Chip-based isothermal amplification devices and methods

Abstract

Disclosed are methods and compositions for isothermal amplification of nucleic acids in a microfabricated substrate. Methods and compositions for the analysis of isothermally amplified nucleic acids in a microfabricated substrate are disclosed as well. The microfabricated substrates and isothermal amplification and detection methods provided are envisioned for use in various diagnostic methods, particularly those connected with diseases characterized by altered gene sequences or gene expression.

What is claimed is:  
     
         1 . An apparatus for use in the isothermal amplification of a selected nucleic acid, comprising a microfabricated substrate defining at least a first channel, said at least a first channel connected to a reaction chamber, and a means for isothermally regulating the temperature of said reaction chamber.  
     
     
         2 . The apparatus of  claim 1 , wherein said microfabricated substrate further defines at least a first entry port connected to said at least a first channel.  
     
     
         3 . The apparatus of  claim 1 , wherein said microfabricated substrate further defines at least a second channel directly or indirectly connected to said reaction chamber.  
     
     
         4 . The apparatus of  claim 3 , wherein said microfabricated substrate further defines at least a second entry port connected to said at least a second channel.  
     
     
         5 . The apparatus of  claim 3 , wherein said at least a second channel is connected to said at least a first channel at a point prior to connection of said at least a first channel to said reaction chamber.  
     
     
         6 . The apparatus of  claim 1 , wherein biological reagents effective to permit an isothermal nucleic acid amplification reaction are disposed in said reaction chamber, or in a first or second channel or reservoir that is directly or indirectly connected to said reaction chamber.  
     
     
         7 . A method for isothermal amplification of a selected nucleic acid, comprising: 
 a) providing a sample comprising said selected nucleic acid, and reagents effective to permit an isothermal amplification reaction, to a microfabricated substrate that defines at least a first channel, said at least a first channel connected to an isothermally regulated reaction chamber; and    b) conducting an isothermal amplification reaction to amplify said selected nucleic acid.    
     
     
         8 . The method of  claim 7 , wherein said microfabricated substrate fuirther comprises a flow-directing means system in operable relation to said at least a first channel.  
     
     
         9 . The method of  claim 8 , wherein said flow-directing means system is separated from said at least a first channel by a liquid barrier.  
     
     
         10 . The method of  claim 9 , wherein said liquid barrier comprises a first silicon oxide layer, a silicon nitride layer and a second silicon oxide layer.  
     
     
         11 . The method of  claim 8 , wherein said flow-directing means system comprises a series of heating elements arrayed along said at least a first channel.  
     
     
         12 . The method of  claim 11 , wherein said heating elements are comprised of aluminum.  
     
     
         13 . The method of  claim 7 , wherein said sample is conveyed from said at least a first channel to said isothermally regulated reaction chamber by differential heating of said sample.  
     
     
         14 . The method of  claim 8 , wherein said flow-directing means system comprises a series of hydrophobic and hydrophilic surface structures arrayed along said at least a first channel.  
     
     
         15 . The method of  claim 14 , wherein said at least a first channel is treated with a hydrophilicity-enhancing compound.  
     
     
         16 . The method of  claim 14 , wherein said at least a first channel is modified to comprise one or more hydrophobic regions.  
     
     
         17 . The method of  claim 8 , wherein said flow-directing means system comprises a gas source in fluid communication with said at least a first channel.  
     
     
         18 . The-method of  claim 7 , wherein said microfabricated substrate further defines at least a first entry port connected to said at least a first channel.  
     
     
         19 . The method of  claim 7 , wherein said microfabricated substrate further defines at least a second channel directly or indirectly connected to said reaction chamber.  
     
     
         20 . The method of  claim 19 , wherein said microfabricated substrate further defines at least a second entry port connected to said at least a second channel.  
     
     
         21 . The device of  claim 19 , wherein said at least a second channel is connected to said at least a first channel at a point prior to connection of said at least a first channel to said reaction chamber.  
     
     
         22 . The method of  claim 7 , wherein said microfabricated substrate further defines at least a first reservoir directly or indirectly connected to said at least a first channel or to said reaction chamber.  
     
     
         23 . The method of  claim 7 , wherein said microfabricated substrate further defines a nucleic acid analysis component operably connected to said isothermally regulated reaction chamber.  
     
     
         24 . The method of  claim 23 , wherein said nucleic acid analysis component is a gel electrophoresis channel.  
     
     
         25 . The method of  claim 24 , wherein said nucleic acid analysis component is a capillary gel electrophoresis channel.  
     
     
         26 . The method of  claim 23 , further comprising a nucleic acid detection means operably connected to said nucleic acid analysis component.  
     
     
         27 . The method of  claim 26 , wherein said nucleic acid detection means is a DNA sensor means.  
     
     
         28 . The method of  claim 27 , wherein said DNA sensor means detects a radiolabel.  
     
     
         29 . The method of  claim 28 , wherein said DNA sensor means is a p-n-type diffusion diode.  
     
     
         30 . The method of  claim 27 , wherein said DNA sensor means detects a fluorescent label.  
     
     
         31 . The method of  claim 30 , wherein said DNA sensor means is a p-n-type diffusion diode combined with a wavelength filter and an excitation source.  
     
     
         32 . The method of  claim 7 , wherein said microfabricated substrate is comprised of silicon, quartz or glass.  
     
