Methods and devices for nucleic acids synthesis
Inventors
Jacobson, Joseph • Kung, Li-yun A. • Wilson, Andrew Kirk • Ramu, Senthil • SCHINDLER, DANIEL • Hudson, Michael E.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11845054-B2
Publication Date
2023-12-19
Expiration Date
Abstract
Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.
Core Innovation
The invention provides systems and methods for high-fidelity synthesis and assembly of predefined polynucleotide sequences on solid supports or nucleic acid arrays. Construction oligonucleotides are generated by primer extension so that overlapping construction oligonucleotides are produced for assembly into longer polynucleotides.
Anchor single-stranded oligonucleotides are provided on aligned array features on a solid support, and construction oligonucleotides are hybridized to the corresponding anchor oligonucleotides for guided assembly. The method positions a first support and a second support so that features of the second support align to corresponding features of the first support, and release of the construction oligonucleotides into solution under conditions promoting hybridization enables hybridization to the plurality of corresponding anchor oligonucleotides.
The approach supports multiple predefined sequence populations by using support-bound template oligonucleotides comprising at least two populations with different predefined sequences and 3′ end complementary sequences. To improve accuracy and yield and reduce miscapture, the method includes mismatch/error removal processing using mismatch recognizing and cleaving components, including mismatch endonucleases such as CEL I, and post-cleavage cleanup.
Claims Coverage
The document contains one independent claim describing a method for synthesizing a plurality of polynucleotides with predefined sequences using aligned solid supports, template-bound primer extension, release into solution for anchor hybridization, and hybridization-promoting conditions. The claim set presents five core inventive features, with dependent claims refining mismatch cleavage, primer removal chemistry, porous membrane transport constraints, stoichiometry relationships, and ligase in the solution.
Aligned first and second supports with anchor complementarity
Positioning the first and the second support such that each feature of the second support is aligned to a corresponding feature of the first support, where each feature of the first support comprises a plurality of support-bound anchor single-stranded oligonucleotides with 5′ ends complementary to 5′ ends of first plurality of construction oligonucleotides.
Template-bound generation of construction oligonucleotides from multiple predefined sequence populations
Generating at least a first and second plurality of construction oligonucleotides by polymerase extension using the plurality of single-stranded support-bound template oligonucleotides as templates, wherein the first plurality of construction oligonucleotides are complementary to the first population of oligonucleotides, and wherein the second plurality of construction oligonucleotides are complementary to the second population of oligonucleotides.
Hybridization-prompting release of construction oligonucleotides to anchors
Releasing the at least first and second pluralities of construction oligonucleotides in a solution under conditions promoting hybridization of the first plurality of construction oligonucleotides to the plurality of corresponding anchor oligonucleotides and hybridization of the second plurality of construction oligonucleotides to the first plurality of construction oligonucleotides.
Support features with multiple anchor oligonucleotides per feature
Providing a first support comprising a plurality of features, wherein each feature comprises a plurality of support-bound anchor single-stranded oligonucleotides.
Support-bound template oligonucleotides comprising at least two predefined sequence populations
Providing a second support having a plurality of features, wherein each feature comprises a plurality of single-stranded support-bound template oligonucleotides, wherein the plurality of single-stranded support-bound template oligonucleotides comprises at least two populations of oligonucleotides having different predefined sequences, the at least two populations of oligonucleotides having 3′ end complementary sequences.
Overall, the claims cover a solid-support assembly workflow where predefined polynucleotides are synthesized by polymerase extension from support-bound templates in a second support, producing first and second pluralities of construction oligonucleotides, aligning a first support having feature-level anchor oligonucleotides, and releasing the construction oligonucleotides into solution so hybridization occurs to the anchors and between construction oligonucleotides. Dependent refinements include mismatch-processing using CEL I, primer-removal using uracil-containing primer and specified glycosylase/endonuclease-lyase reagents, transport constraint using a porous membrane, stoichiometry relationships comparing construction oligonucleotides and anchor oligonucleotides, and inclusion of ligase in the solution.
Stated Advantages
Improve accuracy and yield and reduce miscapture.
Documented Applications
High-fidelity synthesis and assembly of predefined polynucleotide sequences on solid supports or nucleic acid arrays.
Interested in licensing this patent?