating a hybridization step. It is well known to those skilled in the art that PCR is one of the most commonly used methods, and the specificity enhancement is the preferred method according to an embodiment of the present invention.
  • enhanced melting curves and CESB are provided.
  • the enhanced melting curves are due to the additional attractive force the positively charged surface exerts on the DNA.
  • the level of positive change preferably must be optimal and the level of ions in the buffer solution must also be optimal. These levels can vary to some degree.
  • CESB can create hybridization conditions with maximum specificity and without any loss of sensitivity. More preferably, CESB can occur whenever a positively charged surface is present with the correct ion concentration in the buffer. In a preferred embodiment of the present invention, creating enhanced melting curves and CESB preferably requires a positively charged surface and interplay with the ion concentration of the buffer.
  • the consistency of surface charge density of the solid surface is even and consistent.
  • quality control levels be higher than other applications such as classic microarrays, but special handling and packaging methods may be needed to preserve the surface chemistry.
  • a novel method in cancer diagnostic assay for KRAS mutations has been developed and is provided.
  • this assay comprises 12 different mutations occurring within 6 base pairs.
  • this test can be performed by melting curve analysis or by CESB during hybridization, according to the methods disclosed in the present invention.
  • CESB and enhanced melting curves can be performed in the liquid phase with special adaptations that allow a miniature solid surface with positive charge to be attached to a probe or primer. This allows liquid phase methods like PCR to benefit from CESB.
  • the following issues were addressed and disclosed: 1) determining what is causing the inconsistencies in the ability of the expoxysilane sides to produce 2 stepped melting curves, 2) examining the effect of the ion concentration on the shape of the melting curves, 3) refining a model of how the positive charge enhances the melting curves, 4) using the enhanced melting curves to create a molecular diagnostic test, and 5) proposing future applications for the technology.
  • a novel cost-effective method to detect the melting of different sequences of target DNA with one base pair sensitivity for each probe spot of a microarray is disclosed, and represents a powerful genomic analysis tool with the ability to perform a type of DNA sequence determination or low resolution sequencing at the same cost as a microarray.
  • it functions as more than just a microarray, as it allows for the quick and efficient scanning of the entire genome for the few very important genetic differences that exist between samples. Afterwards, if a more detailed analysis is needed, NGS could be performed on the same DNA sample that was hybridized and then selected when melted off the microarray chip.
  • This novel method and application would be ideal for rapid screening of a population for genetic differences at a much lower cost than sequencing the entire genome. It is known to those skilled in the art, that single nucleotide polymorphisms typically are detected by sequencing DNA first before rapid low cost screening tests are developed to detect known SNPs. Therefore, SNP detection would be limiting to the population already sequenced. Screening with a melting curve microarray would reduce costs so low the entire population could be screened and in theory detect all SNPs in the population. Another application might be tracking the progress of an infectious disease outbreak or biological weapons attack. In this scenario, a large number of infected patients might be screened to allow characterization of virulence factors, drug resistance, or just obtain epidemiologic information about how the outbreaks progress. NGS is a shot gun approach providing global information from which specific information can be gleaned and is not practically cheap enough to sequence entire populations. Melting curve microarray screening in turn can focus in on only producing the relevant genomic information needed, saving time, energy and cost.
  • the present invention provides for technology related to surface chemistry of the array that produces enhanced melting curves. Additional research involves identifying improvements in the chemical coating of the microarray slide with the aim of yielding more durable, sensitive, and consistent results. Additionally, the stringency of the hybridization/melting process needs to be documented. The eluted target DNA from each microarray should be sequenced via NGS to confirm exactly what bound the probe DNA and exactly what is being melted away at given temperatures. Confirmatory testing would facilitate developing specific applications.
  • the developed method of the present invention serves to identify one base pair differences between different types of target DNA bound to a single probe spot. This has been accomplished on microarray slides containing 600-800 probe spots. An initial attempt to perform this analysis on commercial microarray chips containing over 30,000 probe spots was made but failed for reasons likely related to labeling of the target DNA and not the actual melting analysis. During initial development simplicity of array target density and composition was chosen to avoid any difficulty with interpretation of results. As such, custom microarray slides with 1000 or more probe spots were not ordered. However, it is likely that this technique will work on slides containing a minimum of 1000 probe spots provided the spots were large enough in diameter (> 150 ⁇ ).
  • the technology bridges the gap between microarray and Next Generation Sequencing (NGS and can achieve the accuracy of NGS systems at microarray prices.
  • NGS Next Generation Sequencing
  • This technology competes with microarrays but can work with or compete against NGS depending on the application.
  • the technology can be used as a capture/enrichment platform, and can more specifically and efficiently capture target DNA than conventional microarray capture systems. More preferably, this technology elutes nonspecific and extraneous DNA at low melting temperatures while retaining stably bound desired target DNA.
  • NGS NGS
  • this can increase the efficacy of enrichment while reducing NGS sequencing cost by avoiding the sequencing of unwanted DNA.
  • actual sequencing data can also be obtained by melting curves analysis on the array, the technology can simultaneously capture and re-sequence DNA by association, thereby obviating NGS sequencing.
  • novel assays to consistently resolve one base pair differences between different types of target DNA in a complex mixture. Typically, these results are unlikely to be accomplished under standard microarray, standard PCR, or variations of PCR such as allele specific PCR.
  • the first challenge is that a 2 base pair mismatch is unlikely to bind under the current hybridization conditions.
  • the second challenge is that typically, assays are unable to differentiate different KRAS activation mutants if the mutation is in the exact same base pair position.
  • the optimal way to clarify this result is to use probes specific for the possible mutations.
  • this was resolved in accordance with a method of the present invention with the KRAS assay using C6 wild type and SI mutant probes. It is known to those skilled in the art, that it may not be possible to obtain by standard methods. Previous techniques typically have known to be unreliable and unable to consistently resolve one base pair differences between different types of target DNA in a complex mixture.
  • cDNA stained with Cy3 dye (Arrayit Corp.) was used as target DNA for hybridization.
  • melting experiments can be carried out in a temperature range from about 0° C to about 100° C.
  • melting experiments were carried out over a temperature range from about 40° C to about 70° C, preferably with temperature increase increments of 1° C and fluidics buffer flush of 600 ⁇ of 2.5x SSC buffer.
  • a 10% solution of 2-Mercaptoethanol in 2.5 X SSC buffer (0.375M NaCl) was made and used as the melting/flush buffer to reduce photobleaching of the Cy3 dye.
  • the hybridization, washing and melting cycles of the melting curve microarray requires a strong and durable covalent attachment of probe molecules to ensure repeatability between assays.
  • the epoxysilane coated microarray slides Nexterion Slide E (Schott, Louisville, KY) were selected as a most durable product to attach the probes.
  • Probe DNA sequences were 25 base pairs in length (bp) and contained a modified amino 5' terminus containing a 6 amino acid linker. All probe sequences were custom synthesized by IDT (Coral ville, IA).
  • Microarrays were fabricated using standard pin printing techniques known to those skilled in the art, producing 150 ⁇ diameter probe spots. However, chemical deactivation with ethanolamine of unreacted epoxy groups after printing was not performed on the first batch of slides from the Functional Genomics Lab and the slides were shipped with an unblocked reactive surface. Subsequent epoxy microarrays were order from Microarray Inc. in both ethanolamine deactivated surfaces and non-deactivated surfaces.
  • Microarrays were fabricated with between 6-8 repeating blocks down the array slide with approximately 100 probes spots per block.
  • the general layout is summarized in Table 1.
  • the first row of each block contained a set of control probe spots. These consisted of positive control spots affixed with Cy3 dye which ranged in concentration from 5 ⁇ to 20 ⁇ , blank space(s), an E. coli gene as a negative control, and the gene sequence of interest, mouse GAPDH, in antisense orientation.
  • the mouse GAPDH probe spot was repeated in sense orientation between rows 1-10.
  • Target DNA consisting of 25 mer synthesized oligos (IDT) with Cy3 modifications added to the 5' terminus are shown in Table 2. A one base pair mismatch or SNP was added to the targeted DNA at position 13 causing a G to A mutation.
