Polynucleotide synthesis method, system and kit
AssigneeOxford Nanopore Technologies PLC
InventorsJohn Milton, Sobia Nayyar, Jan Riedl, Ryosuke Ogaki
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Abstract
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
Claims94
An in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis wherein each cycle comprises cleaving a double-stranded polynucleotide and extending the cleaved double-stranded polynucleotide by incorporating a nucleotide pair, wherein a terminal end of a first strand of the cleaved double-stranded polynucleotide is extended by the addition of a nucleotide of the predefined sequence and a terminal end of the second strand of the cleaved double-stranded polynucleotide which is hybridized to the first strand is extended by the addition of a partner nucleotide thereby forming a nucleotide pair with the incorporated nucleotide of the first strand.
A method according to any one of the preceding claims, wherein a partner nucleotide which pairs with a first/next nucleotide of the predefined sequence is a nucleotide which is complementary with the first/next nucleotide, preferably naturally complementary.
A method according to any one of the preceding claims, wherein both strands of the synthesised double-stranded polynucleotide are DNA strands.
A method according to any one of the preceding claims, wherein synthesis cycles are performed in droplets within a microfluidic system.
A method according to any one of the preceding claims, wherein following synthesis the strands of the double-stranded polynucleotides are separated to provide a single-stranded polynucleotide having a predefined sequence.
A method according to any one of the preceding claims, wherein following synthesis the double-stranded polynucleotide or a region thereof is amplified, preferably by PCR.
A method of assembling a polynucleotide having a predefined sequence, the method comprising performing the method of any one of the preceding claims to synthesise a first polynucleotide having a predefined sequence and one or more additional polynucleotides having a predefined sequence and joining together the first and one or more additional polynucleotides.
Dependencies are inferred from the claim text.