Compositions and methods for nucleotide sequencing

Abstract

The invention provides nucleoside and nucleotide molecules containing cleavable linkers linking a label such as a dye. The invention also provides nucleosides and nucleotide molecules containing a blocking group, either immovable or non-removable. The invention additionally provides methods of using the nucleoside and nucleotide molecules containing a cleavable linker and/or a blocking group.

What is claimed is: 
     
         1 . A nucleoside, comprising a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker and wherein a blocking group is attached at the 2′or 4′ position of said sugar moiety. 
     
     
         2 . The molecule of  claim 1 , wherein said blocking group is removable. 
     
     
         3 . The molecule of  claim 1 , wherein said label is a fluorescent dye. 
     
     
         4 . The molecule of  claim 1 , wherein said linker can be cleaved by a protease. 
     
     
         5 . The molecule of  claim 1 , wherein said label comprises a ligand. 
     
     
         6 . A nucleotide molecule comprising the nucleoside of  claim 1 . 
     
     
         7 . The nucleotide molecule of  claim 6 , wherein said nucleotide molecule is a nucleotide triphosphate. 
     
     
         8 . A nucleic acid molecule comprising the nucleoside of  claim 1 . 
     
     
         9 . The nucleic acid molecule of  claim 8 , wherein said nucleoside is at the 5′ or 3′ terminus of the nucleic acid. 
     
     
         10 . A method for sequencing a nucleic acid molecule, comprising:
 (a) contacting a nucleic acid template, a nucleic acid primer complementary to a portion of said template, a polymerase and an RT-NTP under conditions wherein said primer is extended to incorporate said RT-NTP into a sequence complementary to said nucleic acid template, wherein said RT-NTP comprises a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker and wherein a blocking group is attached at the 2′ or 4′ position of said sugar moiety; and   (b) identifying the RT-NTP in said sequence complementary to said nucleic acid template, wherein identifying said RT-NTP identifies at least a portion of the sequence of said nucleic acid molecule.   
     
     
         11 . The method of  claim 10 , further comprising (c) removing said removable blocking group from said RT-NTP in said sequence complementary to said nucleic acid template. 
     
     
         12 . The method of  claim 11 , wherein said removable blocking group is removed by an enzyme. 
     
     
         13 . The method of  claim 11 , further comprising adding a second HT-NTP and repeating step (a), wherein said complementary sequence is extended and said second RT-NTP is incorporated into said sequence complementary to said nucleic acid template. 
     
     
         14 . The method of  claim 13 , further comprising repeating step (b), wherein identifying said second RT-NTP identifies at least a portion of the sequence of said nucleic acid template. 
     
     
         15 . The method of  claim 14 , further comprising repeating step (c). 
     
     
         16 . The method of  claim 11 , further comprising (d) cleaving said cleavable linker to remove said label from said RT-NTP in said sequence complementary to said nucleic acid template. 
     
     
         17 . The method of  claim 16 , wherein steps (c) and (d) are carried out in the same reaction. 
     
     
         18 . The method of  claim 16 , further comprising adding one or more RT-NTPs and repeating steps (a), (b), (c) and (d) one or more times, wherein at least a portion of the sequence of said nucleic acid template is determined. 
     
     
         19 . A method for extending a primer nucleic acid, comprising contacting a nucleic acid molecule with a nucleotide incorporating catalyst and a nucleotide molecule, wherein said nucleotide molecule comprises a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker and wherein a blocking group is attached at the 2′or 4′ position of said sugar moiety, whereby said catalyst extends the nucleic acid to produce an extended nucleic acid molecule incorporating said nucleotide molecule. 
     
     
         20 . The method of  claim 19 , further comprising removing said label and said blocking group from said nucleotide using enzymes.