Patent ID: 11884950
Assignee: OXFORD NANOPORE TECHNOLOGIES PLC
Field: Biotechnology (Chemistry)
Classification: CPC C  B | IPC B  C

Claim 2:
3. The method according to claim 2, the method comprising:
(1) providing a scaffold polynucleotide comprising a synthesis strand and a support strand hybridized thereto, wherein the synthesis strand comprises a primer strand portion and a helper strand portion separated by a single-strand break, and the support strand comprises a universal nucleotide;
(2) incorporating a first nucleotide of the predefined sequence into the synthesis strand by the action of the polymerase, the first nucleotide comprising a reversible terminator group which prevents further extension by the polymerase;
(3) cleaving the scaffold polynucleotide at a cleavage site, the site defined by a sequence comprising the universal nucleotide in the support strand, wherein cleavage comprises cleaving the support strand and removing the universal nucleotide to provide in the synthesis strand an overhanging end comprising the first nucleotide;
(4) ligating a double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide comprising a support strand, a helper strand and a complementary ligation end, the ligation end comprising in the support strand a universal nucleotide and a partner nucleotide for the first nucleotide which overhangs the helper strand, and in the helper strand a terminal nucleotide lacking a phosphate group, wherein upon ligation of the support strands the first nucleotide pairs with the partner nucleotide,
(5) removing the reversible terminator group from the first nucleotide after step (4) and before step (6), or after step (2) and before step (3), or after step (3) and before step (4);
(6) incorporating the next nucleotide of the predefined nucleotide sequence into the synthesis strand of the scaffold polynucleotide by the action of the polymerase, the next nucleotide comprising a reversible terminator group which prevents further extension by the polymerase;
(7) cleaving the scaffold polynucleotide at a cleavage site, the site defined by a sequence comprising a universal nucleotide in the support strand, wherein cleavage comprises cleaving the support strand and removing the universal nucleotide to provide in the synthesis strand an overhanging end comprising the next nucleotide;
(8) ligating a double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide comprising a support strand, a helper strand and a complementary ligation end, the ligation end comprising in the support strand a universal nucleotide and a partner nucleotide for the next nucleotide which overhangs the helper strand, and in the helper strand a terminal nucleotide lacking a phosphate group, wherein upon ligation of the support strands the next nucleotide pairs with the partner nucleotide;
(9) removing the reversible terminator group from the next nucleotide after step (8) and before step (10), or after step (6) and before step (7), or after step (7) and before step (8); and
(10) repeating steps £6 to M multiple times to provide the double-stranded polynucleotide having a predefined nucleotide sequence.