Telesis Bio Inc.

Codex Dna, Inc.

Lixia Fu

Daniel Glenn Gibson

John E Gill

Krishna Kannan

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.

Demand synthesis of polynucleotide sequences

The invention provides methods of assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA ligase with a plurality of short oligonucleotides to be assembled and performing the ligase chain reaction to thereby generate a set of polynucleotides. Oligonucleotides in the plurality overlap with and are complementary to a sequence of at least one other oligonucleotide in the plurality, and at least 50% of the oligonucleotides in the plurality are 6-30 nucleotides in length. The set of polynucleotides produced are contacted with a DNA polymerase and dNTPs in a mixture to join the set of polynucleotides and thereby create a DNA molecule having a desired sequence by polymerase chain assembly. The method allows for production of oligonucleotides of any length having very high sequence fidelity to a desired sequence.

Methods for assembling nucleic acids

The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3' or a 5&#x2032; primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3&#x2032; flanking sequence and a universal 5&#x2032; flanking sequence, respectively. The methods involve performing a first amplification reaction on the plurality of pairs of oligonucleotides; removing the 3&#x2032; and 5&#x2032; primer binding sequences from the plurality of pairs of oligonucleotides; and subjecting the plurality of pairs of oligonucleotides to an assembly reaction to thereby assemble the dsDNA molecule having the desired sequence.

Growing community of inventors

San Diego, CA, United States of America

Lixia Fu