Demand synthesis of polynucleotide sequences
Inventors
Kannan, Krishna • Gill, John E. • Gibson, Daniel G. • Fu, Lixia
Assignees
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Publication Number
US-12065684-B2
Publication Date
2024-08-20
Expiration Date
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.
Core Innovation
The disclosure describes a method of synthesizing a product DNA molecule having a desired and pre-determined sequence. The method anneals 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 long and short oligonucleotides, within defined binding sets, to form double-stranded DNA molecules with abutting regions suitable for ligation.
After annealing, abutting long and short oligonucleotides are ligated to form double-stranded DNA molecules, and in one pathway blunt end ligation forms a double-stranded DNA molecule. The anchor strand(s) comprise one or more non-standard nucleotides, and the ligated material is contacted with one or more enzymes that degrade DNA comprising non-standard nucleotides to form a single-stranded DNA molecule.
The method then performs amplification and contacts the amplified double-stranded DNA with a Type IIS restriction endonuclease to synthesize the product DNA molecule having the desired and pre-determined sequence. The method is performed without any of the oligonucleotides being bound to a solid phase, and the disclosure further supports scarless and sticky-end formats through the Type IIS restriction endonuclease step.
The disclosure also includes oligonucleotide libraries assembled from defined long and short oligonucleotides, including libraries with less than 20,000 members that can be assembled into all possible DNA sequences. Documented use cases include DNA data storage using encoded messages/bytes, and construction of CRISPR guide RNA constructs that include promoter, Cas9 handle, and terminator elements.
Claims Coverage
The independent claim is clm-00001. It recites five inventive features in combination: anchor-strand-mediated assembly of double-stranded intermediate molecules without solid-phase binding, anchor-strand degradation based on non-standard nucleotides, amplification, and Type IIS restriction endonuclease conversion to a product DNA molecule with a desired and pre-determined sequence.
Anchor-strand annealing of long and short oligonucleotides within defined binding sets
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 long and short oligonucleotides, wherein the annealing occurs within a binding set that forms double-stranded DNA molecules with abutting O1/O2 and O3/O4 (or alternative binding-set arrangement forming a double-stranded DNA molecule).
Ligation of abutting oligonucleotides to form double-stranded DNA intermediates
Ligating the abutting long and short oligonucleotides, and in one pathway blunt end ligation, to form a double-stranded DNA molecule from the annealed binding-set arrangement.
Anchor strands with non-standard nucleotides degraded by sequence-specific enzyme contacting
Anchor strand(s) each comprising one or more non-standard nucleotides, and 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.
Amplification followed by Type IIS restriction endonuclease synthesis of the product DNA sequence
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.
No solid-phase binding of oligonucleotides
Performing the method without any of the oligonucleotides being bound to a solid phase.
Overall, the claim coverage centers on an anchor-strand assembly workflow that anneals long and short oligonucleotides to anchor strands, ligates abutting regions to form double-stranded intermediates, degrades anchor-strand DNA containing non-standard nucleotides, amplifies the resulting material, and then uses a Type IIS restriction endonuclease to generate a product DNA molecule with a desired and pre-determined sequence without solid-phase binding.
Stated Advantages
Not explicitly described in patent.
Documented Applications
DNA data storage, including encoding messages/bytes.
Construction of CRISPR guide RNA constructs including promoter, Cas9 handle, and terminator elements.
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