Enzymatic Nucleic Acid Synthesis: Compositions and Methods for Inhibiting Pyrophosphorolysis

Abstract

Nucleotide triphosphate probes containing a molecular and/or atomic tag on a a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.

1 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formulas:
   Z—OPO 2 O—Z′  (a)
     Z—PO 2 O—Z′  (b)
     Z—OPO 2 —Z′  (c)
     Z—PO 2 —Z′  (d)
     Z—OPO 2 —(OP(EE′)O) n —PO 2 O—Z′  (e)
     Z—OPO 2 —(OP(EE′)O) n PO 2 —Z′  (f)
     Z—PO 2 —(OP(EE′)O) n PO 2 O—Z′  (g)
     Z—PO 2 —(OP(EE′)O) n PO 2 —Z′  (h)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent, E and E′ are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The composition of  claim 1 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         22 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—OPO 2 O—Z′  (a)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent and E and are an oxygen atom or a thermally stable substituent. 
     
     
         23 . The composition of  claim 22 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         24 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—PO 2 O—Z′  (b)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent and E and are an oxygen atom or a thermally stable substituent. 
     
     
         25 . The composition of  claim 24 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         26 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—OPO 2 —Z′  (c)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent and E and F are an oxygen atom or a thermally stable substituent. 
     
     
         27 . The composition of  claim 26 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         28 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—PO 2 —Z′  (d)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent and E and F are an oxygen atom or a thermally stable substituent. 
     
     
         29 . The composition of  claim 28 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         30 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—OPO 2 —(OP(EE′)O) n —PO 2 O—Z′  (e)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent, E and E′ are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5. 
     
     
         31 . The composition of  claim 30 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         32 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—OPO 2 —(OP(EE′)O) n PO 2 —Z′  (f)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent, E and E′ are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5. 
     
     
         33 . The composition of  claim 32 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         34 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—PO 2 —(OP(EE′)O) n PO 2 O—Z′  (g)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent, E and F are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5. 
     
     
         35 . The composition of  claim 34 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         36 . A pyrophosphorolysis inhibiting composition comprising at least one pyrophosphorolysis inhibitor of the general formula:
   Z—PO 2 —(OP(EE′)O) n PO 2 —Z′  (h)
   
       where Z or Z′ is a hydrogen atom or a thermally stable substituent, E and E′ are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5. 
     
     
         37 . The composition of  claim 36 , wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements. 
     
     
         38 . The composition of  claim 1 ,  22 ,  24 ,  26 ,  28 ,  30 ,  32 ,  34 , or  36 , wherein Z and/or Z′ are tags.