Methods and compositions for monitoring polymer array synthesis

Abstract

VLSIPS™ manufacturing processes are of increasing commercial importance. The present invention provides methods and compositions for monitoring the efficiency and quality of polymer synthesis in VLSIPS™ arrays. Methods for monitoring polymer synthesis in an array on a substrate are provided. Mono-isomeric labels for the labeling of synthetic polymer arrays are provided. Methods and compositions for post-synthetically labeling polymers in polymer arrays are also provided.

1 . A detectable monomeric polymer synthesis reagent with the structure A-B, wherein A comprises a detectable chromogenic moiety and B comprises a polymer integration element, said integration element comprising a polymer joining agent selected from the group consisting of an amine, a carboxyl, an oxygen, and a phosphate, and wherein A-B is a single isomer.  
   
   
       2 . The polymer synthesis reagent of  claim 1 , wherein the chromogenic moiety is a fluorophore.  
   
   
       3 . The polymer synthesis reagent of  claim 1 , wherein the polymer synthesis reagent is a nucleic acid synthesis reagent, wherein B comprises the structure  
     
       
         
         
             
             
         
       
     
     and wherein 
 L is a linking chain selected from the group of linking chains consisting of an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally substituted with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation, an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally replaced with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation;  
 R 1  is selected from the group consisting of hydrogen, alkyl, and aryl;  
 R 2  is selected from the group consisting of hydrogen, alkyl, and aryl;  
 X is a nucleic acid integration element comprising a phosphorous atom,  
 Y is selected from the group consisting of hydrogen, alkyl, and aryl;  
 Y 2  is an alkyl chain; and  
 Z comprises a protecting group.  
 
   
   
       4 . The polymer synthesis reagent of  claim 1 , wherein the polymer synthesis reagent is a nucleic acid synthesis reagent, wherein B comprises the structure  
     
       
         
         
             
             
         
       
     
     and wherein 
 L is selected from the group of alkyl linking chains consisting of an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally substituted with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation, and an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally replaced with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation; and  
 R 1  is selected from the group consisting of hydrogen, alkyl, and aryl.  
 
   
   
       5 . A labeled oligonucleotide array attached to a solid substrate, wherein the oligonucleotides of the array comprise a single isomer of a detectable label.  
   
   
       6 . A labeled oligonucleotide array attached to a solid substrate, wherein the label is a mono-isomeric label comprising the structure  
     
       
         
         
             
             
         
       
     
     wherein 
 F comprises a fluorescent group.  
 L is selected from the group of alkyl linking chains consisting of an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally substituted with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation, and an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally replaced with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation;  
 R 1  is selected from the group consisting of hydrogen, alkyl, and aryl;  
 R 2  is selected from the group consisting of hydrogen, alkyl, and aryl;  
 X is a nucleotide or a cleavable linker;  
 Y is selected from the group consisting of hydrogen, alkyl, and aryl;  
 Y 2  is selected from the group consisting of a hydrocarbon chain and a substituted hydrocarbon chain; and,  
 Z is selected from the group consisting of a nucleotide and a nucleic acid.  
 
   
   
       7 . The nucleic acid synthesis reagent of  claim 6 , wherein the nucleic acid synthesis reagent has the structure  
     
       
         
         
             
             
         
       
     
     wherein 
 R 1  is selected from the group consisting of alkyl, aryl, and hydrogen;  
 R 2  is selected from the group consisting of alkyl, and aryl; and  
 FL is a fluorescent moiety.  
 
   
   
       8 . The isomeric nucleic acid synthesis reagent of  claim 6 , wherein the compound has the structure  
     
       
         
         
             
             
         
       
     
   
   
       9 . The array of  claim 6 , wherein the composition further comprises a cleavable linker.  
   
   
       10 . A method of post-synthetically labeling an oligonucleotide array, comprising: 
 (i) providing a polymer array which comprises a plurality of polymers, wherein each polymer comprises a labeling site; and    (ii) attaching a detectable label to the labeling site.    
   
   
       11 . The method of  claim 10 , wherein the detectable label comprises a fluorophore.  
   
   
       12 . The method of  claim 10 , wherein step (i) of said method comprises synthesizing a polymer array, which polymer array comprises polymers attached to a substrate, said polymers comprising a labeling linker, which labeling linker comprises an attachment site for the detectable label.  
   
   
       13 . The method of  claim 10 , wherein step (i) of said method comprises synthesizing a polymer array, which polymer array comprises polymers attached to a substrate, said polymers comprising a cleavable linker and a labeling linker, which labeling linker comprises an attachment site for the detectable label, a site for attachment to the cleavable linker and a protected site for the attachment of the detectable label.  
   
   
       14 . The method of  claim 10 , wherein step (i) of said method comprises synthesizing a polymer array, which polymer array comprises polymers attached to a substrate, said polymers comprising a cleavable linker and a labeling linker, which labeling linker comprises a protected attachment site for the detectable label, and wherein step (ii) of the method comprises deprotecting the labeling linker, thereby making the protected attachment site into an unprotected attachment site, and incubating the polymer array with a detectable labeling reagent, which detectable labeling reagent comprises a site which is reactive with the unprotected attachment site, and which labeling reagent comprises the detectable label.  
   
   
       15 . The method of  claim 14 , wherein said protected attachment site comprises a DMT protective group.  
   
   
       16 . The method of  claim 10 , wherein said labeling site is located proximal to a cleavage site in the polymers of the polymer array.  
   
   
       17 . A post-synthetic labeling linker which comprises a site for polymer elongation, a site for attaching a polymer to a substrate and an attachment site for attaching a detectable label.  
   
   
       18 . The labeling linker of  claim 17 , wherein the linker has the structure  
     
       
         
         
             
             
         
       
     
     wherein: 
 R 1  is selected from the group consisting of hydrogen, alkyl and aryl;  
 R 2  is selected from the group consisting of hydrogen, alkyl and aryl;  
 R 3  is selected from the group consisting of hydrogen, alkyl and aryl,  
 L 1  is a linking chain selected from the group of alkyl linking chains consisting of an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally substituted with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation, and an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally replaced with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation;  
 L 2  is a linking chain selected from the group of alkyl linking chains consisting of an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally substituted with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation, and an alkyl linking chain from 1 to 30 carbons in length, wherein one or more carbon is optionally replaced with a heteroatom selected from the group consisting of N, S, O and P, and wherein the alkyl linking group optionally includes one or more sites of unsaturation;  
 Y is selected from the group consisting of a dimethoxytrityl protecting group and a photocleavable protecting group;  
 Z is selected from the group consisting of a dimethoxytrityl protecting group and a photocleavable protecting group; and  
 X is a nucleic acid integration element comprising a phosphorous atom.  
 
   
   
       19 . The labeling linker of  claim 18 , wherein Z is the photocleavable group MeNPOC.  
   
   
       20 . The labeling linker of  claim 17 , wherein the linker has the structure  
     
       
         
         
             
             
         
       
     
   
   
       21 . The labeling linker of  claim 17 , wherein the linker has the structure