Dna sequencing reaction additive

Abstract

The present invention provides methods, compositions, mixtures and kits utilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencing reactions, and in particular, sequencing reactions where deoxynucleoside triphosphates comprising a 3′-O position capped by a disulfide-based 3′-terminator group are used. In one embodiment, the deoxynucleoside triphosphates comprise 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 .- 26 . (canceled) 
     
     
         27 . A kit comprising one or more DNA sequencing reagents, instructions, 5-Chloro-2-methyl-4-isothiazolin-3-one (CMIT) and a cleave reagent comprising a thiol-containing compound. 
     
     
         28 . The kit of  claim 27 , further comprising a cleave scavenger. 
     
     
         29 . The kit of  claim 28 , wherein said cleave scavenger is cystamine. 
     
     
         30 . The kit of  claim 27 , wherein said one or more DNA sequencing reagents are selected from the group comprising polymerase, primer, template and a plurality of nucleotide analogues. 
     
     
         31 . The kit of  claim 27 , further comprising 2-Methyl-4-isothiazolin-3-one. 
     
     
         32 . The kit of  claim 30 , wherein each nucleotide analogue of the plurality of nucleotide analogues comprises a nucleobase and a sugar, the sugar comprising a cleavable protecting group on the 3′-O, wherein the cleavable protecting group comprises a disulfide-based 3′-terminator group. 
     
     
         33 . The kit of  claim 32 , wherein the disulfide-based 3′-terminator group comprises methylenedisulfide. 
     
     
         34 . The kit of  claim 30 , wherein at least a portion of the plurality of nucleotide analogues nucleotide analogues further comprises a detectable label attached via a cleavable disulfide-containing linker to the nucleobase. 
     
     
         35 . The kit of  claim 27 , further comprising a cleave reagent comprising a thiol-containing compound. 
     
     
         36 . The kit of  claim 35 , wherein the thiol-containing compound comprises 2,3-dimercaptopropanesulfonate, 2,3-dimercapto-1-propanol, or 2,3-dimercaptopropanephosphonate. 
     
     
         37 . A kit for sequencing, comprising
 a plurality of nucleotide analogues, each nucleotide analogue of the plurality of nucleotide analogues comprises a nucleobase and a sugar, the sugar comprising a cleavable protecting group on the 3′-O, wherein the cleavable protecting group comprises a disulfide-based 3′-terminator group, and   5-Chloro-2-methyl-4-isothiazolin-3-one.   
     
     
         38 . The kit of  claim 37 , further comprising 2-Methyl-4-isothiazolin-3-one. 
     
     
         39 . The kit of  claim 37 , wherein the disulfide-based 3′-terminator group comprises methylenedisulfide. 
     
     
         40 . The kit of  claim 37 , wherein at least a portion of the plurality of nucleotide analogues nucleotide analogues further comprises a detectable label attached via a cleavable disulfide-containing linker to the nucleobase. 
     
     
         41 . The kit of  claim 40 , the plurality of nucleotide analogues nucleotide analogues comprises a mixture of labeled nucleotides labeled with the detectable label and unlabeled nucleotides. 
     
     
         42 . The kit of  claim 37 , further comprising a cleave reagent comprising a thiol-containing compound. 
     
     
         43 . The kit of  claim 42 , wherein the cleave reagent comprises a di-mercapto reagent, a mono-mercapto reagent, and/or a helicon reagent. 
     
     
         44 . The kit of  claim 42 , wherein the thiol-containing compound comprises 2,3-dimercaptopropanesulfonate, 2,3-dimercapto-1-propanol, or 2,3-dimercaptopropanephosphonate. 
     
     
         45 . The kit of  claim 37 , further comprising a cleave scavenger. 
     
     
         46 . The kit of  claim 45 , wherein said cleave scavenger is cystamine.