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

  • US 10,378,051 B2
  • Filed: 09/26/2012
  • Issued: 08/13/2019
  • Est. Priority Date: 09/29/2011
  • Status: Active Grant
First Claim
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1. 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, and a deblocking agent having activity to selectively remove a blocking moiety that has been incorporated into a nucleic acidcompared to a blocking moiety attached to a monomeric nucleotide analog, wherein each different nucleotide analog comprises a blocking moiety, and(b) allowing sequential addition of a plurality of the different nucleotide analogs to the nucleic acid to proceed via several reaction cycles in the mixture, wherein the polymerase and the deblocking agent are simultaneously present in the mixture to carry out the reaction cycles,wherein each reaction cycle comprises:

  • (i) the polymerase adding a nucleotide analog from the collection of different nucleotide analogs to the nucleic acid to form a transient nucleic acid species comprising the blocking moiety, and(ii) the deblocking agent modifying the transient nucleic acid species to remove the blocking moiety;

    wherein the blocking moiety is attached to the nucleotide analog at a 3′

    carbon of a pentose moiety of the nucleotide analog, and the blocking moiety comprises a phosphodiester moiety or a phosphotriester moiety.

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