Continuous extension and deblocking in reactions for nucleic acids synthesis and sequencing

A method of synthesizing a polynucleotide, comprising

• (a) providing a mixture comprising a nucleic acid, a collection of different nucleotide analogs, a polymerase having activity to incorporate a nucleotide analog comprising a blocking moiety into a nucleic acid, a phosphotriesterase and a phosphodiesterase, wherein the phosphotriesterase and the phosphodiesterase together have activity to selectively remove a blocking moiety that has been incorporated into a nucleic acid compared to a blocking moiety attached to a monomeric nucleotide analog,
• wherein the different nucleotide analogs each comprise a phosphotriester blocking moiety, and
• (b) sequentially adding a plurality of the different nucleotide analogs to the nucleic acid by several reaction cycles in the mixture, wherein the polymerase, phosphotriesterase and phosphodiesterase are simultaneously present in the mixture to carry out the reaction cycles,
• wherein each reaction cycle comprises:
• (i) the polymerase adding a nucleotide analog of the collection of different nucleotide analogs to the nucleic acid to form a transient nucleic acid species comprising the phosphotriester blocking moiety,
• (ii) the phosphotriesterase converting the phosphotriester blocking moiety to a phosphodiester blocking moiety, and
• (iii) the phosphodiesterase removing the phosphodiester blocking moiety from the nucleic acid.