Demand synthesis of polynucleotide sequences
AssigneeTelesis Bio Inc.
InventorsKrishna Kannan, John E. Gill, Daniel G. Gibson, Lixia Fu
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Abstract
The invention provides methods of synthesizing a product DNA molecule having a desired and/or defined sequence. The methods involve annealing at least one long oligonucleotide and at least one short oligonucleotide to at least one anchor strand having a sequence at least partially complementary to the at least one long and at least one short oligonucleotide. The invention also provides methods of synthesizing DNA molecules by assembling oligonucleotide members of a library that contains less than 20,000 members that can be assembled into all possible DNA sequences.
Claims37
A method of synthesizing a product DNA molecule having a desired and pre-determined sequence comprising:
- annealing at least one long oligonucleotide and at least one short oligonucleotide to at least one anchor strand(s) having a sequence at least partially complementary to the at least one long and at least one short oligonucleotide, wherein,
- (a) the annealing occurs within a binding set comprising at least two long oligonucleotides O1 and O4 of 9-35 nucleotides in length, two short oligonucleotides O2 and O3 of 5-7 nucleotides in length, and at least two anchor strands O5 and O6, wherein after annealing O1 abuts O2, and O3 abuts O4, and O1 and O2 are bound to the same anchor strand O5, and O3 and O4 are bound to the same anchor strand O6, to form double-stranded DNA molecules O1-O2-O5 and O3-O4-O6;
- ligating the abutting O1 and O2, and the abutting O3 and O4 to form double-stranded DNA molecules O1-O2-O5 and O3-O4-O6;
- and
- ligating double-stranded DNA molecules O1-O2-O5 and O3-O4-O6 by blunt end ligation to form a double-stranded DNA molecule;
- or
- (b) the annealing occurs within a binding set comprising at least two long oligonucleotides O1 and O3 of 9-35 nucleotides in length, one short oligonucleotide O2 of 5-7 nucleotides in length, and at least one anchor strand O4, wherein after annealing O2 abuts O1 and O3, and O1-O3 are bound to the same anchor strand O4 to form a double-stranded DNA molecule O1-O4;
- ligating the abutting O1 and O2, and the abutting O2 and O3 to form a double-stranded DNA molecule;
- wherein the anchor strand(s) each comprise one or more non-standard nucleotides,
- contacting the double-stranded DNA molecule with one or more enzymes that degrade DNA comprising one or more non-standard nucleotides to form a single-stranded DNA molecule;
- performing a step of amplification to produce an amplified double-stranded DNA molecule;
- and
- contacting the amplified double-stranded DNA molecule with a Type IIS restriction endonuclease to thereby synthesize the product DNA molecule having the desired and pre-determined sequence;
- and wherein the method is performed without any of the oligonucleotides being bound to a solid phase.
Dependencies are inferred from the claim text.