Methods and reagents for synthesising polynucleotide molecules

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 to create compatible termini, and joining together the first and one or more additional double-stranded polynucleotides by ligation;

• wherein the first double-stranded polynucleotide and the one or more additional double-stranded polynucleotides are synthesized by an in vitro method comprising performing cycles of synthesis wherein in each cycle, a first strand is extended by incorporation of a nucleotide of the predefined sequence and the second strand which is hybridized to the first strand is extended by incorporation of a nucleotide thereby forming a nucleotide pair with the incorporated nucleotide of the first strand;
• wherein each cycle comprises extending the first strand by incorporating the nucleotide of the predefined sequence together with an attached reversible terminator group followed by extending the second strand;
• further wherein in each cycle the nucleotides are incorporated into a scaffold polynucleotide and wherein each cycle comprises:
• (1) providing a scaffold polynucleotide;
• (2) incorporating into the scaffold polynucleotide by action of a polymerase a nucleotide of the predefined sequence, the nucleotide comprising a reversible terminator group which prevents further extension by the polymerase;
• (3) cleaving the scaffold polynucleotide at a cleavage site;
• (4) ligating a ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide comprising a partner nucleotide for the nucleotide of the predefined sequence, wherein upon ligation, the nucleotide of the predefined sequence pairs with the partner nucleotide;
• and
• (5) removing the reversible terminator group from the nucleotide of the predefined sequence after step (4) or removing the reversible terminator group from the nucleotide of predefined sequence after step (2) and before step (3), or after step (3) and before step (4).