Materials and methods for the synthesis of error-minimized nucleic acid molecules

Inventors

Gibson, Daniel G • Caiazza, Nicky • Richardson, Toby H.

Assignees

Telesis Bio Inc

Abstract

The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches. Also provided are novel nucleic acid sequences encoding mismatch endonucleases, polypeptides encoded thereby, as well as nucleic acid constructs, transgenic cells, and various compositions thereof.

Core Innovation

The patent describes error correction for nucleic acids by denaturing and annealing to form mismatches in double-stranded nucleic acids. Nucleotide mismatches are identified and processed by a mismatch endonuclease that has unidirectional mismatch endonuclease activity, and the mismatch endonuclease cleaves at or near the mismatch site and leaves a mismatched terminal site or a near-terminal site on the nucleic acid fragment.

After cleavage, a unidirectional exonuclease having unidirectional exonuclease activity of the same directionality removes mismatched nucleotides from the mismatched terminal or near-terminal site. Remaining gaps are filled by a polymerase, and assemblies or amplifications are repeated to reduce mismatch frequency. The disclosed approach includes mismatch endonucleases and exonucleases operating with consistent directionality across endonuclease cleavage and exonuclease removal.

The document identifies mismatch endonucleases and exonucleases used in the error-correction process, including RES I and CEL I/CEL II, and exonuclease III with DNA polymerases having exonuclease/proofreading activity. The patent further discloses nucleic acid sequences encoding mismatch endonucleases with SEQ ID NOs, including recombinant constructs and host cells for producing chimeric endonucleases.

Claims Coverage

The claims cover one inventive combination and related packaging: a unidirectional mismatch endonuclease paired with a unidirectional exonuclease of the same directionality, with the mismatch endonuclease limited by SEQ ID NO identity thresholds, and kit variants containing the defined composition.

The claims are directed to compositions and kits that pair a unidirectional mismatch endonuclease, limited to specified SEQ ID NOs with an identity threshold of at least 80% or at least 90%, with a unidirectional exonuclease having the same directionality. Dependent refinements narrow the permissible exonuclease-type molecule, including exonuclease III alone or exonuclease III, a DNA polymerase, or their combination.

Stated Advantages

Documented Applications