Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis

Abstract

Nucleotide triphosphate probes containing a molecular and/or atomic tag on 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 method for sequencing nucleic acids, comprising:
 a) flowing labeled deoxynucleotide triphosphates onto a plurality of template nucleic acids which are hybridized to primer nucleic acids, wherein the plurality of template nucleic acids or the primers are attached to a support in an addressable array format, wherein the labeled nucleotide triphosphates include a molecular tag attached to their base moiety and the molecular tag emits a detectable signal, and wherein the labeled nucleotide triphosphates lack a quencher moiety that absorbs the detectable signal emitted by the molecular tag;   b) incorporating with a polymerase the labeled nucleotide triphosphates into the primers; and   c) detecting incorporation of the labeled nucleotide triphosphates.   
     
     
         2 . The method of  claim 1 , wherein the molecular tag which is attached to the base moiety comprises a fluorophore moiety. 
     
     
         3 . The method of  claim 1 , wherein the labeled deoxynucleotide triphosphates further comprise a molecular tag attached to their sugar moiety. 
     
     
         4 . The method of  claim 3 , wherein the molecular tag is attached to the 3′ position of the sugar moiety. 
     
     
         5 . The method of  claim 4 , wherein the molecular tag is removable from the 3′ position of the sugar moiety. 
     
     
         6 . The method of  claim 3 , wherein the molecular tag comprises an alkyl group, aryl group, alkaryl group, aralkyl group. 
     
     
         7 . The method of  claim 1 , wherein the labeled deoxynucleotide triphosphates further comprise a molecular tag attached to their beta phosphate or gamma phosphate. 
     
     
         8 . The method of  claim 7 , wherein the molecular tag comprises an alkyl group, aryl group, alkaryl group, aralkyl group. 
     
     
         9 . The method of  claim 7 , wherein the molecular tag which is attached to the beta phosphate or gamma phosphate comprises a fluorophore moiety. 
     
     
         10 . The method of  claim 1 , wherein the flowing in step (a) includes a single type of labeled deoxynucleotide triphosphates selected from the group consisting of deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate and deoxyuridine triphosphate. 
     
     
         11 . The method of  claim 10 , wherein the different types of labeled deoxynucleotide triphosphates are labeled at their base moiety with a fluorophore that distinguishes the different types of deoxynucleotide triphosphates. 
     
     
         12 . The method of  claim 1 , wherein the flowing in step (a) includes a mixture of different types of labeled deoxynucleotide triphosphates which include deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate. 
     
     
         13 . The method of  claim 12 , wherein the different types of labeled deoxynucleotide triphosphates are labeled at their base moiety with a fluorophore that distinguishes the different types of deoxynucleotide triphosphates. 
     
     
         14 . The method of  claim 1 , wherein the support comprises a micro-fabricated chip. 
     
     
         15 . The method of  claim 1 , further comprising: washing away non-incorporated labeled nucleotide triphosphates after step (b). 
     
     
         16 . The method of  claim 1 , wherein the detecting comprises optically detecting the incorporation of the labeled nucleotide triphosphates. 
     
     
         17 . The method of  claim 1 , wherein the detecting comprises detecting in parallel incorporation of the labeled nucleotide triphosphates into the primers. 
     
     
         18 . The method of  claim 1 , wherein the plurality of template nucleic acids comprise an oligonucleotide adaptor attached to both ends, and wherein one of the oligonucleotide adaptors contains a sequencing primer binding site. 
     
     
         19 . The method of  claim 1 , further comprising: (d) repeating steps (a)-(c) at least once. 
     
     
         20 . The method of  claim 1 , further comprising: flowing apyrase onto the plurality of template nucleic acids after step (b) or (c). 
     
     
         21 . The method of  claim 1 , further comprising: flowing onto the plurality of template nucleic acids in step (a) at least one polyphosphate compound selected from the group consisting of compounds of the following general formulas:
   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 and Z′ are each independently a hydrogen atom or a thermally stable substituent, E and E′ are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of 2, 3, 4 or 5.