Methods for nucleic acid assembly and high throughput sequencing
Inventors
Hudson, Michael E. • Kung, Li-yun A. • SCHINDLER, DANIEL • Archer, Stephen • Saaem, Ishtiaq
Assignees
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Publication Number
US-11072789-B2
Publication Date
2021-07-27
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 provides a method of producing a nucleic acid having a predefined sequence by assembling internal sequences derived from a first pool of double-stranded oligonucleotides. Each double-stranded oligonucleotide includes 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 5′ flanking sequences and 3′ flanking sequences that include a common primer recognition site and a Type IIS restriction endonuclease recognition site.
The method exposes the first pool of double-stranded oligonucleotides to a ligase and a Type IIS restriction enzyme under conditions suitable to promote concurrent restriction enzyme digestion and ligation. This concurrent restriction digestion and ligation generates the first target nucleic acid, which comprises an internal sequence identical to a portion of a final target nucleic acid and includes 5′ and 3′ flanking sequences, each containing a restriction enzyme recognition site.
Further aspects include reducing errors in the assembled nucleic acids using mismatch-binding agents such as MutS family mismatch recognizing agents, confirming assembled products by sequencing and/or isolating correctly assembled products, and supporting hierarchical or recursive assembly into larger constructs by leveraging reuse of restriction sites while removing common flanking sequence elements.
Claims Coverage
The independent claim is directed to producing a nucleic acid with a predefined sequence using an oligonucleotide pool that combines overlapping internal payload sequences with flanking primer recognition and Type IIS restriction endonuclease recognition sites, followed by concurrent Type IIS digestion and ligase ligation to generate a target nucleic acid. Dependent claims refine the method for forming multi-part final constructs, producing the oligonucleotide pool via amplification using common primer recognition sites, removing errors using mismatch-binding agents including MutS, and confirming sequence accuracy by sequencing and/or isolating.
Concurrent Type IIS digestion and ligation to assemble a predefined sequence
Providing a first pool of double-stranded oligonucleotides with internal overlapping regions and 5′ and 3′ flanking sequences that each include a common primer recognition site and a Type IIS restriction endonuclease recognition site arranged so that Type IIS restriction enzyme digestion will remove the flanking sequences and expose the internal sequence; and exposing the first pool to a ligase and a Type IIS restriction enzyme under conditions suitable to promote concurrent restriction enzyme digestion and ligation, thereby generating the first target nucleic acid with internal sequence identical to a portion of a final target nucleic acid.
Common primer recognition sites and Type IIS restriction sites flanking internal payload sequences
Each double-stranded oligonucleotide comprises internal sequences identical to different portions of a first target nucleic acid with an overlapping region in common with another double-stranded oligonucleotide in the first pool, and 5′ flanking sequences and 3′ flanking sequences each comprise a common primer recognition site and a Type IIS restriction endonuclease recognition site oriented so that Type IIS restriction enzyme digestion will remove the flanking sequences and expose the internal sequence.
Multi-part final target construction from first and second target nucleic acids
Producing a final target nucleic acid from a mixture of a first target nucleic acid and a second target nucleic acid, wherein the second target nucleic acid comprises an internal sequence that differs from the internal sequence of the first target nucleic acid and is identical to a portion of the final target nucleic acid, and each of the first and second target nucleic acids includes 5′ and 3′ flanking sequences comprising a restriction enzyme recognition site, such that exposure to a ligase and a restriction enzyme recognizes the restriction endonuclease recognition site to generate the final target nucleic acid containing both internal sequences.
Producing the double-stranded oligonucleotide pool by amplification with common primer recognition sites
Producing the double-stranded oligonucleotides in the first pool by amplifying a plurality of single-stranded oligonucleotides, each representing one strand of a double-stranded oligonucleotide, using common primer recognition sites.
Error removal using a mismatch binding agent
Removing errors by contacting amplified oligonucleotides with a mismatch binding agent.
Using MutS as the mismatch binding agent
Performing the error removal using MutS as the mismatch binding agent.
Confirming sequence accuracy by sequencing and/or isolating
Confirming the sequence accuracy of the final target nucleic acid by sequencing and/or isolating it.
Across the independent and dependent claims, the core coverage centers on assembling an internal-sequence-defined nucleic acid using a pool of overlapping double-stranded oligonucleotides with common primer recognition sites and oriented Type IIS restriction endonuclease recognition sites, then generating the target via concurrent Type IIS digestion and ligase ligation. Dependent claims cover multi-part construction using first and second target nucleic acids, pool generation by amplification using common primer recognition sites, error removal using mismatch binding agents including MutS, and confirming sequence accuracy by sequencing and/or isolating.
Stated Advantages
Higher yield
Lower cost/time
Enabling reuse of restriction sites for hierarchical assembly
Scalable throughput for nucleic acid libraries
Documented Applications
Production of nucleic acid constructs including vector entry and long target nucleic acid constructs up to 10 kb to 100 kb scale as part of hierarchical/concurrent processing for recursive assembly into larger constructs.
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