  • Table 2 List of Target Sequences
  • Probe DNA sequences were 25 base pairs in length (bp) and contained a modified amino 5 ' terminus containing a 6 amino acid linker. All probe sequences were custom synthesized by IDT (Coralville, IA) and in sense orientation. Slides were professionally printed using a Nexterion Slide E protocol by Microarray Inc. (Huntsville, AL). Each probe spot block consisted of 21 probe spots in a layout of 10 blocks. The probe spot layout and probe sequences of each block are shown in Table 3. A batch of 100 slides was fabricated using standard pin printing techniques known to those skilled in the art, producing 150 ⁇ diameter probe spots. However, chemical deactivation with ethanolamine of unreacted epoxy groups after printing was not performed and the slides shipped with a reactive surface.
  • Table 3 KRAS Probes for Codons 12 and 13 (Sense Orientation) and Probe Spot Block Layout for KRAS Arrays (Mutated bases in bold font).
  • Target KRAS DNA consisting of 25 mer synthesized oligos (IDT) with Cy3 dye modifications added to the 5' terminus are shown in Table 4. Mutations in bold font.
  • a 100 ⁇ hybridization mixture was made from 76 ⁇ hybridization buffer with formamide (Array It Hybit® 2, Arrayit Corp.), 16 ⁇ dH20, and 8 ⁇ of target DNA at 250 ⁇ concentration. If the target sample consisted of a mixture of 50% perfect match and 50% 1 bp mismatch, then 4 ⁇ of each was combined. Between 33 ⁇ to 50 ⁇ , of hybridization mixture was applied to each slide before placing a cover slip over the sample. Microarrays were placed in hybridization chambers (Arrayit, Corp.) and incubated at 45° C for 16-24 hours with mild agitation via a rotating shaker in a hybridization oven without humidification.
  • PEI polyethyleneimine
  • a modified Axon 4000a microarray scanner operated using custom software that interfaced the existing GenePix software included with the Axon reader.
  • the plumbing system was flushed with 2.5x SSC buffer, the thermal control chamber was pre warmed to 44° C, and the scanner focused.
  • the general programmed parameters for the experiment called for successive temperature incubations and washes over a range of 40° C to 70° C with temperature increase increments of 1° C, a temperature hold time of 1 minute, and a 2.5x SSC buffer flush of 600 ⁇ .
  • a scan was then made at 532nm with starting PMT settings ranging between 600-700 and scan files saved to the hard drive of the computer.
  • As the experiment progressed after each temperature increase there was an automatic PMT increase of 3 units followed by an automatic focus adjustment increment. These cycles were continued until the last temperature was reached for the range of the experiment.
  • a scan file was produced. Typical experiments generated over 20 scan files. Each scan was analyzed using the GenePix software according to the manufacturer's instructions. Briefly, for the first scan at a temperature of 40° C, the file was analyzed with the microarray manufacturers GAL file and GenePix software with a fixed surface area of the spot circle. Once the first scan was analyzed, a GPS file was generated by the GenePix software. The GPS file contained the software parameters used for analysis of the first scan. In order to insure consistency of data analysis, the same GPS file was used to analyze all remaining scans from the experiment.
  • the resulting scan file produced for each 1° C increment of temperature contained all the statistical data in a GenePix spreadsheet format termed a GPR file.
  • GPR file For each GPR file the column containing the Mean F532-B532 (Mean Fluorescence 532-Background 532) was copied and transferred to a Microsoft Excel Spread sheet. This procedure may be computed by hand but data compiling software was written to automate the task. All graphs were generated with the Excel software program.
  • an improved microarray reader including a redesigned cassette port (FIG. 8B) and cassette (FIG. 9A).
  • FIG. 9B shows a loaded cassette in place with the objective lens of the reader below the cassette.
  • Three novel software programs were written to operate the machine and help analyze the data (FIG. 2).
  • computerized Z-axis was added to the thermal control block to speed up the focusing procedure and allow automatic incremental adjustments of focus during experiments (figure not shown).
  • the microarray chips produced by the Keck Center at University of Illinois at Urbana-Champaign were, composed entirely of probes to detect the binding of the mouse GAPDH gene sequence.
  • GAPDH is a housekeeping gene that is normally expressed at high levels within mouse cells because of its involvement with glycolysis. Eight blocks or about 800, 150 ⁇ probe spots were replicated on a microarray slide. Duplicate blocks allowed verification of the consistency of results and the large probe spots made scanning detection easier. Arrays were hybridized with excess of target DNA which contained a complementary 25 mer labeled with Cy3 dye.
  • the Keck Center slides may have bound target via bonds other than hydrogen bonds or had the capacity to react with abundant levels of ozone and hydrocarbons present in the tropical air of developed areas of Hawaii and certainly present in the room air of the laboratory.
  • the type A curve may possibly be related to the reaction of the chip surface with unwanted agents in the air causing background and unreadable results. Also, it may be possible that the type B results were attained if the chips were processed very quickly and placed in liquid buffer without allowing the room to air to react with the surface.
  • the active surface of the Keck Center chips contained an epoxide exposed to the surface.
  • the epoxide was intended to react with the amino terminus of the modified probed DNA via a nucleophilic addition where the epoxide functions as an electrophile and the probe molecule as a nucleophile. Since the epoxide is un-reacted, the surface is coated with a strong electrophile, which generally can have a positive chemical charge.
  • the deactivation of epoxide with ethanolamine via a nucleophilic addition changes the surface composition of the slide from epoxide to that of a hydrocarbon with attached hydroxyl group.
  • Testing was conducted using a proprietary chemical treatment for changing the surface charge of a biosensor.
  • This proprietary mixture comprises of off the shelf chemicals with the primary ingredient being the positively charged polymer polyethyleneimine. Solutions of polyethyleneimine ranging in concentration from 1% to 10% in 2.5x SSC buffer were used to coat the deactivated surface of Microarray Inc. slides before the hybridization mixture was added.
  • FIG. 5 shows the graph of 10 different probe spots and displays a very steep drop off curve with a Tm approximated by an arrow at 64.5° C. All results are raw unadjusted data. Note the consistency of the slope with graphs starting at different intensities, indicating different concentrations of target had bound to these probe spots, but melting away at exactly the same Tm.
  • This graph resembles the shape of type B data from the Keck Center chips shown in FIG. 3B and is distinctly different from Microarray Inc, slides not treated with polyethyleneimine. Therefore, polyethyleneimine treatment appears to have some type of an effect on the binding/melting characteristics of the DNA.
  • the presence of a positively charged surface in close proximity to the nucleic acids melting or dissociating can cause the characteristics of the melting to change.
  • the two complimentary nucleic acids melt apart over a broad temperature range and not a specific temperature point (FIG. 18B).
  • the melting curves of each species can overlap making the detection of the two species not possible as seen in FIG. 18B.
  • the binding partners containing the one base pair mismatch begin to melt apart.
  • the perfect match is already melting apart, generating a melting curve with a smooth ski slope masking the presence of two species of nucleic acid binding partners.
  • the reason for the change of melting behavior in the presence of a positively charged surface may not be well known.
  • the presence of a positively charged surface can add an additional force to the melting nucleic acids.
  • hydrogen bounding is holding the double stranded nucleic acids tighter.
  • the hydrogen bonding is the force that must be overcome during dissociation.
  • the presence of a positively charged surface now becomes an additional force together with the hydrogen bonding that may play a role in affecting how the nucleic acids melt apart and sharpening the melting curve.
  • FIG. 18, graph A produced a stair step type curve with easy identification of the Tm's for both mismatched and perfectly matched data.
  • Type B data shown in FIG. 3 has only one very steep melting curve which confirms that only one type of perfectly matched target was present.
  • FIG. 5 displays two slopes, following from left to right, the slope begins with a shallow convex shape which then turns into an almost vertical line straight down.
  • graph B breaks the melting curve into sections based on the slope of the curve. If the shallow slope represented the one base pair mismatched target melting away and the steep slope the perfect match melting, then relative amounts of each product could be calculated based on the percentage of fluorescence associated with each slope of the graph.
  • FIG. 6, graph A depicts a graph of a combination of several probe spots and shows a stair step type curve but with a much shallower slope than shown in FIG. 3 type B data or FIG. 5.
