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US 9,752,176 B2Granted
Methods for preparative in vitro cloning
AssigneeGinkgo Bioworks, Inc.
InventorsLi-yun A. Kung, Daniel Schindler, Joseph Jacobson
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Abstract
Methods and devices relate to the isolation of nucleic acids of interest from within a population of nucleic acids such as libraries of nucleic acid sequences.
Claims25
A method of isolating a target nucleic acid molecule, the method comprising:
- (a) providing a plurality of nucleic acid molecules;
- (b) providing a library of oligonucleotide tags, each oligonucleotide tag comprising a region having a different sequence than the other tags in the library and comprising a primer binding site;
- (c) ligating at least a subset of the plurality of nucleic acid molecules with the library of oligonucleotide tags, thereby generating a population of conjugates, wherein each conjugate has a unique oligonucleotide tag that is different in sequence than that of the other conjugates;
- (d) amplifying in a pool at least a subset of the population of conjugates using one or more primers each having sequence complementarity to the primer binding site thereon;
- (e) isolating at least one conjugate of the amplified conjugates from the pool, wherein the isolated conjugate comprises a target nucleic acid molecule having a predetermined sequence;
- and
- (f) removing the oligonucleotide tag attached thereto to recover the target nucleic acid molecule.
4 more elements
A method of isolating a target nucleic acid molecule, the method comprising:
- (a) providing a population of nucleic acid molecules having different predetermined sequences;
- (b) providing a plurality of oligonucleotide sequences that are different from one another in sequence and are immobilized on microparticles, each microparticle having a single copy of oligonucleotide sequence immobilized on its surface, wherein each single copy of oligonucleotide sequence is uniquely complementary to a corresponding nucleic acid molecule of the population;
- (c) hybridizing the population of nucleic acid molecules to the plurality of oligonucleotide sequences, thereby forming a population of hybridized microparticles, each hybridized microparticle having a single nucleic acid molecule attached thereon via hybridization with its corresponding uniquely complementary oligonucleotide sequence;
- (d) separating the population of hybridized microparticles into samples, each sample comprising a single hybridized microparticle;
- and
- (e) isolating at least one hybridized microparticle comprising a target nucleic acid molecule having a predetermined sequence.
3 more elements
A method of isolating a target nucleic acid molecule, the method comprising:
- (a) providing a population of nucleic acid conjugates, each having a distinct predetermined sequence and a oligonucleotide tag that is different in sequence than that of the other conjugates, wherein each predetermined sequence and each oligonucleotide tag are conjugated via ligation;
- (b) amplifying in a pool at least a subset of the population of nucleic acid conjugates using primers having sequence complementarity to the oligonucleotide tags conjugated thereto;
- (c) isolating a target nucleic acid molecule having a predetermined sequence from the amplified nucleic acid molecules in the pool;
- and
- (d) removing the oligonucleotide tag conjugated thereto to recover the target nucleic acid molecule.
2 more elements
Dependencies are inferred from the claim text.