Four-color dna sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

Abstract

This invention provides a process for sequencing single-stranded DNA employing modified nucleotides.

1 - 33 . (canceled) 
     
     
         34 . A method, comprising:
 a) providing a plurality of primer strands hybridized to nucleic acid templates immobilized in a plurality of spots on a solid surface,
 wherein each spot comprises a plurality of nucleic acid templates having the same sequence, 
 wherein the plurality of nucleic acid templates in each spot differs in sequence from the plurality of nucleic acid templates in another spot, 
 wherein a fraction of said plurality of primer strands comprises a free 3′—OH and another fraction of said plurality of primer strands comprises an incorporated labeled nucleotide analogue reversible terminator, 
 wherein each incorporated labeled nucleotide analogue reversible terminator comprises a cleavable moiety bound to the 3′-oxygen of the deoxyribose, a base, and a label bound to the base via a linker; and 
   b) extending with a polymerase one or more of said primer strands that has a free 3′—OH with an unlabeled nucleotide analogue reversible terminator, wherein each unlabeled analogue comprises a cleavable moiety bound to the 3′-oxygen of the deoxyribose.   
     
     
         35 . The method of  claim 34 , further comprising the step of detecting the incorporated labeled nucleotide analogue reversible terminator. 
     
     
         36 . The method of  claim 35 , wherein label is bound to the base of the incorporated labeled nucleotide analogue reversible terminator via a chemically cleavable linker, and said method further comprises the step of chemically cleaving the chemically cleavable linker of said incorporated labeled nucleotide analogue reversible terminator so as to remove the label. 
     
     
         37 . The method of  claim 35 , wherein the moiety cleavably linked to the 3′-oxygen of the deoxyribose in said incorporated labeled nucleotide analogue reversible terminator is chemically cleavable. 
     
     
         38 . The method of  claim 37 , wherein the cleavable moiety attached to the 3′-oxygen of the deoxyribose of the incorporated labeled nucleotide analogue reversible terminator is chemically cleavable with Na 2 PdCl 4 /P(PhSO 3 Na) 3 . 
     
     
         39 . The method of  claim 38 , wherein the cleavable moiety attached to the 3′-oxygen of the deoxyribose of the unlabeled nucleotide analogue reversible terminator is chemically cleavable with Na 2 PdCl 4 /P(PhSO 3 Na) 3 . 
     
     
         40 . The method of  claim 39 , wherein the label is bound by a chemically cleavable linker which is chemically cleavable with Na 2 PdCl 4 /P(PhSO 3 Na) 3 . 
     
     
         41 . The method of  claim 40 , wherein the chemically cleavable linker, the cleavable moiety attached to the 3′-oxygen of the deoxyribose of the incorporated labeled nucleotide analogue reversible terminator, and the cleavable moiety attached to the 3′-oxygen of the deoxyribose of the unlabeled nucleotide analogue reversible terminator are chemically cleaved simultaneously. 
     
     
         42 . The method of  claim 41 , wherein the incorporated labeled nucleotide analogue reversible terminator has a base which is a thymine. 
     
     
         43 . The method of  claim 41 , wherein the incorporated labeled nucleotide analogue reversible terminator has a base which is selected from adenine, cytosine, and guanine. 
     
     
         44 . The method of  claim 41 , wherein the unlabeled nucleotide analogue reversible terminator has a base which is a guanine. 
     
     
         45 . The method of  claim 41 , wherein the unlabeled nucleotide analogue reversible terminator has a base which is selected from adenine, thymine, and cytosine. 
     
     
         46 . The method of  claim 39 , wherein label is bound to the base of the incorporated labeled nucleotide analogue reversible terminator via a chemically cleavable linker, and said method further comprises the step of chemically cleaving the chemically cleavable linker of said incorporated labeled nucleotide analogue reversible terminator so as to remove the label. 
     
     
         47 . The method of  claim 46 , wherein after the label is removed, the 3′—OH groups of the incorporated labeled nucleotide analogue reversible terminator and the unlabeled nucleotide analogue reversible terminator are chemically regenerated. 
     
     
         48 . The method of  claim 47 , wherein the incorporated labeled nucleotide analogue reversible terminator has a base which is an adenine. 
     
     
         49 . The method of  claim 47 , wherein the incorporated labeled nucleotide analogue reversible terminator has a base which is selected from guanine, thymine, and cytosine. 
     
     
         50 . The method of  claim 47 , wherein the unlabeled nucleotide analogue reversible terminator has a base which is a guanine. 
     
     
         51 . The method of  claim 47 , wherein the unlabeled nucleotide analogue reversible terminator has a base which is selected from adenine, thymine, and cytosine. 
     
     
         52 . The method of  claim 34 , wherein the solid surface is a chip. 
     
     
         53 . The method of  claim 34 , wherein the nucleic acid templates are produced using PCR.