  • the shape of this graph was consistent as multiple probe spots had similar but not exactly the same shape (FIG. 6, graph A).
  • Two distinct Tm's can be observed in the slope of the graph at 51° C and 59° C which resembles the shape of curves found in FIG. 18, graph A.
  • the approximate amounts of each target (FIG. 6, graph B) are 20.3% of one base pair mismatch and 79.2% perfect match.
  • whole human genome chips contained over 30,000 probe spots and probes sequences of 60 base pairs in length. Since the number of probe spots was much higher, the individual spots were much smaller in diameter at 80 ⁇ .
  • One chip was processed with a mixture of Cy3 labeled human liver and Cy5 labeled heart cDNA. This was the first experiment with a large number probe spots and the first two color gene expression profiling microarray experiment. An objective for the scanning machine was to be able to read this chip and that the melting curve data would reduce the noise in the measurement. For example, if a given gene was expressed at a 2: 1 ratio between two different samples, noise might alter the measured ratio to 1 : 1. It was hoped that the melting procedure would remove non-specifically bound cDNA at lower melting temperatures.
  • a working instrument has been provided and method demonstrated for using thermal melting analysis in a microarray format as a novel low cost genomics analysis tool.
  • This technique both improves the accuracy of microarrays and fills gaps not covered well by NGS.
  • Microarray chips manufactured by the W. M. Keck Center for Comparative and Functional Genomics produced some of the most amazing melting curves but not in a consistent manner (FIGS. 3 and 4).
  • an improved microarray reader has been developed.
  • the improved microarray reader according to an embodiment of the present invention comprises improvements to the machine and its operating system, including the addition of an automatic focusing system which greatly enhanced the consistency and quality of data acquired.
  • microarray surface chemistry of the microarray has on the actual melting analysis.
  • experiments conducted using the Keck Center chips revealed unexpected results, which were determined to be affected by a non standard active epoxide surface coating on these chips.
  • conventional off the shelf microarrays and supplies are not suitable for melting curve microarray analysis. This is confirmed by the very consistent melting curve data produced by the Microarray Inc. chips which were ethanolamine deactivated but unable to distinguish between different types of bound target (FIG. 18, graph B).
  • ethanolamine (as seen in FIGS. 18 and 19), how the surface of the microarray affects the melting of DNA might involve effects on the melted target DNA.
  • polyethyleneimine was chosen to coat the slides.
  • polyethyleneimine is frequently used as a cationic lipid for the formation of liposomes used in transfection of mammalian cells. It has been determined that the cationic properties of polyethyleneimine can cause dsDNA with a net negative charge to condense within the liposome as well as causing some limited denaturation of the double stranded helix.
  • the present invention provides for the novel use of polyethyleneimine for enhancement of DNA microarray melting curve analysis. Since polyethyleneimine is a solid with a melting temperature of approximately 75° C, it is known to those skilled in the art, that the polymer stays on the surface of the glass slide at temperatures below 75° C allowing interaction with the dsDNA located immediately above the microarray surface. The precise mechanics of this interaction are unknown but the polymers association with denaturation when used in liposomes, suggest conditions on the microarray are promoting denaturation in a manner that allows one base pair resolution of detection. This in turn may imply an effect on the strength of hydrogen bonds between base pairs.
  • the Tm of the perfectly matched targeted DNA decreased as the concentration of polyethyleneimine increased from 1% to 10% suggesting a weakening of hydrogen bonds between base pairs (FIGS. 18, 5, and 6).
  • the positively charged surface is having an effect on the localization of the melted targeted DNA either causing it to be repelled or attracted to the surface as seen in FIG. 19.
  • a repelling effect might push the target DNA away from the surface and would give the appearance of faster melting or a lower melting temperature.
  • DNA generally carries a net negative charge and would likely be attracted to the positive charge of polyethyleneimine (FIGS. 18 and 19).
  • Experiments with microarray s coated with 5% and 10% solution of polyethyleneimine showed incomplete melting of target DNA (FIGS. 6 and 6) suggesting that the target might be binding the surface of the microarray via other non hydrogen bonding mechanisms.
  • the observed changes in DNA melting characteristics may be complex and involve more than one type of chemical interaction.
  • positive charge on the solid surface typically can attract the negatively charged nucleic acids directly above the charged surface.
  • the exact confirmation of the nucleic acids has not been determined but it is reasonable to assume that the attractive force would cause the negatively charged nucleic acids to bend over into a position which allows it to be in close proximity to the positively charged surface providing the nucleic acids and the method of attachment to the surface is flexible (FIG. 19).
  • the enhancement is provided by positively charged surface and the composition of the buffer solution. The attractive force between the nucleic acids and the positively charged surface is likely modulated by the composition of the buffer solution.
  • a buffer containing a high ionic strength typically would contain many positively and negatively charged ions that would be attracted towards the positively charged surface and the nucleic acids, therefore reducing the perceived attractive forces between the surface and nucleic acids.
  • a buffer with a low ionic strength typically would have fewer ions to be attracted to the surface and nucleic acids and therefore less effect on the attractive forces between the surface and nucleic acids. This effect could be utilized to modulate the attractive force between the surface and nucleic acids by either reducing or strengthen the attractive force by changing the ionic strength of the buffer (see FIG. 19).
  • the microarray is normally read at room temperature completely dry.
  • a special microarray reader was built (see FIG. 8).
  • a heating element and fluidics system was added. This is shown in a schematic diagram of FIG. 8A.
  • the fluidics system included a buffer heating system capable of heating the buffer to a desired temperature and then distributing this buffer across the array to wash the array. Buffer runoff is then taken to a waste bottle.
  • the temperature of the microarray was raised one degree Celsius.
  • buffer of the same temperature was flowed across the surface of the microarray and then the microscope was focused before a picture was taken. This cycle was repeated multiple times per experiment.
  • FIGS. 8A-B depict a microarray to perform DNA melting curve analysis.
  • the actual completed machine was a modified Axon 4000a microarray scanner and operated using custom software that interfaced the existing GenePix software included with the Axon reader.
  • This machine was a compact unit with the main component, termed the slide card, containing both the heating element and fluidics system in the upper module (see FIG. 8B).
  • FIGS. 9AB and 9B an array cassette and array reader are shown.
  • the microarray was placed DNA facing down into a plastic cassette with both in and out ports (see FIG. 9A.
  • FIG. 9B When the slide card was closed the ports of the slide card aligned with the ports of cassette and allowed for buffer flow (FIG. 9B).
  • FIG. 9B The entire microarray reader machine with the slide card in both a closed and open position is shown in FIG. 9B.
  • the microscope was in an inverted orientation below the microarray and not clearly visible in the photo. The raster scanning process took place by moving the slide card in the Y axis and the microscope objective lens back and forth in the X axis.
  • the scanning machine plumbing system was flushed with SSC buffer that was the same concentration used during the melting phase of the experiment and ranged from 2.0x-4.0x SSC. Then the thermal control chamber was pre- warmed to 44° C, and the scanner focused.
  • the general programmed parameters for the experiment called for successive temperature incubations and washes over a range from about 40° C to 70° C with temperature increase increments of 1° C, a temperature hold time of 1 minute, and a SSC buffer flush of 600 ⁇ .
  • a scan was then made with an excitation wavelength of 532nm with starting PMT settings ranging between 600-700 volts and scan files saved to the hard drive of the computer. As the experiment progressed, after each temperature increase there was an automatic PMT increase of 3 units followed by an automatic focus adjustment increment. These cycles were continued until the last temperature was reached for the range of the experiment.
  • a scan file was produced. Typical experiments generated over 20 scan files. Each scan was analyzed using the GenePix software according to the manufacturer's instructions. Briefly, for the first scan at a temperature of 40° C, the file was analyzed with the microarray manufacturers GAL file and GenePix software with a fixed surface area of the spot circle. Once the first scan was analyzed, a GPS file was generated by the GenePix software. The GPS file contained the software parameters used for analysis of the first scan. In order to insure consistency of data analysis, the same GPS file was used to analyze all remaining scans from the experiment.
  • the resulting scan file produced for each 1° C increment contained all the statistical data in a GenePix spreadsheet format termed a GPR file.
