Thiol-containing cleave reagents and oxidative wash

Abstract

The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3′-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. In addition, thiol-containing compounds and scavengers of thio-containing compounds are described. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.

1 .- 81 . (canceled) 
     
     
         82 . A method for detecting labeled nucleotides in a DNA sequence comprising the steps of
 a) providing a nucleic acid template and primer capable of hybridizing to said template so as to form a primer/template hybridization complex, a cleave reagent comprising di-mercaptopropanesulfonate, DNA polymerase, and 4 differently labeled populations of deoxynucleoside triphosphate compounds representing A, G, C and either T or U, wherein each of said labeled deoxynucleoside triphosphate compounds comprise a nucleobase and a sugar, said sugar comprising a cleavable protecting group on the 3′-O, wherein said cleavable protecting group comprises methylenedisulfide, wherein said labeled deoxynucleoside triphosphate comprises a label attached via a cleavable oxymethylenedisulfide-containing linker to the nucleobase;   b) adding said DNA polymerase and said labeled populations of deoxynucleoside triphosphate compounds to said primer and template so as to create a reaction mixture;   c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, wherein a first labeled deoxynucleoside triphosphate is incorporated;   d) detecting said label of said first labeled deoxynucleoside triphosphate in said modified primer/template hybridization complex, wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate;   e) introducing said cleave reagent under conditions so as to remove said cleavable protecting group and said detectable label from said modified primer/template hybridization complex, wherein the incidence of identifying the wrong base is reduced in comparison to other thiol cleave reagents.   
     
     
         83 . The method of  claim 82 , wherein the method further comprises repeating steps b)-e), wherein a second deoxynucleoside triphosphate is incorporated in step b) and wherein said second deoxynucleoside triphosphate comprises a second detectable label. 
     
     
         84 . A method for detecting labeled nucleotides in a DNA sequence comprising the steps of
 a) providing a nucleic acid template and primer capable of hybridizing to said template so as to form a primer/template hybridization complex, a cleave reagent comprising di-mercaptopropanesulfonate in a CHES buffer, wherein the pH of said cleave reagent is between 9.0 and 10.0, a cleave scavenger reagent comprising an oxidative scavenger, DNA polymerase, and 4 differently labeled populations of deoxynucleoside triphosphate compounds representing A, G, C and either T or U, wherein each of said labeled deoxynucleoside triphosphate compounds comprise a nucleobase and a sugar, said sugar comprising a cleavable protecting group on the 3′-O, wherein said cleavable protecting group comprises methylenedisulfide, wherein said labeled deoxynucleoside triphosphate comprises a label attached via a cleavable oxymethylenedisulfide-containing linker to the nucleobase;   b) adding said DNA polymerase and said labeled populations of deoxynucleoside triphosphate compounds to said primer and template so as to create a reaction mixture;   c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, wherein a first labeled deoxynucleoside triphosphate is incorporated;   d) detecting said label of said first labeled deoxynucleoside triphosphate in said modified primer/template hybridization complex, wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate;   e) introducing said cleave reagent under conditions so as to remove said cleavable protecting group and said detectable label from said modified primer/template hybridization complex, wherein the incidence of identifying the wrong base is reduced in comparison to other thiol cleave reagents; and   f) introducing said cleave scavenger reagent.   
     
     
         85 . The method of  claim 84 , wherein said pH of said cleave reagent is 9.5. 
     
     
         86 . The method of  claim 84 , wherein said oxidative scavenger is hydrogen peroxide. 
     
     
         87 . The method of  claim 86 , wherein said hydrogen peroxide is in a buffer. 
     
     
         88 . The method of  claim 87 , wherein said buffer is TRIS. 
     
     
         89 . The method of  claim 88 , wherein said TRIS buffer is at a pH of between 8.5 and 9.0. 
     
     
         90 . The method of  claim 89 , wherein said TRIS buffer is at a pH of 8.8. 
     
     
         91 . The method of  claim 84 , wherein said oxidative scavenger is tert-butyl peroxide. 
     
     
         92 . The method of  claim 84 , wherein said reaction mixture of step b) is in a flow cell. 
     
     
         93 . The method of  claim 92 , wherein said flow cell is positioned on a moving support. 
     
     
         94 . The method of  claim 93 , wherein said moving support is a rotary stage. 
     
     
         95 . The method of  claim 92 , wherein at least a portion of said flow cell is transparent. 
     
     
         96 . The method of  claim 92 , wherein said flow cell is incorporated within an instrument. 
     
     
         97 . The method according to  claim 84 , wherein the method further comprises repeating steps b)-f), wherein a second deoxynucleoside triphosphate is incorporated in step b). 
     
     
         98 . The method of  claim 97 , wherein the nucleobase of said second deoxynucleoside triphosphate is different from the nucleobase of said first deoxynucleoside triphosphate. 
     
     
         99 . A method for detecting labeled nucleotides in a DNA sequence comprising the steps of
 a) providing a nucleic acid template and primer capable of hybridizing to said template so as to form a primer/template hybridization complex, and a flow cell, said flow cell in fluidic communication with a first reservoir and second reservoir, said first reservoir comprising a cleave reagent comprising di-mercaptopropanesulfonate in a CHES buffer, wherein the pH of said cleave reagent is between 9.0 and 10.0, a cleave scavenger reagent comprising an oxidative scavenger, said second reservoir comprising an oxidative wash, DNA polymerase, and 4 differently labeled populations of deoxynucleoside compounds representing A, G, C and either T or U, wherein each of said labeled deoxynucleoside triphosphate compounds comprise a nucleobase and a sugar, said sugar comprising a cleavable protecting group on the 3′-O, wherein said cleavable protecting group comprises methylenedisulfide, wherein said labeled deoxynucleoside triphosphate comprises a label attached via a cleavable oxymethylenedisulfide-containing linker to the nucleobase;   b) adding said DNA polymerase and said labeled populations of deoxynucleoside triphosphate compounds to said primer and template so as to create a reaction mixture in said flow cell;   c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, wherein a first deoxynucleoside triphosphate is incorporated;   d) detecting said label of said first labeled deoxynucleoside triphosphate in said modified primer/template hybridization complex wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate;   e) introducing said cleave reagent from said first reservoir into said flow cell under conditions so as to remove said cleavable protecting group and said detectable label from said modified primer/template hybridization complex, wherein the incidence of identifying the wrong base is reduced in comparison to other thiol cleave reagents; and   f) introducing said oxidative wash from said second reservoir into said flow cell.   
     
     
         100 . The method of  claim 99 , wherein said pH of said cleave reagent is 9.5. 
     
     
         101 . The method of  claim 99 , wherein said oxidative wash comprises hydrogen peroxide. 
     
     
         102 . The method of  claim 101 , wherein said hydrogen peroxide is in a buffer. 
     
     
         103 . The method of  claim 102 , wherein said buffer is TRIS. 
     
     
         104 . The method of  claim 99 , wherein said oxidative wash comprises tert-butyl peroxide.