Polynucleotide synthesis method, kit and system

A method of assembling a polynucleotide having a predefined sequence, the method comprising synthesizing a first double-stranded polynucleotide having a predefined sequence and one or more additional double-stranded polynucleotides having a predefined sequence, cleaving the first double-stranded polynucleotide and the one or more additional double-stranded polynucleotides, and joining together the first and one or more additional double-stranded polynucleotides, wherein the first double-stranded polynucleotide and the one or more additional double-stranded polynucleotides are synthesized by an in vitro method comprising performing repeating cycles of synthesis wherein in each cycle:

• (A) a first strand of the first double-stranded polynucleotide and a first strand of the one or more additional double-stranded polynucleotides are each extended by the addition of a first nucleotide of the predefined sequence and a universal nucleotide by the action of a ligase enzyme, wherein the universal nucleotide defines a cleavage site;
• (B) a second strand of the first double-stranded polynucleotide which is hybridized to the first strand of the first double-stranded polynucleotide and a second strand of the one or more additional double-stranded polynucleotides which is hybridized to the first strand of the one or more additional double-stranded polynucleotides are each extended by the addition of a second nucleotide of the predefined sequence by a nucleotide transferase or polymerase enzyme;
• and
• (C) the first double-stranded polynucleotide and the one or more additional double stranded polynucleotides are then cleaved at the cleavage site;
• wherein the first and second nucleotides of the predefined sequence of each cycle are retained in the first double-stranded polynucleotide and the one or more additional double stranded polynucleotides following cleavage, and wherein the first nucleotide and the second nucleotide become partner nucleotides in different nucleotide pairs in the synthesised first double-stranded polynucleotide and the one or more additional double stranded polynucleotides.