  • GPR file For each GPR file the column containing the Mean F532-B532 (Mean Fluorescence 532-Background 532) was copied and transferred to a Microsoft Excel Spread sheet. This procedure may be computed by hand but data compiling software was written to automate the task. All graphs were generated with the Excel software program. All data graphed were raw non-normalized data unless otherwise indicated.
  • SSC buffer Commercially available stock solutions of SSC buffer were used from Sigma Life Sciences (Sigma-Aldrich, St. Louis, MO) in a 20X concentration. Dilutions were made with distilled water in the ranges of 2.0X to 4.0X SSC buffer and placed into 250ml GL medium storage bottles (Kimble-Chase, Vineland, NJ.). Buffers were used within 24 hours of dilutions to avoid evaporation of the solution which would alter the ion concentrations.
  • FIGS. 11A-B depict results obtained from amine coated microarrays.
  • the first set of experiments was done on commercially fabricated amine coated microarray slides from Arrayit named the "Check It Chips". These microarray chips were designed as a calibration chip for calibrating microarray readers.
  • the Check It Chips kit comes with a universal target DNA which can bind all probe spots.
  • the universal target was not used and human cDNA stained with Cy3 dye (Arrayit Corp) was instead used as the target DNA for hybridization.
  • the melting buffer selected was SSC buffer at a 2.5x concentration which contained 0.375M NaCl. Representative results of these experiments are shown in FIGS. 11A-B. On FIG.
  • 11A are 2 actual scans of the microarray at the beginning of the experiment at 45° C and the end of the experiment at 65 °C.
  • the scan at 45 °C shows the square shaped probes spots of both blocks of the microarray which are replicates of each other with many of the probes fluorescing due to the binding of the target human cDNA.
  • the positive control probe spots are Cy3 dye bound directly to the glass and have a rounded shape. These are located in the upper left and lower right corners of each block. In the 65 °C scan most of the target DNA bound to test probes was melted away leaving the round shaped positive control spots fluorescing.
  • FIG. 1 IB melting curves obtained on unblocked amine coated surface plates with 2.5 X SSC buffer for Cadherin 1 (FIG. 11B1) and Beta Actin (FIG. 11B2).
  • the first set of experiments was done on commercially fabricated amine coated microarray slides from Arrayit named the "Check It Chips". These microarray chips were designed as a calibration chip for calibrating microarray readers.
  • the Check It Chips kit comes with a universal target DNA which can bind all probe spots.
  • the universal target was not used and human cDNA stained with Cy3 dye (Arrayit Corp) was instead used as the target DNA for hybridization.
  • the melting buffer selected was SSC buffer at a 2.5x concentration which contained 0.375M NaCl. Representative results of these experiments are shown in FIG.
  • FIG. 11 On the left side of FIG. 11 are two actual scans of the microarray at the beginning of the experiment at 45° C and the end of the experiment at 65° C.
  • the scan at 45° C shows the square shaped probes spots of both blocks of the microarray which are replicates of each other with many of the probes fluorescing due to the binding of the target human cDNA.
  • the positive control probe spots are Cy3 dye bound directly to the glass and have a rounded shape. These are located in the upper left and lower right corners of each block.
  • the 65° C scan most of the target DNA bound to test probes was melted away leaving the round shaped positive control spots fluorescing.
  • graphs of the melting curves generated depict the most striking feature of the graphs show the generation of sharp melting curves.
  • the section of the melting curves where the DNA melting took place is marked with an arrow which approximates the Tm.
  • the results of the melting curve for the probe spot Cadherin 1 is shown. Between the start of the curve at about 40° C and the beginning of the melting curve at about 59° C the graph is almost horizontal with a slight upward slope. At about 60° C, there is a sharp downward slope that is the melting curve.
  • the presence of one melting curve in the graph suggests that there is one type of target DNA which has bound the probe spot and is melting away.
  • the target is likely to be the true perfect match target of the probe and not mismatched target.
  • the graft depicts two melting curve Tms obtained from the Beta Actin probe spot, one at about 62° C and another at about 46° C.
  • the true power of the melting curves becomes apparent in this graph as it was able to detect the presence of perfectly matched target melting at 62° C and mismatch target melting at about 46° C both on the same probe spot.
  • the Check It Chips kit did not include a blocking agent that would normally neutralize the positive charged amine coated array surface. So it can be assumed that the surface was positively charged.
  • Microarray surfaces are normally chemically blocked and would be expected to have a near neutral surface charge.
  • the blocking is performed in order to prevent nonspecific binding of target DNA to the array surface which would produce background noise.
  • the detection of both perfect match and mismatch target on the sample probe spot is not normally possible.
  • FIG. 12 depicts the effects of 10% 2-Mercaptoethanol on DNA melting curves.
  • the use of unblocked amine coated slides in the presence of 2.5x SSC buffer was capable of producing unusually sharp DNA melting curves with the ability to detect the presence of both perfect match and mismatch melting away from the same probe spot.
  • the use of 10% 2-Mercaptoethanol helped to reduce photo bleaching of the Cy3 dye but also altered the characteristics of the melting curve by lowering the Tm and changing the slope of the melting curve making it a shallower curve. The change in the slope indicates that the temperature range of melting was wider in comparison to the melting curves performed using only 2.5x SSC buffer.
  • microarray Inc Epoxy microarray chips were ordered which were identical to the previous batch in both surface chemistry and probe sequences. Experiments with these new chips and perfectly matched target DNA produced ski slope type melting curves for all probe spots on the array. This was repeated several times consistently.
  • FIG. 3 shows types of melting curves obtained on epoxide-coated microarray slides.
  • the top graph of FIG. 3 shows a ski slope melting curve and the bottom graph depicts a stair step melting curve.
  • FIGS. 14A-B depict epoxy silane attachment to a glass microarray.
  • the epoxide that is commonly attached to the glass microarray is in the form of epoxy silane.
  • the structure of epoxy-silane is shown in FIG. 14A.
  • the silicon (Si) in the epoxide silane molecule is bonded to three oxygens (O).
  • the 3 oxygens each with an electronegativity value of 3.44 (on the Pauling Scale) act to withdraw or hog electrons shared by the bonds with 1 silicon atom with an electronegativity value of 1.90. This creates a strong dipole moment in which the silicon is the positively charged side of the dipole and the oxygens are on the negatively charged side of the dipole.
  • the dipole When the epoxy silane molecule is attached to the glass microarray surface (FIG. 14B), the dipole is oriented such that the negatively charged side of the dipole is nearest to the glass surface. Therefore, the positively charged silicon is oriented above the negatively charged oxygens.
  • the microarrays are printed, single stranded DNA modified with amino acids at one terminus can become attached to the epoxide group via a nucleophilic substitution reaction. Once the DNA is attached, the silicon oxygen dipole is oriented with the positively charged silicon nearest the DNA. Therefore, although the epoxide coating is neutral in charge, there is a layer of positive charge below the DNA and above the microarray slide.
  • FIGS. 18A-B depict coating microarray slides with polyethyleneimine.
  • PEI cationic polymer polyethyleneimine
  • FIG. 18 A This melting curve produced a 2 stepped curve confirming the binding and then melting of both the perfectly matched target with a Tm of about 67° C and the 1 bp mismatched target with a Tm of 59° C. It was noticed that the temperature range of melting of the mismatched target does not overlap with the temperature range of melting of the perfectly matched target. This is evidenced by the change in slope of the graph to a flat spot with no slope at about 60° - 62° C. In this short section of the graph no melting is taking place. An estimate of the percentage of each target that bound the probe can be made by measuring the amount of relative fluorescence lost during the melting curves. Estimates are 81.6% perfect match and 18.4% mismatch (FIG. 18 A). This shows that perfectly matched target has a preference for binding but mismatch can also bind under these conditions.
  • the Tms are different with a 1% PEI treatment producing a 1 bp mismatch Tm of 59° C and perfect match of 67° C.
  • the Tms for the 10% PEI are approximately 51° C for the 1 bp mismatch and 59° C for perfectly matched target.
