Non-Emulsion Methods And Masked Biomolecules

Abstract

The present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions. In a preferred embodiment, a plurality of groups of amplified product are generated on the surface, each group positioned in different (typically predetermined) locations on said surface so as to create an array.

1 . A method for making an array, comprising:
 a) providing:
 i) a first element comprising a surface; 
 ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; and 
 iii) a solution comprising molecules selected from the group consisting of biomolecules and anchoring molecules; 
   b) contacting the surface of said first element with said solution under conditions such that at least a portion of said molecules attach to at least a portion of said surface so as to create a modified surface comprising attached molecules; and   c) positioning said bottom surface of said second element on top of said modified surface, so as to create first regions defined by said channels, said first regions comprising unmasked attached molecules, and second regions comprising masked attached molecules, thereby making an array.   
   
   
       2 . The method of  claim 1 , whereby said positioning of step c) causes at least a portion of said solution on said surface of said first element to move off of said surface. 
   
   
       3 . The method of  claim 1 , further comprising:
 d) providing a third element comprising a surface;   e) bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create a device comprising sealed first regions.   
   
   
       4 . The method of  claim 1 , wherein said contacting of step b) results in the entire surface being contacted with said solution. 
   
   
       5 . The method of  claim 1 , wherein said channels of said second element are evenly spaced. 
   
   
       6 . The method of  claim 5 , wherein said attached molecules after step b) are attached over substantially the entire surface at a substantially even density. 
   
   
       7 . The method of  claim 1 , wherein said molecules are biomolecules. 
   
   
       8 . The method of  claim 7 , wherein said biomolecules are nucleic acid molecules. 
   
   
       9 . The method of  claim 8 , wherein said nucleic acid molecules comprise PCR primers. 
   
   
       10 . The method of  claim 8 , wherein said nucleic acid molecules comprise oligonucleotides. 
   
   
       11 . The method of  claim 8 , wherein said nucleic acid molecules comprise probes for RCA. 
   
   
       12 . The method of  claim 8 , wherein said nucleic acid molecules comprise hairpins. 
   
   
       13 . The device of  claim 3 . 
   
   
       14 . A method for making an array, comprising:
 a) providing:
 i) a first element comprising a surface; 
 ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; and 
 iii) a solution comprising forward and reverse PCR primers; 
   b) positioning said bottom surface of said second element on top of said surface of said first element, so as to create unmasked regions defined by said channels, and masked regions; and   c) contacting the surface of said first element with said solution under conditions such that at least a portion of said PCR primers attach to at least a portion of said surface so as to create a modified surface comprising attached molecules, said attached molecules positioned in said unmasked regions, thereby making an array.   
   
   
       15 . The method of  claim 14 , wherein said masked regions are free of attached molecules. 
   
   
       16 . The method of  claim 14 , further comprising:
 d) providing a third element comprising a surface;   e) bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create a device comprising sealed unmasked regions.   
   
   
       17 . The method of  claim 14 , wherein said channels of said second element are evenly spaced. 
   
   
       18 . The method of  claim 17 , wherein said attached molecules after step b) are attached over substantially the entire surface at a substantially even density. 
   
   
       19 . The device of  claim 16 . 
   
   
       20 . A method for making an array, comprising:
 a) providing:
 i) first and third elements each comprising a substantially flat surface; 
 ii) a second element comprising substantially flat bottom and top surfaces, said second element further comprising a plurality of substantially evenly spaced channels extending from said bottom to said top surfaces; and 
 iii) a solution comprising molecules selected from the group of biomolecules and anchoring molecules; 
   b) contacting the substantially flat surface of said first element with said solution under conditions such that at least a portion of said molecules attach over substantially the entire surface at a substantially even density so as to create a modified surface comprising attached molecules;   c) positioning said bottom surface of said second element on top of said modified surface, so as to create first regions defined by said channels, said first regions comprising unmasked attached molecules, and second regions comprising masked attached molecules, thereby making an array; and   d) bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create a device comprising sealed first regions.   
   
   
       21 . The device created by process of  claim 20 . 
   
   
       22 . The method of  claim 20 , wherein said second element comprises a polymer film treated with a laser to create said channels. 
   
   
       23 . The method of  claim 22 , wherein said polymer comprises a polyimide. 
   
   
       24 . The method of  claim 22 , wherein said channels are approximately 20 microns in diameter and are spaced approximately 30 microns apart. 
   
   
       25 . The method of  claim 20 , wherein said molecules are biomolecules. 
   