     
         33 . The method of  claim 7 , wherein said reagents comprise reagents for conducting a Strand Displacement Amplification reaction.  
     
     
         34 . The method of  claim 7 , wherein said reagents comprise reagents for conducting a self-sustained sequence replication amplification reaction.  
     
     
         35 . The method of  claim 7 , wherein said reagents comprise reagents for conducting a Qβ replicase amplification reaction.  
     
     
         36 . The method of  claim 7 , wherein said reagents are prefabricated into said reaction chamber, or into a first or second channel or reservoir that is directly or indirectly connected to said reaction chamber.  
     
     
         37 . The method of  claim 36 , wherein said reagents are prefabricated into said reaction chamber, first or second channel or reservoir in a lyophilized form.  
     
     
         38 . The method of  claim 7 , wherein said reagents further comprise a DNA ligase.  
     
     
         39 . The method of  claim 7 , wherein said reagents further comprise a nuclease.  
     
     
         40 . The method of  claim 39 , wherein said reagents further comprise a restriction endonuclease.  
     
     
         41 . The method of  claim 7 , wherein said sample is derived from an animal having or suspected of having a disease.  
     
     
         42 . The method of  claim 41 , wherein said sample is derived from a human subject.  
     
     
         43 . A method for detecting the presence of a selected nucleic acid, comprising: 
 a) introducing a sample suspected of containing said selected nucleic acid, and reagents effective to permit an isothermal amplification reaction, into a microfabricated substrate defining at least a first channel, said at least a first channel connected to an isothermally regulated reaction chamber;    b) conducting an isothermally regulated amplification reaction to amplify said selected nucleic acid; and    c) detecting the presence of the amplified selected nucleic acid, wherein the presence of the amplified selected nucleic acid confirms the presence of said selected nucleic acid in said sample.    
     
     
         44 . The method of  claim 43 , wherein said sample is derived from an animal or human subject.  
     
     
         45 . The method of  claim 43 , wherein said sample is derived from an animal having or suspected of having a disease, and the presence of said selected nucleic acid is indicative of the disease state.  
     
     
         46 . The method of  claim 43 , wherein said sample is derived from an animal having or suspected of having a disease, and the absence of said selected nucleic acid is indicative of the disease state.  
     
     
         47 . The method of  claim 43 , wherein said selected nucleic acid comprises a mutation not present in the wild-type version of the selected nucleic acid.  
     
     
         48 . The method of  claim 43 , wherein said selected nucleic acid is detected by means of a detectable label incorporated into the amplified selected nucleic acid by said isothermal amplification reaction.  
     
     
         49 . The method of  claim 43 , wherein said selected nucleic acid is detected by means of a labeled nucleic acid probe.  
     
     
         50 . The method of  claim 48 , wherein said detectable label is a radioisotopic, enzymatic or fluorescent label.  
     
     
         51 . A method of making a device for use in isothermal nucleic acid amplification, comprising preparing at least a first microfabricated device, chip or wafer defining at least a first channel that is operably connected to an isothermally regulated reaction chamber.  
     
     
         52 . A kit for conducting isothermal amplification of a selected nucleic acid, comprising, in suitable container means: 
 a) at least a first microfabricated substrate defining at least a first channel, said at least a first channel connected to an isothermally regulated reaction chamber; and    b) reagents effective to permit an isothermal nucleic acid amplification reaction.    
     
     
         53 . The kit of  claim 52 , wherein said at least a first microfabricated substrate further defines a nucleic acid analysis component operably connected to said isothermally regulated reaction chamber.  
     
     
         54 . The kit of  claim 53 , wherein said at least a first microfabricated substrate further comprises a nucleic acid detection means operably connected to said nucleic acid analysis component.  
     
     
         55 . The kit of  claim 52 , wherein said reagents effective to permit an isothermal nucleic acid amplification reaction are disposed in said reaction chamber, or in a first or second channel or reservoir that is directly or indirectly connected to said reaction chamber.  
     
     
         56 . A diagnostic system for identifying a selected nucleic acid, comprising at least a first microfabricated substrate defining at least a first channel that is connected to at least a first isothermally regulated reaction chamber; said diagnostic system further comprising a nucleic acid analysis component and a nucleic acid detection means in operable association with the reaction chamber of said at least a first microfabricated substrate.  
     
     
         57 . The diagnostic system of  claim 56 , further comprising, in operable association, at least a second microfabricated substrate defining at least a second channel that is connected to at least a second isothermally regulated reaction chamber.  
     
     
         58 . The diagnostic system of  claim 57 , wherein said at least a first and at least a second microfabricated substrates are operably connected in series to a single nucleic acid analysis component and nucleic acid detection means.  
     
     
         59 . The diagnostic system of  claim 57 , wherein said at least a first and at least a second microfabricated substrates are operably connected in parallel to at least two distinct nucleic acid analysis components and nucleic acid detection means.  
     
     
         60 . The diagnostic system of  claim 56 , further comprising reagents effective to permit an isothermal nucleic acid amplification reaction.  
     
     
         61 . The diagnostic system of claim  60 , wherein said reagents are disposed in said reaction chamber, or in a first or second channel or reservoir that is directly or indirectly connected to said reaction chamber.  
     
     
         62 . A method of making a nucleic acid diagnostic system, comprising preparing at least a first microfabricated substrate defining, in a series of operable associations, at least a first channel, an isothermally regulated reaction chamber, a nucleic acid analysis component and a nucleic acid analysis detection means.