  • PEI is a positively charged polymer and it can be assumed that the concentration of PEI that is used to coat the microarray surface can also be used to change the level of positive charge on the surface. It is reasonable to assume that the level of positive charge on PEI coated arrays of 1%, 5%, and 10% were different, with the higher concentrations having higher levels of positive charge. While positive charge is needed to produce the enhanced 2 stepped melting curves, the amount of positive charge needed appears to not be limited to just one specific level of positive charge but can vary to some degree. This is evidenced by the ability of both 1% and 10% PEI coatings being able to produce 2 stepped melting curves but with different characteristics. However, using PEI coatings as method to create a positively charged microarray surface has significant limitations.
  • FIGS. 6A-B depicts a blocked epoxy slide coated with 10% polyethyleneimine.
  • FIGS. 15A-E depict DNA melting curves on unblocked epoxy coated microarrays with glass cover slips. The results of the first set of experiments are show in FIGS. 15A-E. Each graph FIGS. 15A-E shows the melting curves of three separate probe spots near to each other on the same microarray.
  • the 2.8x SSC buffer graph shows a gradual slope upward at the beginning due to the focus of the microarray being slightly out focus at the start and then going into focus as the temperature of the experiment increased. This occurred because changes in temperature change the refractive index of the SSC buffer, which in turn changes the focal point.
  • the machine was designed to compensate for changes in refractive index by increasing the PMT gain with each temperature increase. However, if the first scan was not in good focus then compensational changes in gain will not work as well. Next there is a slight downward slope before the slope of the graph become almost vertical.
  • This graph is similar to the melting curve graph generated on blocked epoxy slides with 5% PEI (FIG. 3). There were obvious changes in the slope of the curve but no flat step in the graph. If the change in downward slope is used as an indicator of the melting of 1 bp mismatch and perfectly matched target then the relative percentages of each can be estimated to be 30% 1 bp mismatch and 70% perfectly matched (FIGS. 15A-E). This result was consistent over the entire microarray surface.
  • FIGS. 16A-D depict stability comparisons of unblocked epoxy slides before and after one month. About one month had elapsed since starting experiments with the new microarrays ordered from Microarray Inc. In an effort to clarify these results more melting curves experiments were done at the 2.8x, 2.9x and 3.0x SSC buffer concentrations. None of these experiments could repeat the enhanced stair step type melting curves generated with 2 stepped curves or even steep melting curves. Two example results are shown in FIGS. 16A- D. SSC buffer concentration 2.8x and 3.0x were both able to produce sharp enhanced melting curves before one month had passed after first opening the microarray slides from the vacuum sealed bag and being placed in the dissector.
  • unblocked epoxy microarrays were stored as individual microarrays and transferred to vacuum sealed bags inside an argon glove box. This was accomplished by taking a second box of microarrays in a 25 pack fabricated by Microarray Inc. that was still sealed in a nitrogen purged bag. This bag was opened inside an argon gas glove box and each microarray transferred to an individual slide box and vacuum resealed in an individual vacuum bag while still inside the argon glove box. This procedure insured that the slides were not exposed to room air at any time. Once the transfer had been completed, experiments were resumed by varying the concentration of SSC buffer in each experiment as before. Another small change to note was the switch from glass cover slips on the microarray cassette to plastic cover slips.
  • FIGS. 17A-E depicts melting curves on unblocked epoxy coated slides with plastic cover slips.
  • the experiments resumed starting with a 2.6x SSC buffer concentration and ending at a 3.3x SSC buffer concentrations. The results of these experiments are shown FIG. 17 and all graphs depict three different probe spots near each other in the same block.
  • the SSC buffer concentrations of 2.6x, 2.7x, and 2.8x all showed a ski slope type melting curves.
  • the result of the 2.8 x SSC (0.420 M NaCl) buffer concentrations is representative of the 2.6x and 2.7x concentrations and is shown in FIG. 17. It is not possible to detect the presence of two different DNA targets melting away from the probe in this graph as the slope is rather smooth and without abrupt slope changes.
  • the graphs As the concentration of the SSC buffer is increased to 3.0x and 3.1x SSC, the graphs also have a flat step marked by a dashed circle around the change in slope (FIGS. 17A-E). These types of melting curves occurred in 3 blocks out of 6 blocks or about half the probe spots on the microarray. Notice that the flat step occurs between 51° to 53° C at the SSC buffer concentrations of 2.9x, 3.0x, and 3.1x SSC. As the buffer concentration is increased to 3.2x SSC (0.480M NaCl) the flat step or abrupt change in the slope of the graph is lost. There are some small irregularities in the graph of the 3.2x SSC buffer concentration in the temperature range of 47° to 51° C.
  • FIGS. 19A-B depict a model of how a positively charged surface enhances DNA melting.
  • the ability of the positively charged microarray surface to enhance nucleic acid melting curves was discovered by trial and error. This effect was first observed on unblocked amine coated microarrays without knowing that the positive surface charge was important.
  • the use of blocked epoxy microarrays with a coating of positively charged PEI confirmed the observations with unblocked amine coated slides.
  • PEI coated microarray arrays also showed that multiple concentration of PEI ranging from 1% to 10% could produce enhanced melting curves. This indicates that multiple levels of positive charge can produce the effect and that this phenomenon and it is not limited to a single level of positive charge.
  • Unwanted changes to the chemical surface of unblocked epoxy microarrays prevented the enhanced melting curves from occurring. This suggests that the surface chemistry is very sensitive to changes and must be carefully preserved to insure repeatability.
  • the chemistry of the buffer also plays a large role in creating enhanced nucleic acid melting curves.
  • the use of 10% 2-Mercaptoethanol in 2.5x SSC buffer completely prevents any enhanced melting curves from being detected.
  • the buffer concentration of 2.5x SSC was selected as a starting point for all experiments. Experiments to determine the optimal SSC buffer concentration revealed that the enhanced melting curves are produced within a range of concentration that is higher than 2.5x. Early experiments with unblock epoxy microarrays using 2.5x SSC buffer that produced enhanced melting curves, likely had a higher concentration of ions in solution. This occurred because of the evaporation of water in the buffer, due to the use of containers with poor sealing caps and a hold time of days to a week or more.
  • a model to describe the conditions needed to produce sharp enhanced two stepped melting curves would have two main components. These components are the surface charge of the microarray and the buffer composition.
  • these components are the surface charge of the microarray and the buffer composition.
  • the DNA is shown as having a net negative charge due to the phosphate backbone.
  • the target strand of DNA has a chemical dye attached which is shown as a star. Since the microarray surface is not positively charged, the negatively charged DNA is not attracted to the surface and is just tethered to the solid surface without folding over on the surface (FIG. 19A). No additional chemical interactions are taking place that would alter the kinetics of the DNA melting in this situation.
  • Probes for the microarray can be attached by several chemical methods. Two of the most common methods known to those skilled in the art, are binding the DNA directly to the surface or to use linkers to tether the DNA to the microarray. The surface binding method is often used with amine microarray surface chemistry as the positively charged amine surface can attract the negatively charged DNA. The linker bound method is commonly used on epoxy coated microarray surfaces.
  • both unblocked amine and unblocked epoxy surfaces have a layer of positive charge facing the DNA that can attract the DNA. This is shown in FIG. 19B as an attractive electrostatic force occurs between the DNA and positively charged surface. If the DNA is linker bound, it is expected to bend over and towards the positively charged surface due to the attraction. This is shown with a double arrow (FIG. 19B). The surface bound DNA experiences the same attractive force but is already attached to surface and does not bend over.
  • FIG. 19B In solid phase DNA melting environments with positive charge on the surface, there are at least two significant chemical interactions acting on the target DNA holding it place.
  • the target strand of DNA is shown with a chemical dye attached which is depicted as a star (FIG. 19B).
  • This strand of DNA is bound to the complementary probe strand of DNA which is bound to the microarray surface.
  • FIG. 19A When the microarray surface is not positively charged (FIG. 19A) hydrogen bonding with its complementary probe hold the target strand in place.
  • FIG. 19A hydrogen bonding with the complementary probe plus the attraction to the surface holds the target strand in place.
  • both the hydrogen bonds with the probe and the attraction to positively charged surface must be overcome to allow the target to melt away.
