Compositions and methods for multiplex nucleic acids synthesis
Inventors
Jacobson, Joseph • SCHINDLER, DANIEL • Saaem, Ishtiaq E. • Lawton, Scott S. • Goldberg, Martin J. • Hudson, Michael E. • Kung, Li-yun A.
Assignees
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Publication Number
US-11130948-B2
Publication Date
2021-09-28
Expiration Date
Abstract
Aspects of the invention relate to methods, compositions for designing and producing a target nucleic acid. In particular, aspects of the invention relate to the multiplex synthesis of target polynucleotides.
Core Innovation
The invention relates to producing a plurality of double-stranded oligonucleotides having a plurality of overhangs. The method includes melting a first plurality of blunt-ended double-stranded oligonucleotides and a second plurality of blunt-ended double-stranded oligonucleotides to form a plurality of single stranded oligonucleotides, and re-annealing the plurality of single-stranded oligonucleotides to form double-stranded oligonucleotides having overhangs. The process is directed to creating overhang-bearing double-stranded products from blunt-ended inputs via a melt-and-reanneal conversion.
The produced double-stranded oligonucleotides have overhangs that are designed to be relatively mutually orthogonal to each other. This orthogonal overhang design supports multiplexing, including serial MPS, hierarchical MPS, and parallel multiplexed polynucleotide synthesis. The approach is described as ligase-based stepwise assembly in which cohesive overhanging oligonucleotides are used as assembly intermediates.
In described embodiments, anchor oligonucleotides and construction oligonucleotides are used in support-immobilized assembly, including bead support and stem-loop polynucleotide support. Strategies are also described to mitigate termination/trapped states using fragment pooling and concentration distribution approaches, including offset dimer concentration distribution and bead-based titration, and by using tethered ligase. The disclosed concepts further include computer-assisted fragment design/parsing from target sequences and error reduction using shuffling together with mismatch binding proteins or nucleases from MutS/MutS homologs, MutHLS, and CEL I/CEL II families.
Claims Coverage
The independent claim covers producing double-stranded oligonucleotides with overhangs by melting two blunt-ended double-stranded oligonucleotide pluralities into single strands and re-annealing to generate relatively mutually orthogonal overhangs. Dependent refinements cover mismatch binding protein-based error correction, handling the two pluralities in separate reaction volumes and combining them, and immobilization on the same support, including stem-loop polynucleotide support.
Mutually orthogonal overhang re-annealing from blunt ends
Melting a first plurality of blunt-ended double-stranded oligonucleotides and a second plurality of blunt-ended double-stranded oligonucleotides to form a plurality of single stranded oligonucleotides, and re-annealing the plurality of single-stranded oligonucleotides to form a plurality of double-stranded oligonucleotides having a plurality of overhangs, wherein the plurality of overhangs are designed to be relatively mutually orthogonal to each other.
Mismatch binding protein-based error correction
Correcting or reducing errors in first and second pluralities of blunt-ended double-stranded oligonucleotides using a mismatch binding protein.
Separate reaction volumes for two blunt-ended pluralities
Placing a first plurality of blunt-ended double-stranded oligonucleotides in a first reaction volume and a second plurality of blunt-ended double-stranded oligonucleotides in a second reaction volume.
Combining separate reaction volumes
Combining the first and second reaction volumes.
Same support immobilization of oligonucleotides
Immobilizing each blunt-ended double-stranded oligonucleotide from the plurality onto the same support.
Stem-loop polynucleotide support
Wherein the support is a stem-loop polynucleotide.
Overall, claim coverage centers on creating double-stranded oligonucleotides with relatively mutually orthogonal overhangs by melting blunt-ended double-stranded inputs into single strands and re-annealing. Dependent coverage further narrows how errors may be corrected, how the two input pluralities may be partitioned by reaction volumes, and how oligonucleotides may be immobilized on specific support types, including stem-loop polynucleotide support.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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