Generating nucleic acids with modified bases using recombinant terminal deoxynucleotidyl transferase
AssigneesIllumina, Inc., Illumina Singapore Pte. Ltd.
InventorsSaurabh Rajendra Nirantar, Sergio Peisajovich, Eric Brustad
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Abstract
Disclosed herein include methods of generating a single stranded deoxyribonucleic acid (ssDNA) scaffold comprising nucleotides with modified bases using a recombinant terminal deoxynucleotidyl transferase (TdT). The recombinant TdT can comprise an amino acid sequence that is at least 80% identical to a Bos taurus TdT, or a fragment thereof, and for example comprise one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His420 in the Bos taurus TdT.
Claims24
A method of modifying a nucleic acid, the method comprising:
- (a) providing a single stranded deoxyribonucleic acid (ssDNA) and a nucleoside triphosphate comprising a modified base;
- and
- (b) contacting the ssDNA and the nucleoside triphosphate comprising the modified base with a recombinant terminal deoxynucleotidyl transferase (TdT) to generate a ssDNA scaffold, wherein the ssDNA scaffold comprises the ssDNA incorporated with one or more nucleotides comprising the modified base from the nucleoside triphosphate,
- wherein the recombinant TdT comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 1, and wherein the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions corresponding to Glu191, Glu194, Asp242, Lys287, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO:
- 12.
A method of modifying a nucleic acid, the method comprising:
- (a) providing a first nucleic acid and a first nucleoside triphosphate comprising a first modified base;
- and
- (b) contacting the first nucleic acid and the first nucleoside triphosphate comprising the first modified base with a recombinant terminal deoxynucleotidyl transferase (TdT) for a first reaction time at a first temperature in a first reaction to generate a second nucleic acid, wherein the second nucleic acid comprises the first nucleic acid incorporated with one or more first nucleotides comprising the first modified base from the first nucleoside triphosphate,
- wherein the recombinant TdT comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 1, and wherein the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions corresponding to Glu191, Glu194, Asp242, Lys287, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO:
- 12.
Dependencies are inferred from the claim text.