Chemically cleavable 3'-o-allyl-dntp-allyl-fluorophore fluorescent nucleotide analogues and related methods

Abstract

This invention provides a nucleotide analogue comprising (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine and uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′-oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker, and methods of nucleic acid sequencing employing the nucleotide analogue.

1 . A nucleotide analogue comprising (i) a base selected from the group consisting of adenine or an analogue of adenine, guanine or an analogue of guanine, cytosine or an analogue of cytosine, thymine or an analogue of thymine and uracil or an analogue of uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′-oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker. 
     
     
         2 . The nucleotide analogue of  claim 1 , wherein the nucleotide analogue is an analogue of DATP, dGTP, dCTP or dUTP. 
     
     
         3 . The nucleotide analogue of  claim 1 , wherein the fluorophore is selected from the group consisting of ROX, Bodipy-FL-510, Bodipy-650 and R6G. 
     
     
         4 . The nucleotide analogue of  claim 1 , wherein the fluorophore is bound to the base via an iso-allyl linker. 
     
     
         5 . The nucleotide analogue of  claim 1 , wherein the nucleotide analogue is selected from the group consisting of 3′-O-allyl-dGTP-iso-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-iso-allyl-Bodipy-650, 3′-O-allyl-dATP-iso-allyl-ROX and 3′-O-allyl-dUTP-iso-allyl-R6G. 
     
     
         6 . The nucleotide analogue of  claim 1 , wherein the nucleotide analogue is selected from the group consisting of 3′-O-allyl-dGTP-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-allyl-Bodipy-650, 3′-O-allyl-dATP-allyl-ROX and 3′-O-allyl-dUTP-allyl-R6G. 
     
     
         7 - 18 . (canceled) 
     
     
         19 . A method for determining the sequence of a DNA comprising performing the following steps for each residue of the DNA to be sequenced:
 (a) contacting the DNA with a DNA polymerase in the presence of (i) a primer and (ii) four fluorescent nucleotide analogues under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein (1) the nucleotide analogues consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of DATP, (2) each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′-oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker, so that a nucleotide analogue complementary to the residue being sequenced is bound to the DNA by the DNA polymerase, and (3) each of the four analogues has a predetermined fluorescence wavelength which is different than the fluorescence wavelengths of the other three analogues;   (b) removing unbound nucleotide analogues;   (c) determining the identity of the bound nucleotide analogue; and   (d) following step (c), except with respect to the final DNA residue to be sequenced, chemically cleaving from the bound nucleotide analogue the fluorophore and the allyl moiety bound to the 3′-oxygen atom of the deoxyribose, thereby determining the sequence of the DNA.   
     
     
         20 . The method of  claim 19 , wherein chemically cleaving the fluorophore and the allyl moiety bound to the 3′-oxygen atom is performed using Na 2 PdCl 4 . 
     
     
         21 . The method of  claim 19 , wherein the primer is a self-priming moiety. 
     
     
         22 . The method of  claim 19 , wherein the DNA is bound to a solid substrate. 
     
     
         23 . The method of  claim 22 , wherein the DNA is bound to the solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry. 
     
     
         24 . The method of  claim 22 , wherein about 1000 or fewer copies of the DNA are bound to the solid substrate. 
     
     
         25 . The method of  claim 19 , wherein the four fluorescent nucleotide analogues are 3′-O-allyl-dGTP-iso-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-iso-allyl-Bodipy-650, 3′-O-allyl-dATP-iso-allyl-ROX and 3′-O-allyl-dUTP-iso-allyl-R6G. 
     
     
         26 . The method of  claim 19 , wherein the four fluorescent nucleotide analogues are 3′-O-allyl-dGTP-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-allyl-Bodipy-650, 3′-O-allyl-dATP-allyl-ROX and 3′-O-allyl-dUTP-allyl-R6G. 
     
     
         27 . The method of  claim 19 , wherein the DNA polymerase is a 9°N polymerase. 
     
     
         28 . A kit for performing the method  claim 19 , comprising, in separate compartments,
 (a) a nucleotide analogue of (i) GTP, (ii) ATP, (iii) CTP and (iv) TTP or UTP, wherein each analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, or an analogue thereof, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′-oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker,   (b) reagents suitable for use in DNA polymerization; and   (c) instructions for use.   
     
     
         29 . The kit of  claim 28 , wherein the kit comprises 3′-O-allyl-dGTP-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-allyl-Bodipy-650, 3′-O-allyl-dATP-allyl-ROX and 3′-O-allyl-dUTP-allyl-R6G. 
     
     
         30 . The kit of  claim 28 , wherein the kit comprises 3′-O-allyl-dGTP-iso-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-iso-allyl-Bodipy-650, 3′-O-allyl-dATP-iso-allyl-ROX and 3′-O-allyl-dUTP-iso-allyl-R6G. 
     
     
         31 - 32 . (canceled)