Materials and methods for the synthesis of error-minimized nucleic acid molecules
Inventors
Gibson, Daniel G. • Caiazza, Nicky • Richardson, Toby H.
Assignees
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Publication Number
US-9771576-B2
Publication Date
2017-09-26
Expiration Date
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 described invention is an error-correction method for nucleic acid molecules, in particular double-stranded nucleic acid molecules with at least one nucleotide mismatch relative to a desired sequence. The approach amplifies a desired double-stranded nucleic acid sequence, then obtains a first plurality that includes both nucleic acid molecules of the desired sequence and nucleic acid molecules having mismatches.
The method subsequently fragments the mismatched double-stranded molecules using at least one molecule having unidirectional mismatch endonuclease activity. After fragmentation, the method removes the nucleotide mismatch by reacting the fragmented double-stranded nucleic acid molecules with at least one molecule having unidirectional exonuclease activity of the same directionality as the unidirectional mismatch endonuclease activity, to provide a fragmented error-free double-stranded nucleic acid molecule of the desired sequence.
The fragmented error-free molecules are then assembled to produce a second plurality of double-stranded nucleic acid molecules. The second plurality is characterized by a higher proportion of nucleic acid molecules having the desired sequence and a decreased frequency of nucleotide mismatches compared to the first plurality.
Claims Coverage
The partial claim set provides one independent claim covering a sequential error-correction workflow with unidirectional mismatch endonuclease fragmentation, same-direction unidirectional exonuclease mismatch removal, and assembly to increase the desired-sequence proportion while decreasing mismatch frequency.
Sequential error correction via unidirectional mismatch endonuclease and same-direction unidirectional exonuclease
A method for error correction of nucleic acid molecules with steps carried out in sequential order as listed: amplifying a desired double-stranded sequence; obtaining a first plurality including desired-sequence and mismatch-containing molecules; fragmenting the first plurality using at least one molecule having unidirectional mismatch endonuclease activity; removing the nucleotide mismatch by reacting fragmented molecules with at least one molecule having unidirectional exonuclease activity of the same directionality as the mismatch endonuclease activity; and assembling a second plurality whose desired-sequence proportion is higher and mismatch frequency is decreased compared to the first plurality.
The independent claim centers on a sequential workflow that couples unidirectional mismatch endonuclease fragmentation with same-direction unidirectional exonuclease mismatch removal, followed by assembly to increase desired-sequence recovery while reducing nucleotide mismatch frequency.
Stated Advantages
Provides a second plurality with a higher proportion of nucleic acid molecules having the desired sequence.
Decreases the frequency of nucleotide mismatches in the second plurality compared to the first plurality.
Documented Applications
Error correction of synthetic HA and NA genes with measured improvements in correct clone fractions/error rates.
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