  • the additional attractive force with the surface is the fundamental difference in the chemical environments between the positively charged surface and non-positively charged surface that is responsible for changing the kinetics of the way the DNA dissociates or melts apart.
  • the breaking of hydrogen bonds is the principle factor that determines the kinetics of the DNA melting reaction.
  • the temperature ranges of melting of perfect match and 1 bp mismatch do not overlap during solid phase melting on a positively charged surface. This may be due to the additional attractive force the positively charged surface places on the target DNA during dissociation.
  • Exactly how the positive charge changes the melting kinetics may be related to the combination of hydrogen bonds with probe DNA and the attraction to the positively charge surface acts in a way that makes it slightly more difficult for dissociation to take place holding the target DNA in place longer till a slightly higher temperature is reached. Then when melting takes place, the unbinding event happens much more rapidly which in effect narrows the temperature range of melting.
  • the enhanced melting curves take place over a range of PEI concentrations. This indicates that they take place over a range of positive surface charge densities and are not limited to just one specific charge density.
  • different PEI concentrations produced different enhanced graphs with different characteristics meaning that different levels of surface charge can produce graphs with different characteristics. In order to obtain melting curve graphs with ideal characteristics the exact level of surface charge would likely need to be determined.
  • the enhanced DNA melting curves can occur with different levels of positive surface charge, there is likely a range of positive surface charge density that this effect occurs although it may be a wide range.
  • the second component needed to obtain enhanced DNA melting curves is the optimal buffer composition.
  • SSC buffer stands for saline sodium citrate buffer and this term is used here on after.
  • the most prominent chemical in the buffer is sodium chloride, NaCl.
  • NaCl sodium chloride
  • Increasing the concentration of NaCl in a buffer used for DNA melting can increase the Tm of the sequence being melted by acting to lessen the repulsive effects between the two negatively charged phosphate backbones.
  • reducing the concentration of NaCl in the buffer can reduce the Tm of a particular DNA sequence by increasing the repulsive effect between the negatively charged phosphate backbones.
  • NaCl will dissolve into the ions Na + and CI " .
  • the concentration of NaCl can be used as a means to modulate the attractive force between the DNA and positively charged surface.
  • the positive charge on the microarray surface is the most important factor for producing enhanced melting curves and the level of positive charge that can produce this effect likely occurs over a range. Enhanced DNA melting curves will not be produced if the positive charge surface density is not within the appropriate range.
  • One method for adjusting the level of surface charge on the microarray is by adjusting the level of positively charged chemical or chemicals coating the surface. This has been accomplished by using different levels of PEI on a blocked epoxy microarray.
  • a second method of adjusting the level of positive charge on the surface of microarray is by changing the level of ions in the buffer.
  • the ion combination is NaCl or Na + and CI " .
  • the level of positive surface charge density combined with the buffer concentration of ions work together to produce enhanced melting curves. They produce an overall level of charge attraction between the negatively charged DNA and the positively charged surface. If the level of positive surface charge density is too low to produce enhanced melting curves and cannot be increased, the buffer concentration can be decrease which can lower the level of ions in solution. Lowering the level of ions in solution can decrease the shielding effect the ions have on the attraction between the negatively charged DNA and the positively charged surface. This in turn can increase the attractive forces between the DNA and surface. If on the other hand the positive charge on the surface is too strong to produce enhanced melting curves and cannot be reduced, the buffer concentration can be increased which can increase the number of ions in solution.
  • the increase in the number of ions in solution can increase the shielding effect and decrease the attractive forces between the DNA and the surface.
  • concentration of the buffer can be adjusted to optimize the ability of the positively charged surface to produce enhanced melting curves. This optimization may require an increase or decrease in the level of positive surface charge on the microarray surface and can be accomplished by changing the buffer concentration.
  • the amount of shielding from the buffer concentration is hypothesized to be too high to produce enhanced melting curves.
  • the amount of attractive force between the DNA and positively charged surface is too weak.
  • concentration of SSC buffer is reduced to 3.1x, this is hypothesized to reduce the amount of shielding taking place and increase the level of attractive force between the DNA and microarray surface.
  • the level of attractive force between DNA and the surface is not ideal but it does allow enhanced melting curves to occur. Further adjustment of the buffer concentration by lowering the ions level can produce even better results.
  • enhanced melting curves by controlling the interplay between the positively charged surface and the buffer ion concentration are disclosed.
  • This can be a sensitive system.
  • Earlier experiments with blocked epoxy microarray coated with PEI were able to produced enhanced meting curves but not over the whole microarray surface only part of it. It was believed that the PEI did not coat the surface evenly or washed off in certain areas.
  • the unblocked epoxy microarrays produce more repeatable results.
  • the epoxy coatings were made as an attachment surface for binding the probes molecules and not for consistency in charge density across the entire surface of the microarray. Higher quality control levels may be needed to completely optimize the unblocked epoxy microarray for melting curve analysis.
  • Another component that may be important in optimizing the enhanced melting curve analysis is the surface charge of other parts of the microarray cassette. Experiments using PEI coated microarrays would not work after several reuses of the microarray cassette. This was thought to be due to the buildup of PEI on the internal surfaces of the cassette. Furthermore, the use of plastic cover slips on the cassette used for unblocked epoxy microarrays changed the optimal range of SSC buffer concentration needed to produce enhanced melting curves. Other factors that may be important in optimizing the melting curves are the length and sequence of the probes and target. Still another factor may be the length of the linker molecule used to attach the probe the surface of the microarray.
  • optimizing the ability of microarrays to produce enhanced melting curves can depend on uniformity of the surface charge density, high quality control during manufacturing, and special storage procedures to preserve the surface chemistry.
  • the interplay between the positively charged surface and the concentration of ions in solution is very sensitive and requires that the buffer concentration be adjusted to each microarray surface chemistry used.
  • buffer concentrations may also need to be optimized for surface charge density of the cassette material, the sequence and length of both probes and target DNA, and the length of the linker molecule used to attach the probe to the microarray surface.
  • liquid phase DNA melting curves typically are not able to produce enhanced melting curves.
  • the use of a positively charged microarray surface is counterintuitive.
  • a positively charged microarray surface is known to cause nonspecific binding with target DNA and increases the levels of background noise which is why blocking of the microarray surface is performed before hybridization.
  • analyzing software can subtract the amount of background noise from the signal giving a relatively accurate result.
  • FIGS. 20A-C depict an overview of the KRAS Mutation Assay.
  • the KRAS gene is an oncogene that codes for the K-Ras protein.
  • K-Ras is a GTPase in a signal transduction pathway known as the RAS/MAPK pathway. This pathway controls cell growth, division, and differentiation.
  • Mutations in the KRAS gene can cause an activating mutation in the GTPase protein, causing the enzyme to be always activated. This in turn can cause disregulation in the RAS/MAPK pathway leading to abnormal cell behavior with the potential to cause cancer.
  • Activating mutations in KRAS can occur in codons 12 and 13.
  • a class of cancer treatment drugs known as epidermal growth factor receptor (EGFR) inhibitors work by blocking the receptor and interfering with the growth and cell division of cancer cells.
  • K-Ras is a downstream component of the signal transduction pathway activated by the EGFR. If mutations occur in KRAS that cause the protein to be always activated, the signal transduction pathway will be always on and EGFR inhibitor drugs will no longer work.
  • a KRAS mutation test must be given. If KRAS activating mutations are present the patent is not likely to respond to EGFR inhibitors. And if KRAS is not mutated then the patent is likely to respond to EGFR inhibitors.
  • KRAS mutations can be detected by DNA sequencing which includes both Sanger sequencing and targeted next generation sequencing methods. DNA sequencing costs have had an incremental drop over the last 10 years but the price per test of older technologies such as PCR and microarray analysis are still more cost effective. Methods of analysis for KRAS mutations that use hybridization and melting are not able to reliably detect each of the 12 mutations in the 6 base pairs of codons 12 and 13. Several KRAS mutation diagnostic kits are commercially available and FDA approved in the United States, however, they are only able to detect the presence of a mutation and not confirm the sequence of the mutation.
  • kits using PCR followed by liquid hybridization are known to exist, however, these kits are only able to detect a change from the wild type sequence but are not able to determine which mutation is present.
