Materials and methods for the synthesis of error-minimized nucleic acid molecules
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.
1. A composition comprising: (i) a molecule having a unidirectional mismatch endonuclease activity having at least 80% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and (ii) a molecule having unidirectional exonuclease activity of the same directionality as the unidirectional mismatch endonuclease activity of (i).
2. A composition according to claim 1 , wherein the molecule of (ii) is selected from the group consisting of exonuclease III, a DNA polymerase, and a combination of exonuclease III and a DNA polymerase.
3. A composition according to claim 2 , wherein the molecule of (ii) is selected from the group consisting of exonuclease III.
4. The composition of claim 1 , wherein the molecule having a unidirectional mismatch endonuclease activity has at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.
5. The composition according to claim 4 , wherein the molecule of (ii) is selected from the group consisting of exonuclease III, a DNA polymerase, and a combination of exonuclease III and a DNA polymerase.
6. The composition according to claim 4 wherein the molecule of (ii) is exonuclease III.
7. A kit comprising a composition according to claim 1 .
8. A kit comprising a composition according to claim 2 .
9. A kit comprising a composition according to claim 3 .
10. A kit comprising a composition according to claim 4 .
11. A kit comprising a composition according to claim 5 .
12. A kit comprising a composition according to claim 6 .