Methods and apparatuses for chip-based DNA error reduction
Inventors
Ramu, Senthil • Jacobson, Joseph
Assignees
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Publication Number
US-10829759-B2
Publication Date
2020-11-10
Expiration Date
Abstract
Methods and apparatus relate to reduction of sequence errors generated during synthesis of nucleic acids on a microarray chip. The error reduction can include synthesis of complementary stands (to template strands), using a short universal primer complementary to the template strands and polymerase. Heteroduplex can be formed be melting and re-annealing complementary stands and template strands. The heteroduplexes containing a mismatch can be recognized and cleaved by a mismatch endonuclease. The mismatch-containing cleaved heteroduplexes can be removed from the microarray chip using a global buffer exchange. The error free synthetic nucleic acids generated therefrom can be used for a variety of applications, including synthesis of biofuels and value-added pharmaceutical products.
Core Innovation
The invention relates to a method for producing a population of double-stranded oligonucleotides having high fidelity on a solid support. Error-containing oligonucleotides and error-free oligonucleotides are used to produce homoduplexes and mismatch-containing heteroduplexes through denaturing and hybridizing steps. The method includes cleaving mismatch-containing heteroduplexes using a mismatch recognizing and cleaving component, and then removing cleaved mismatch-containing heteroduplexes to leave high-fidelity duplexes on the solid support.
A key feature is that one or more discrete features of the solid support are selectively hydrated such that template-bound oligonucleotides are present within one or more droplets. Template-directed chain extension is performed in the selectively hydrated features, thereby producing a first plurality of duplexes. After chain extension, the duplexes are denatured to release oligonucleotides, and the released first plurality is hybridized with the second plurality bound to the discrete features to form mismatch-containing heteroduplexes.
In a related embodiment, the invention produces at least one double-stranded support-bound error-free oligonucleotide having a predefined sequence on a solid support. The method synthesizes a first plurality of oligonucleotides on the solid support using second plurality oligonucleotides as templates, with selectively hydrated features that place the templates within droplets. Released oligonucleotides are hybridized to form double-stranded products including mismatches, and mismatch binding agent contacting and selective cleavage are used to remove mismatched products, thereby producing support-bound error-free oligonucleotides.
In another embodiment, the invention produces single-stranded high fidelity oligonucleotides by template-dependent synthesis on support-bound single-stranded oligonucleotides within droplets, denaturation, reannealing to form homoduplexes and heteroduplexes, mismatch recognizing and cleaving, removal of cleaved heteroduplexes, and selective denaturation of the resulting error-free support-bound double-stranded oligonucleotides.
Claims Coverage
The document includes three independent claims. Across the independent claims, the core inventive approach centers on solid support-bound oligonucleotides in selectively hydrated, droplet-present features, followed by denaturing or reannealing to generate homoduplexes and mismatch-containing heteroduplexes, mismatch-recognizing cleavage, and removal to obtain high-fidelity products.
Template-directed duplex synthesis on selectively hydrated solid support features in droplets
A method for producing a population of double-stranded oligonucleotides having high fidelity on a solid support by synthesizing a first plurality of oligonucleotides in a chain extension reaction using a second plurality of oligonucleotides as templates, wherein the second plurality is bound at their 3′ end to one or more discrete features of the solid support and the discrete features are selectively hydrated such that the second plurality is present within one or more droplets.
Denaturing and re-hybridizing to form homoduplexes and mismatch-containing heteroduplexes
Denaturing the first plurality of duplexes to form a released first plurality of oligonucleotides, and hybridizing the released first plurality with the second plurality of oligonucleotides so that the second plurality of duplexes comprises one or more homoduplexes and one or more mismatch-containing heteroduplexes formed between error-containing and error-free oligonucleotides.
Mismatch-recognizing cleavage and removal to produce high-fidelity duplex population on the solid support
Cleaving the one or more mismatch-containing heteroduplexes by a mismatch recognizing and cleaving component to form cleaved mismatch-containing heteroduplexes, and removing the cleaved one or more mismatch-containing heteroduplexes thereby producing the population of double-stranded oligonucleotides having high fidelity on the solid support.