  • Other technology utilizes assays using PCR followed by a liquid phase DNA melting assay. Although these assays are able to detect a change from the wild type sequence, they are not able to determine the sequence of the mutation. Confirming the changes in KRAS sequence is important during cancer treatment. Additionally, it would be beneficial to address the question, that if a cancer patient responds to treatment only to have a reoccurrence of cancer later in life, is this a new cancer or the reoccurrence of an old cancer coming back? DNA sequence data would be needed to address and solve the aforementioned question.
  • the microarray assay is a type of hybridization assay and in its current evolution is unable to detect and confirm the presence of all of the 12 different activating mutations that can occur in KRAS.
  • the detection of activating mutations in KRAS is challenging due the close proximity of codons 12 and 13. Up to 12 different mutations can occur within 6 base pairs.
  • a probe molecule designed to capture the KRAS sequence works best if it overlaps both upstream and downstream of the mutation.
  • Hybridization can detect a mutation within the region of the probe binding the target but is not be able to distinguish which base is mutated, only that a mutation is present. This is the same capability of the commercially produced KRAS mutation tests.
  • FIGS. 21A-C show the layout of the microarray and results of an experiment performed with 2.6x SSC buffer.
  • FIG. 21 A is a table showing all the 12 activating mutation of codons 12 and 13 and an identification code given to each mutations ranging from SI to S12.
  • Figure 21B shows the layout of the microarray.
  • Probe spots CI to C3 are positive Cy3 dye controls ranging from 20M to 5M in concentration. Spots C4 and C5 are negative controls.
  • Probe spot C6 is the wild type sequence and SI to S 12 are the mutant probe spots.
  • Probe spots C7 to C9 are Cy5 dye positive control spots but the excitation wavelength to excite this dye was not used.
  • FIG. 22C The first set of results for the KRAS mutation assay are shown in FIG. 22C.
  • the pictures are scans at different temperatures. The scan at 45° C was the very start of the experiment.
  • FIG. 21B as a guide, positive control spots CI to C3 all fluoresced brightly. Negative controls spots C4 and C5 do not fluoresce at all. Positive control C6 and mutants SI to S12 all fluoresce brightly. Differences in the measured fluorescence intensity between spots were observed but these differences were relatively small. Using the naked eye it is not possible to determine which mutations is present since all mutant probes spots display a fluorescence signal of similar intensity. This is the type of result a standard microarray would produce.
  • FIG. 21C As the temperature was raised 1° C a new scan was completed. The cycles of temperature increase and scans continued until the last scan of 65° C. The scan of 65° C is shown in FIG. 21C right side. At this temperature all 3 positive control spots CI to C3 are fluorescing and none of the negative control spots are fluorescing. The wild type probe spot C6 is fluorescing and mutant SI. All other mutant probe spots are not fluorescing and the slight signal emitted is a residual background signal. So, FIG. 21C at 65° C produces a correct answer in that sample had two targets present, wild type and S 1 which is mutation in codon 12 of the first base.
  • FIGS. 22A-C depicts results of the KRAS Mutation Assay.
  • a more detailed analysis of the results is shown in the graph of FIG. 22C.
  • This graph shows probe spots C6 to S12 and all data is raw non- normalized. Positive controls CI to C3 showed relatively constant fluorescence over the 45° to 65° C temperature range and was not included in the graph.
  • the table of activating mutations is shown in FIG. 22A and microarray layout in FIG. 22B. There are so many graphs depicted in one figure that interpretation is difficult.
  • FIGS. 23A-E depict results of the KRAS Mutation Assay by Codon in 5 different graphs.
  • Graph A shows the melting curves of both C6 wild type and the S 1 mutant. These slopes are relatively similar with a flat step at about the half-way point marked with a dashed circle. Both graphs have a similar starting intensity and the ending intensity is almost the same at about half the stating intensity. The first half of both graphs is the melting of one base pair mismatch and the second part of the graph is the melting of perfect match.
  • perfect match is the wild type target and mismatch is SI target which can bind a one base pair mismatch.
  • the SI mutant target has a mutation in codon 12 first base. If SI target had bound any of the S4, S5, or S6 probes it would bind as a 2 base pair mismatch since these probes have mutations in the second base of codon 12. However, there is no major change in the slope of the melting curves to form a flat section for any of the graphs in FIG. 23C. It can be inferred that the 1 base pair mismatch target wild type bound the probes and that the 2 base pair mismatch SI did not bind any probes. The hybridization temperature was 45° C and this was likely too high to allow 2 base pair mismatches to bind.
  • the graphs of melting curves of probes for codons 13 with mutations in the first base are shown in FIG. 32D and the graphs of melting curves of probes of codon 13 with mutations in the second base are shown in FIG. 23E.
  • the melting curves of probes for codons 13 both first and second bases all show a sloped line with no flat section in the middle of the curve indicating that one type of target bound the probes and melted away.
  • the wild type target binds all probes as a 1 base pair mismatch. If the SI target bound any of the probes it would bind as a 2 base pair mismatch since SI has a mutation codon 12 of the first base position.
  • FIGS. 24A-B depict the DNA Melting Kinetics on Standard Microarray surface chemistry.
  • a review of the principles of specificity and sensitive of hybridization and melting on a standard non-positively charges surface must be described.
  • the characteristics of sensitivity versus specificity during DNA hybridization is a tradeoff. This tradeoff is best demonstrated in FIGS. 24A and 24B.
  • FIG. 24A is a DNA melting curve on a blocked epoxy microarray slide. The microarray was printed with 25 bp probes to capture a perfectly matched target and probes to capture a 1 base pair mismatched target. So, a 1 bp mutation was built into some of the probe molecules.
  • the target was a perfectly matched sequence of 25 bp (Table 2).
  • the melting curves for a perfectly matched probe and 1 bp mismatched probe are displayed together. Both curves display a classic ski slope shape with flat section at the lower temperature range and then a slope. The Tms of both slopes are distinctly different.
  • the Tm for the perfectly matched probe is approximately 55° C and the Tm for 1 bp mismatch is approximately 53° C.
  • the melting temperature ranges of the perfect match probe and 1 bp mismatch overlap significantly.
  • the temperature range of melting for the perfectly match probe is approximately 49° C to 60° C and the temperature range of melting of the 1 bp mismatch is approximately 48° C to 59° C.
  • the optimal hybridization temperature is 49° C or lower. This is marked with an arrow in FIG. 24A. However, hybridizing at this temperature can also allow significant amounts of target to bind the 1 bp mismatch probe reducing the level of specificity. If high levels of specificity are desired, the optimal hybridization temperature needs to be higher. At a hybridization temperature of 59° C (marked with an arrow in FIG.
  • the amount of target that binds the 1 bp mismatched probe is much less but also the total amount of target that binds the perfectly matched target is less reducing sensitivity.
  • increases in specificity or sensitivity of binding that occur from the adjustment of NaCl concentrations in the buffer produce similar results. Decreasing the concentration of NaCl in the buffer increases the repulsive forces between the negatively charged phosphate backbones of the DNA double helix and destabilizes the structure. This lowers the Tm and increases the specificity of binding at a temperature below the Tm but also lowers the sensitivity of binding at the same time.
  • Solid phase melting curves on a positively charged surface can narrow the temperature ranges of melting of perfect match and mismatch so that these ranges no longer overlap.
  • both perfect match and mismatch can be easily detected binding the same probe by melting curves.
  • Being able to detect and confirm the presence of perfectly matched target and mismatched target DNA binding a probe spot increases the accuracy of any test being performed.
  • diagnostic tests such as the KRAS mutation assay can now be performed with a high level of accuracy not possible using a standard microarray hybridization without DNA melting.
  • FIGS. 25A-C depict DNA melting curves obtained from optimized charge enhanced specificity surface chemistry.
  • a positively charged microarray surface When a positively charged microarray surface is used, the temperature ranges of melting for perfectly matched target and 1 bp mismatched target no longer overlap.
  • FIGS. 25A, 25B, and 25C graphs that shows graphs of melting curves generated on unblocked epoxy microarray slides.
  • FIGS. 25A and 25B show the melting curves for perfectly matched and 1 bp mismatched target respectively. In both graphs multiple probe spots were graphed which had an identical sequence.
