Methods for nucleic acid assembly and high throughput sequencing
Inventors
Hudson, Michael E. • Kung, Li-yun A. • SCHINDLER, DANIEL • Archer, Stephen • Saaem, Ishtiaq
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12241057-B2
Publication Date
2025-03-04
Expiration Date
Abstract
Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.
Core Innovation
The invention relates to producing a nucleic acid having a predefined sequence by using a first pool of double-stranded oligonucleotides. The double-stranded oligonucleotides include internal sequences identical to different portions of a first target nucleic acid, with an overlapping region with another double-stranded oligonucleotide in the first pool, and flanking sequences that comprise a common primer recognition site and a first restriction enzyme recognition site oriented so digestion removes the flanking sequences and exposes the internal sequence.
The method generates the first target nucleic acid by exposing the first pool of double-stranded oligonucleotides to a ligase and the first restriction enzyme under conditions suitable for concurrent restriction enzyme digestion and ligation. The resulting first target nucleic acid comprises an internal sequence identical to a portion of a final target nucleic acid and also comprises flanking sequences that include a second restriction enzyme recognition site, where the first restriction enzyme recognition site and the second restriction enzyme recognition site have different sequences.
The documented platform emphasizes producing high-fidelity target nucleic acids using multiplex oligonucleotides that carry internal payload sequences between common primer-recognition and restriction-enzyme recognition sites. It supports recursive or hierarchical assembly into larger nucleic acid constructs, vector assembly followed by host transformation and sequencing-based sequence verification, and error reduction strategies that include denaturation and reannealing to remove mismatches and enrichment or filtration using mismatch-binding agents such as MutS / MutS homologs and related mismatch/repair proteins.
Claims Coverage
The independent claim provides a core workflow with three key inventive elements: a multiplex pool of double-stranded oligonucleotides with common primer recognition and a first restriction enzyme recognition site oriented to remove flanks; concurrent restriction digestion and ligation using a ligase and the first restriction enzyme to generate a predefined target nucleic acid; and a first target nucleic acid whose flanking sequences include a second restriction enzyme recognition site having a different sequence from the first. Dependent claims refine assembly by adding additional target nucleic acids and incorporating error removal using mismatch-binding agents.
Multiplex first pool with common primer and oriented first restriction site
Providing a first pool of double-stranded oligonucleotides comprising internal sequences identical to different portions of a first target nucleic acid, the internal sequences comprising an overlapping region with another double-stranded oligonucleotide in the first pool, wherein 5′ flanking sequences and/or 3′ flanking sequences each comprise a common primer recognition site and a first restriction enzyme recognition site, the first restriction enzyme recognition site being oriented so that digestion with a first restriction enzyme that recognizes the first restriction enzyme recognition site will remove the flanking sequences and expose the internal sequence.
Concurrent restriction digestion and ligation to generate predefined target
Exposing the first pool of double-stranded oligonucleotides to a ligase and the first restriction enzyme under conditions suitable to promote concurrent restriction enzyme digestion and ligation, thereby generating the first target nucleic acid.
First and second restriction recognition sites with different sequences
The first target nucleic acid comprises a 5′ flanking sequence and/or a 3′ flanking sequence, each of the flanking sequences comprising a second restriction enzyme recognition site, wherein the first restriction enzyme recognition site and the second restriction enzyme recognition site have different sequences.
Two-target mixture assembly into final target nucleic acid
The method further provides a mixture of a first target nucleic acid and a second target nucleic acid having an internal sequence different from the first but matching part of the final target, and flanking sequences containing a second restriction enzyme recognition site, then exposes the mixture to a ligase and the second restriction enzyme to generate a final target nucleic acid containing internal sequences from both targets.
Mismatch error removal using MutS mismatch binding agent
Producing double-stranded oligonucleotides in a second pool by amplifying corresponding single-stranded oligonucleotides using their common primer recognition sites, and then performing error removal by contacting the amplified oligonucleotides with a MutS mismatch binding agent.
Overall claim coverage centers on producing predefined target nucleic acids from multiplex double-stranded oligonucleotides by orienting a common primer and first restriction recognition architecture to expose internal sequences upon restriction digestion, and then performing concurrent restriction digestion and ligation with a ligase. Dependent refinements include assembling a final target nucleic acid from a mixture of first and second target nucleic acids and introducing error removal via a MutS mismatch binding agent, along with constraints involving flanking restriction site architectures.
Stated Advantages
Eliminating purification bead/capture steps between digestion and ligation to provide throughput/cost/time advantages.
Concurrent restriction digestion and ligation to concurrently remove common flanking sequences and ligate exposed cohesive ends into defined constructs.
High-fidelity production of predefined target nucleic acids, including error reduction through denaturation and reannealing and mismatch enrichment/filtration using mismatch-binding agents such as MutS / MutS homologs.
Sequence verification of constructs using sequencing-based sequence verification, including next-generation sequencing.
Documented Applications
Recursive/hierarchical assembly into large (kb–100 kb) constructs, including assembly into vectors followed by host transformation and sequencing-based sequence verification.
Production of predefined sequence target nucleic acids using multiplex oligonucleotides containing internal payload sequences flanked by common primer recognition and restriction-enzyme recognition sites.
Interested in licensing this patent?