US 2026/0132437 A1 · Methods of Synthesizing Nucleic Acid Molecules

Abstract

The invention provides methods for synthesizing a product DNA molecule of any DNA sequence from a universal library of overlapping oligonucleotides. The method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule. The method also involves annealing the plurality of oligonucleotides, performing a ligation step, and performing an amplification step to thereby synthesize a sub-sequence of the product DNA molecule. The invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme. In one embodiment the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence.

Claims30

Dependent claims

A method of synthesizing a DNA molecule having a desired sequence comprising:

a) annealing at least two oligonucleotides to an anchor strand so that the at least two oligonucleotides annealed to the anchor strand abut one another on the anchor strand;
wherein the at least two oligonucleotides each comprise a primer binding site on a 3′ or 5′ end, and a variable sequence on the opposing 5′ or 3′ end, and a conserved flanking sequence in between the primer binding site and the variable sequence;
and
wherein the anchor strand comprises conserved flanking sequences complementary to those on the at least two oligonucleotides, and further comprises at least one variable sequence, wherein at least a portion of the at least one variable sequence on the anchor strand is complementary to at least a portion of the variable sequences on the at least two oligonucleotides;
b) ligating the at least two oligonucleotides annealed to the anchor strand to produce a first dsDNA molecule;
c) performing an amplification step on the first dsDNA molecule having a desired sequence and comprising a conserved flanking sequence inside each of the 3′ and 5′ ends, and a variable sequence inside the conserved flanking sequences.

3 more elements

Dependencies are inferred from the claim text.

Methods of Synthesizing Nucleic Acid Molecules

Life of this patent

Methods of Synthesizing Nucleic Acid Molecules

Life of this patent

Abstract

Claims30