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

Claim 7:
8. The method according to claim 3, wherein:
a) in step (1) the scaffold polynucleotide is provided in the support strand with a nucleotide (position n) which is the partner nucleotide for the first nucleotide of step (2), and the universal nucleotide in the support strand is positioned next to the partner nucleotide (position n+1, in the direction proximal to the helper strand and distal to the primer strand);
b) in step (2) the first nucleotide is incorporated into the synthesis strand at the position opposite the partner nucleotide in the support strand (position n), whereupon the first nucleotide pairs with the partner nucleotide and in step (6) the next nucleotide is incorporated into the synthesis strand at the position opposite the partner nucleotide in the support strand (position n), whereupon the next nucleotide pairs with the partner nucleotide;
c) in step (3) the support strand is cleaved at a position between the first nucleotide (position n) and the second nucleotide (position n−1) from the universal nucleotide in the support strand in the direction distal to the helper strand and proximal to the primer strand, wherein cleavage removes the universal nucleotide and creates a single-nucleotide overhang in the scaffold polynucleotide comprising the first nucleotide overhanging the support strand and in step (7) the support strand is cleaved at a position between the first nucleotide (position n) and the second nucleotide (position n−1) from the universal nucleotide in the support strand in the direction distal to the helper strand and proximal to the primer strand, wherein cleavage removes the universal nucleotide and creates a single-nucleotide overhang in the scaffold polynucleotide comprising the next nucleotide overhanging the support strand; and
d) in step (4) and (8), the complementary ligation end of the ligation polynucleotide comprises a single-nucleotide overhang and wherein:
i. the universal nucleotide in the support strand is positioned opposite the penultimate nucleotide of the helper strand (position n+1) and is paired therewith;
ii. the universal nucleotide is positioned next to the penultimate nucleotide of the support strand (position n);
iii. the penultimate nucleotide of the support strand (position n) is paired with the terminal nucleotide of the helper strand and is a partner nucleotide for the next nucleotide in step (6) of the next synthesis cycle; and
iv. the terminal nucleotide of the support strand (position n−1) overhangs the terminal nucleotide of the helper strand and is a partner nucleotide for the first nucleotide of step (2) or is a partner nucleotide for the newly-incorporated nucleotide of step (6) of the current synthesis cycle.