Density modification in arrays of surface-attached nucleic acid molecules

Abstract

Substrates having nucleic acid polymers attached at varying surface densities and methods for creating substrates having nucleic acid polymers attached at varying surface densities are provided. Methods according to embodiments of the invention are adapted to the rapid synthesis of arrays of DNA polymers on a substrate surface. In embodiments of the invention an array of DNA molecules on a substrate comprises a plurality of DNA polymers attached to a trifunctional linker such that at least two DNA polymers are attached to one trifunctional linker that is attached to the surface of the substrate. By coupling trifunctional linkers to trifunctional linkers that are attached to a substrate surface, the density of DNA polymers on a substrate surface is increased.

1 . An array of DNA molecules on a substrate comprising,
 a substrate that has a surface wherein the surface comprises regions and the regions comprise a plurality of DNA polymers attached to the surface of the substrate,   wherein at least one region comprises a plurality of DNA polymers that are attached to a trifunctional linker such that at least two DNA polymers are attached to one trifunctional linker,   wherein the one trifunctional linker has three sites for attachment, and wherein a first of the two DNA polymers is attached to one of the three sites, a second of the two DNA polymers is attached to a second of the three sites, and a third site is attached to the surface of the substrate.   
   
   
       2 . The array of  claim 1  wherein the array comprises 1,000 to 10,000 regions. 
   
   
       3 . The array of  claim 1  wherein the array comprises 100 to 1,000 regions. 
   
   
       4 . The array of  claim 1  wherein a region of the array is less than 100 μm 2 . 
   
   
       5 . The array of  claim 1  wherein the DNA polymers each comprise from 5 to 50 nucleic acids. 
   
   
       6 . The array of  claim 1  wherein the DNA polymers each comprise from 10 to 30 nucleic acids. 
   
   
       7 . The array of  claim 1  wherein the DNA polymers are attached to the trifunctional linker through a spacer group and the spacer group comprises a polyethylene glycol. 
   
   
       8 . An array of DNA molecules on a substrate, comprising
 a substrate that has a surface wherein the surface comprises regions and the regions comprise a plurality of DNA polymers attached to the surface of the substrate,   wherein at least one region comprises a plurality of DNA polymers that are attached to a first and a second trifunctional linker such that at least two DNA polymers are attached to the first trifunctional linker and at least two DNA polymers are attached to the second trifunctional linker, and the first and the second trifunctional linker each have three sites for attachment and a first DNA polymer is attached to one of the three sites, a second DNA polymer is attached to a second of the three sites, and a third site is attached to a third trifunctional linker, and   wherein the third trifunctional linker molecule has three sites for attachment and one of the three sites for attachment is attached to the first trifunctional linker, a second of the three sites for attachment is attached to the second trifunctional linker, and the third site for attachment is attached to the surface of the substrate.   
   
   
       9 . The array of  claim 8  wherein array contains 1,000 to 10,000 regions. 
   
   
       10 . The array of  claim 8  wherein the array contains 100 to 1,000 regions. 
   
   
       11 . The array of  claim 8  wherein the DNA polymers each comprise from 5 to 50 nucleic acids. 
   
   
       12 . The array of  claim 8  wherein the DNA polymers are attached to the trifunctional linker through a spacer group and the spacer group comprises a polyethylene glycol. 
   
   
       13 . An array of DNA molecules on a substrate, comprising
 a substrate that has a surface and the surface comprises regions, wherein the regions comprise electrodes and wherein at least one region comprises a plurality of DNA polymers,   wherein the electrodes are connected to a source capable of providing voltage;   and wherein the DNA polymers are attached to a trifunctional linker such that at least two DNA polymers are attached to one trifunctional linker and the one trifunctional linker has three sites for attachment and wherein a first of the two DNA polymer is attached to one of the three sites, a second of the two DNA polymers is attached to a second of the three sites, and a third site is attached to the surface of the substrate.   
   
   
       14 . The array of  claim 13  wherein the electrodes comprise platinum. 
   
   
       15 . The array of  claim 13  wherein the DNA polymers each comprise from 5 to 50 nucleic acids. 
   
   
       16 . The array of  claim 13  wherein a feature size of the array is less than 100 μm 2 . 
   
   
       17 . The array of  claim 13  wherein the DNA polymers are attached to the trifunctional linker through a spacer group and the spacer group comprises a polyethylene glycol. 
   
   
       18 . An array of DNA molecules on a substrate, comprising
 a substrate that has a surface and the surface comprises regions and at least one region comprises a plurality of DNA polymers,   and wherein the region that comprises a plurality of DNA polymers comprises a molecule according to the structure:   
     
       
         
         
             
             
         
       
       wherein when X and Y are different, X and Y are selected from the group consisting of CH 2 , CHR 1 , phenyl, O, S, NH, and NR 2 , and in the case where X and Y are the same X and Y are selected from the group consisting of CH 2 , CHR 2 , and phenyl wherein R 1  is selected from the group consisting of —OH, —CH 3 , —CH 2 OH, —CH 2 CH 3 , and —CH 2 CH 2 OH and R 2  is selected from the group consisting of —CH 3 , —CH 2 CH 3 , and —CH 2 CH 2 CH 3 , n is an integer from 0 to 10, and A is a DNA polymer, and 
       wherein the molecule is attached to the surface of the substrate. 
     
   
   
       19 . The array of  claim 18  wherein the DNA polymers each comprise from 5 to 50 nucleic acids. 
   
   
       20 . The array of  claim 18  wherein n is an integer from 0 to 5. 
   
   
       21 . The array of  claim 18  wherein the regions comprise electrodes. 
   
   
       22 . The array of  claim 18  wherein the regions comprise platinum metal. 
   
   
       23 . The array of  claim 18  wherein A comprises a DNA polymer and a spacer 
   
   
       24 . A method for synthesizing a DNA polymer on a substrate comprising,
 a) providing a substrate wherein the substrate has a surface and the surface has attached hydroxyl groups,   b) supplying a solution to the substrate surface comprising a trifunctional linker molecule wherein the trifunctional linker molecule has three sites for attachment, and wherein a first and second site for attachment are protected by protecting groups, under conditions that allow the trifunctional linker molecule to attach to the hydroxyl groups attached to the surface, wherein a third site for attachment is attached to the surface of the substrate and wherein a second and third site comprise protecting groups,   c) removing the protecting groups attached to the first and second sites for attachment,   d) supplying a solution to the substrate surface comprising nucleotides having a 5′ protecting group under conditions that allow a first nucleotide to attach to the first attachment site of the trifunctional linker and a second nucleotide to attach to the second attachment site of the trifunctional linker,   e) removing the 5′ protecting groups,   f) supplying a solution to the substrate surface comprising nucleotides having a 5′ protecting group under conditions that allow a third nucleotide to attach to the first nucleotide and a fourth nucleotide to attach to the second nucleotide, and   repeating e) and f) a plurality of times.   
   
   
       25 . The method of  claim 24  wherein the substrate is an array that comprises 1,000 to 10,000 regions. 
   
   
       26 . The method of  claim 24  wherein the substrate is an array that comprises 100 to 1,000 regions. 
   
   
       27 . The method of  claim 24  wherein the substrate is an array having regions and the regions comprise electrodes. 
   
   
       28 . The method of  claim 24  wherein the substrate is an array having regions and the regions comprise electrodes and wherein the protecting groups are removed by an electrogenerated acid.