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Publication Number
US-11071963-B2
Publication Date
2021-07-27
Expiration Date
Abstract
Methods and apparatus relate to the synthesis of high fidelity polynucleotides and to the reduction of sequence errors generated during synthesis of nucleic acids on a solid support. Specifically, design of support-bound template oligonucleotides is disclosed. Assembly methods include cycles of annealing, stringent wash and extension of polynucleotides comprising a sequence region complementary to immobilized template oligonucleotides. 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 provides a method of removing error-containing polynucleotides by hybridizing input polynucleotides to predefined single stranded oligonucleotides having a predefined sequence. The single stranded oligonucleotides include a 5′ end sequence region, a 3′ end sequence region, and at least two different sequences regions between the 5′ end and the 3′ end sequence regions. Complementary sequence regions at the 3′ end of the input polynucleotides hybridize to the sequence regions to generate duplexes.
The duplexes are then subjected to melt conditions sufficient to denature duplexes having at least one mismatch in a complementary region without denaturing the duplexes that do not comprise a mismatch in the complementary region. This selective denaturation releases a population of error-containing input polynucleotides, and the method removes the error-containing input polynucleotides.
The approach is described in the context of feature-addressable assembly using cycles of primer extension with hybridization and stringent melt/wash steps to denature and remove mismatched duplexes while retaining matched duplexes. Additional sequence-verification and junction QC is disclosed using additional template regions and oligonucleotide segment layouts, including a screening sequence region, junction QC sequence region, and a spacer sequence region.
Claims Coverage
One independent claim defines the central inventive concept. Dependent claims refine oligonucleotide region layouts, melting-temperature uniformity, the production of the input polynucleotide, and optional shuffling logic involving denature and re-anneal steps.
Selective melt denaturation releasing mismatched duplexes
Subjecting duplexes to melt conditions sufficient to denature duplexes having at least one mismatch in a complementary region without denaturing duplexes that do not comprise a mismatch in the complementary region, thereby releasing error-containing input polynucleotides.
Hybridizing predefined single stranded oligonucleotides to input polynucleotides
Providing single stranded oligonucleotides with a 5′ end sequence region, a 3′ end sequence region, and at least two different sequence regions between the 5′ end and the 3′ end sequence regions, and hybridizing input polynucleotides to the oligonucleotides to generate duplexes.
Removing released error-containing input polynucleotides
Removing error-containing input polynucleotides after melt conditions release a population of error-containing input polynucleotides.
Uniform melting temperature across duplexes
Performing the method such that the melting temperature difference between the plurality of duplexes is less than 1°C.
Relaxed melting temperature uniformity threshold across duplexes
Performing the method where the melting temperatures among the plurality of duplexes differ by less than 10°C.
Consecutive extension chain reactions using specified templates
Using an input polynucleotide produced by at least two consecutive extension chain reactions where templates are the sequences (N1-252) and (N1-251).
Shuffling by denaturing and re-annealing extension products
Denaturing extension duplexes to release single-stranded extension products, re-annealing them to oligonucleotides to form re-annealed duplexes, applying melt conditions to dissociate error-containing duplexes, removing the resulting error-containing single-stranded extension products, and dissociating error-free duplexes to release error-free extension products in solution.
At least three different sequence regions for hybridization
Using oligonucleotides that include at least three different sequence regions between the 5′ end and 3′ end, where the input polynucleotide hybridizes to those regions.
Overall, the claim coverage centers on selective melt conditions that denature duplexes with mismatches in complementary regions while preserving non-mismatched duplexes, followed by removal of the released error-containing input polynucleotides. The dependent claims further cover melting-temperature uniformity constraints, defined oligonucleotide region layouts for hybridization, production of input polynucleotides via consecutive extension chain reactions using specified templates, and optional shuffling workflows using denature and re-anneal followed by selective dissociation and release of extension products.
Stated Advantages
Denaturing duplexes having at least one mismatch in a complementary region without denaturing duplexes that do not comprise a mismatch in the complementary region.
Releasing and removing error-containing input polynucleotides while retaining non-mismatched duplexes.
Reducing error-containing polynucleotides by selective melting discrimination.
Documented Applications
Feature-addressable assembly of predetermined high-fidelity polynucleotides using cycles of primer extension with hybridization and stringent melt/wash steps.
Sequence verification and junction QC using additional template regions and junction quality control sequence regions with selective melt discrimination.
Droplet-based processing involving droplet movement and transfer for assembly and dissociation workflows.
Use of microarrays and microfluidic or DMD-based localized heating for assembly and sequence verification.
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