   
       26 . The method of  claim 25 , wherein said biomolecules are nucleic acid molecules. 
   
   
       27 . The method of  claim 26 , wherein said nucleic acid molecules are probes for RCA. 
   
   
       28 . The method of  claim 26 , wherein said nucleic acid molecules are primers for PCR. 
   
   
       29 . The method of  claim 26 , wherein said nucleic acid molecules comprise hairpins. 
   
   
       30 . A method of amplifying nucleic acid template, comprising:
 a) providing:
 i) first and third elements, each comprising a surface; 
 ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; 
 iii) a first solution comprising primers; and 
 iv) a second solution comprising template, polymerase and dNTPs; 
   b) contacting at least a portion of said surface of said first element with said first solution under conditions such that at least a portion of said primers attach so as to create a modified surface comprising attached primers;   c) positioning said bottom surface of said second element on top of said modified surface, so as to create first regions defined by said channels, said first regions comprising unmasked attached primers, and second regions comprising masked attached primers, thereby making an array;   d) introducing said second solution into said first regions;   e) bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create sealed first regions of a device; and   f) treating said sealed first regions under conditions such that said template is amplified.   
   
   
       31 . The method of  claim 30 , wherein said treating of step (f) comprises thermally cycling the device of step (e). 
   
   
       32 . The method of  claim 30 , wherein said first solution comprises forward primers for PCR. 
   
   
       33 . The method of  claim 30  wherein said first solution further comprises reverse primers for PCR. 
   
   
       34 . The method of  claim 30 , wherein said third element comprises a polymer film treated with a laser to create said channels. 
   
   
       35 . The method of  claim 34 , wherein said channels are approximately 20 microns in diameter and are spaced approximately 30 microns apart. 
   
   
       36 . The device of  claim 30 . 
   
   
       37 . A method of amplifying nucleic acid template, comprising:
 a) providing:
 i) first and third elements, each comprising a surface; 
 ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; 
 iii) a first solution comprising primers; and 
 iv) a second solution comprising template, polymerase and dNTPs; 
   b) positioning said bottom surface of said second element on top of said surface of said first element, so as to create unmasked regions defined by said channels, and masked regions;   c) contacting at least a portion of said surface of said first element with said first solution under conditions such that at least a portion of said primers attach in said unmasked regions so as to create a modified surface comprising attached primers;   d) introducing said second solution into said unmasked regions;   e) bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create sealed unmasked regions of a device; and   f) treating said sealed first regions under conditions such that said template is amplified.   
   
   
       38 . The method of  claim 37 , wherein said treating of step (f) comprises thermally cycling the device of step (e). 
   
   
       39 . The method of  claim 37 , wherein said first solution comprises forward primers for PCR. 
   
   
       40 . The method of  claim 39 , wherein said first solution further comprises reverse primers for PCR. 
   
   
       41 . The method of  claim 37 , wherein said third element comprises a polymer film treated with a laser to create said channels. 
   
   
       42 . The method of  claim 41 , wherein said channels are approximately 20 microns in diameter and are spaced approximately 30 microns apart. 
   
   
       43 . The device of  claim 37 . 
   
   
       44 . A method of amplifying nucleic acid template, comprising:
 a) providing:
 i) first and third elements, each comprising a surface; 
 ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; 
 iii) a first solution comprising a plurality of forward PCR primers; 
 iv) a second solution comprising a plurality of reverse PCR primers; and 
 v) a third solution comprising template, polymerase and dNTPs; 
   b) contacting at least a portion of said surface of said first element with said first solution under conditions such that at least a portion of said forward primers attach so as to create a first modified surface comprising attached forward primers;   c) contacting at least a portion of said surface of said third element with said second solution under conditions such that at least a portion of said reverse primers attach so as to create a second modified surface comprising attached reverse primers;   d) positioning said bottom surface of said second element on top of said first modified surface, so as to create first regions defined by said channels, said first regions comprising unmasked attached forward primers, and second regions comprising masked attached forward primers;   e) introducing said second solution into said first regions;   f) bringing said second modified surface into contact with the top surface of said second element, thereby sandwiching said second element between the modified surfaces of said first and third elements, so as to create sealed first regions of a device, said sealed first regions comprising unmasked attached forward and reverse primers; and   g) treating said sealed first regions under conditions such that said template is amplified.   
   
   
       45 . The method of  claim 44 , wherein said treating of step (g) comprises thermally cycling the device of step (f). 
   
   
       46 . The device of  claim 44 .