Producing support-bound error-free predefined sequence oligonucleotides using mismatch binding agent selective cleavage
A method for producing at least one double-stranded support-bound error-free oligonucleotide having a predefined sequence on a solid support by synthesizing a first plurality of oligonucleotides on a solid support using a second plurality of oligonucleotides as templates with at least one primer, wherein the second plurality is bound at their 3′ end to one or more discrete features and the discrete features are selectively hydrated such that the second plurality is present within one or more droplets, and wherein at least one second plurality oligonucleotide comprises a sequence error and at least one is error-free.
Hybridize released products and selectively cleave mismatched double-stranded oligonucleotides
Releasing the first plurality of oligonucleotides, hybridizing the released first plurality with the second plurality to form a plurality of double-stranded oligonucleotides comprising at least one double-stranded support-bound error-free oligonucleotide and at least one double-stranded oligonucleotide having a mismatch with the sequence error, and contacting the plurality with a mismatch binding agent that selectively binds and cleaves the double-stranded oligonucleotide having the mismatch.
Removing cleaved mismatched product to yield support-bound error-free predefined sequence
Removing the cleaved double-stranded oligonucleotide having the mismatch, thereby producing the at least one double-stranded support-bound error-free oligonucleotide having the predefined sequence on the solid support.
Single-stranded high fidelity oligonucleotide production via droplet synthesis, heteroduplex reannealing, mismatch cleavage, removal, and selective denaturation
A method for producing single-stranded high fidelity oligonucleotides by contacting, on a solid support, a plurality of support-bound single-stranded oligonucleotides comprising error-free and error-containing oligonucleotides with a solution comprising a primer, nucleotides and a polymerase enzyme under conditions suitable for a template-dependent synthesis reaction, wherein the support-bound oligonucleotides are bound at their 3′ end to one or more discrete features and the discrete features are selectively hydrated such that the support-bound single-stranded oligonucleotides are present within one or more droplets, thereby producing double-stranded oligonucleotides comprising synthesized complementary oligonucleotides base paired with the support-bound oligonucleotides.
Denaturation, reannealing to mismatch-containing heteroduplexes, and mismatch recognizing cleavage
Denaturing the plurality of double-stranded oligonucleotides to release synthesized complementary oligonucleotides into a solution, reannealing the synthesized complementary oligonucleotides to the plurality of support-bound single-stranded oligonucleotides to produce reannealed double-stranded oligonucleotides comprising homoduplexes and heteroduplexes wherein each heteroduplex comprises a mismatch, and exposing the reannealed double-stranded oligonucleotides to a mismatch recognizing and cleaving component to form cleaved heteroduplexes.
Removal of cleaved heteroduplexes and selective denaturation to yield single-stranded high fidelity oligonucleotides
Removing the cleaved heteroduplexes to produce a population of error-free support-bound double-stranded oligonucleotides and selectively denaturing the population of error-free support-bound double-stranded oligonucleotides thereby producing the single-stranded high fidelity oligonucleotides.
Across the independent claims, the approach uses selectively hydrated discrete solid support features that place oligonucleotide templates within droplets, creates duplexes via template-dependent synthesis, forms homoduplexes and mismatch-containing heteroduplexes through denaturing or reannealing, cleaves mismatch-containing species using a mismatch recognizing or binding component, removes cleaved mismatch-containing products, and yields high-fidelity double-stranded and single-stranded oligonucleotides on the solid support.
Stated Advantages
Produces a population of double-stranded oligonucleotides having high fidelity on a solid support.
Produces at least one double-stranded support-bound error-free oligonucleotide having a predefined sequence on a solid support.
Produces single-stranded high fidelity oligonucleotides.
Documented Applications
Downstream assembly and producing higher-fidelity oligos for value-added products is described.
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