  • the stacked line function of the graphing software was used to spread out the lines so that they did not lie directly on top of each other. The loss of fluorescence in the graph as shown in FIG.
  • the Tm for perfectly matched target in FIG. 25A is 57° C and is marked by line b.
  • the Tm of 1 bp mismatch is about 49° C and is marked by line "a" in FIG. 25B.
  • the difference in temperature in Tms between the perfectly matched target and 1 bp mismatched targeted is approximately 8 degrees C.
  • FIG. 25C shows the melting curve of a 50:50 mixture of perfectly matched target and 1 bp mismatch target.
  • This melting curve of 3 probe spots is an enhanced melting curve with two Tms.
  • the Tm for 1 bp mismatch is approximately 50° C and is marked by line a and the Tm for the perfectly matched target is approximately 57° C and is marked by line b.
  • the temperature difference between Tms is 7° C.
  • the narrowing of the temperature ranges of melting for both the perfectly matched and 1 bp mismatched target is significant but not as narrow as when either sample is tested alone.
  • the narrowing is sufficient to create a change of slope in the graph also called a step which marks the high end of the temperature range of melting of the 1 bp mismatch and the low end of the temperature range of melting of the perfectly matched.
  • This change of slope is not only important as a marker of the temperature ranges of melting of the two target types but also creates a temperature of hybridization that can allow maximum levels of specificity for the detection of perfectly matched target without any loss of sensitivity of detection. This temperature would be approximately 54° C and is marked with a star. It is only possible to create these conditions with a positively charged surface and a blocked epoxy microarray surface will not narrow the temperature ranges of melting of the targets so they no longer overlap (FIG. 24B).
  • the advantages of using the positively charged microarray surface not only create an enhanced melting curve that can detect the binding and melting of perfectly matched and 1 bp mismatched target, but also create conditions that separate the temperature ranges of melting leading to a temperature of hybridization with maximum levels of specificity for the detection of perfectly matched target DNA without loss of any sensitivity.
  • This novel discovery is termed "Charged Enhanced Specificity of Binding" (CESB).
  • FIGS. 26A-C depict binding mechanisms of liquid phase and solid phase PCR.
  • Charge enhanced specificity of binding can be used to improve the specificity of any hybridization reaction provided the reaction can be done in a solid phase format.
  • a list of techniques that would benefit from CESB may include but is not limited to southern blots, northern blots, microarray, PCR and some forms of next generation DNA sequencing with a hybridization step. It is well known to those skilled in the art that PCR is one of the most commonly used methods in the life sciences, and the specificity enhancement is the preferred method according to an embodiment of the present invention.
  • PCR is a liquid phase reaction and has 3 steps per cycle as shown in FIG. 26A.
  • the second step of PCR is called the annealing step and has the most potential for mis-binding target DNA with a similar sequence but is not a perfectly matched sequence. It is known to those skilled in the art, that most of the errors that occur during PCR occur at the annealing step. If PCR could be changed into a solid phase assay, CESB could be applied which in turn would improve the accuracy of PCR by preventing any mis-binding during annealing.
  • a form of solid phase PCR does exist and has been called "Bridge Amplification” or "Bridge PCR” as shown in FIG. 26B. In this format all 3 steps per cycle of PCR occur on a solid surface which can be flat or round in the shape of a micro bead.
  • the target Since the target is bound to the surface (FIG. 26B, a) it can only bind a primer within reach (FIG. 26B, b). If the primer binds its target in the presence of a positively charged surface, with the optimal level of buffer ions, and at the predicted temperature where the temperature ranges of melting of perfect match and 1 bp mismatch do not overlap, then maximum specificity of hybridization without loss of sensitivity would take place. As amplification continues, there is a buildup of PCR product near the area where the first PCR cycle took place called a DNA colony. As more PCR cycles are completed, the larger the colony becomes.
  • logarithmic amplification of target may not be possible in the later cycles of PCR if the primer in a particular surface area is depleted.
  • Another limitation is the need to remove the PCR product from the solid surface for further analysis which would require cleaving it from the surface. This adds an additional step to the procedure.
  • FIGS. 27A-C depicts a custom synthesized oligo bound to a nano particle.
  • an example of a nano-particle attached to an oligo is as a probe or primer that is attached to a positively charged nano-particle by a flexible cleavable linker molecule.
  • the probe primer section is predicted to be attracted to the positively charged nano-particle and form a hair pin like structure.
  • the double stranded section of primer and target DNA is predicted to bend and be attracted to the positively charged nano-particle as shown in FIG. 27B. In this configuration, the specificity of binding can be improved by CESB provided by the positively charged particle.
  • the positively charged nano-particle primer is intended for liquid phase applications. However, this structure can also be adapted to solid phase applications.
  • the same nano-particle probe can be attached to a solid surface with a linker as shown in FIG. 27C.
  • the advantage of using this combination of nano-particle and probe is that the probe brings along its own positively charged surface. This eliminates the need for special chemical coatings on the microarray slides to produce the positive charge and simplifies the microarray fabrication procedure.
  • the positively charged nano-particle can be removed by cleaving the flexible linker. The removal of the nano-particle may be needed in special applications such as a PCR reaction when the PCR product is further analyzed by DNA sequencing or cloned into a plasmid.
  • FIG. 28 depicts a schematic of hybrid liquid-solid phase PCR.
  • An example of how the nano-particle PCR primer operates in a hybrid liquid-solid phase PCR reaction is shown involving three steps.
  • step 1 of denaturation double stranded DNA is melted into single strands.
  • step 2 primer annealing, the nano-particle primers bind the 3' end of the target strand of DNA which puts the primer in a 5' to 3' orientation and ready for primer extension.
  • This annealing step is able to use CESB conditions since the positively charged nano-particle is present with optimal ion buffer concentrations.
  • the annealing temperature can be higher than classical PCR and prevent the binding of any 1 bp mismatch target without loss of PCR sensitivity.
  • polymerase can extend the PCR primers in the 5' to 3' direction creating a complimentary strand of DNA.
  • the 3 steps are repeated.
  • Some of the target DNA may have nano- particle attached to the 5' end of the target strand of DNA. This does not create a problem since the nano-particle primer attaches to the 3' and of the target DNA. So as more PCR cycles are completed more target DNA can have the nano-particle attached to the 5' terminus. When the PCR reaction is complete, removal of the nano-particle may or may not be needed.
  • a chemical method to cleave the linker section of the nano- particle can be used.
  • linker chemistries and chemical cleavage methods to accomplish this task.
  • differential centrifugation can be used to separate the nano- particle from the DNA.
  • the key advantages of the hybrid liquid-solid phase PCR method are the maximized specificity of amplification and the greater flexibility of primer design. Since CESB is increasing the specificity of binding and 1 base pair mismatched target cannot bind, the criteria for primer design can be relaxed to allow for primers that have as little as albp different in sequence.

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Abstract

Les systèmes, méthodes et compositions selon l'invention peuvent être utilisés dans la capture/l'enrichissement, le profilage d'expression génique et le séquençage ciblé. L'invention concerne des systèmes, des méthodes et des compositions concernant l'amélioration de la spécificité d'hybridation des acides nucléiques et la maîtrise des formes des courbes de fusion révélées par des paires d'hybride d'acides nucléiques pour optimiser l'analyse des acides nucléiques. Ces systèmes, méthodes et compositions comprennent la production d'une surface ou d'un revêtement de surface chargé(e) positivement, sur la surface de lames en microréseau ou d'autres types de surfaces ayant la même finalité, ce qui améliore l'analyse de courbe de fusion au point de permettre la détection ou la différenciation de petites modifications de séquences entre les partenaires de liaison des acides nucléiques. La précision ou la résolution de l'analyse de courbe de fusion devait être suffisante pour permettre la distinction entre la fusion d'ADN double brin parfaitement apparié et d'ADN double brin présentant la modification de séquence la plus petite possible, et un mésappariement d'une paire de bases.
EP16874070.2A 2015-12-11 2016-12-12 Systèmes, méthodes et compositions pour améliorer la spécificité de l'hybridation des acides nucléiques Withdrawn EP3387431A4 (fr)

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