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 recombinant nucleic acid molecule comprising:
a) a nucleic acid sequence exhibiting 70% or greater sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 09, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 32, a complement thereof or a fragment of either; or
b) a nucleic acid sequence encoding a polypeptide exhibiting 80% or greater sequence identity to an amino acid 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;
wherein the nucleic acid sequence is operably linked to a heterologous nucleic acid.
2. A nucleic acid molecule according to claim 1 , wherein said nucleic acid sequence encodes a molecule having mismatch endonuclease activity.
3. A recombinant nucleic acid construct according to claim 2 , wherein said heterologous nucleic acid is a heterologous transcriptional control element.
4. A recombinant nucleic acid construct according to claim 2 , wherein said heterologous nucleic acid comprises a nucleic acid sequence encoding a polypeptide sequence.
5. A recombinant nucleic acid construct according to claim 4 , wherein said polypeptide sequence comprises a secretion signal or an epitope tag.
6. A recombinant host cell comprising a nucleic acid construct comprising a nucleic acid molecule according to claim 2 .
7. A recombinant host cell according to claim 6 , wherein said host cell is an insect cell, a mammalian cell, a microbial cell, or a plant cell.
8. An isolated polypeptide, wherein said polypeptide is expressed by a nucleic acid molecule comprising a nucleic acid sequence according to claim 1 .
9. An isolated polypeptide according to claim 8 , wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, amino acid residues 1 to 297 of SEQ ID NO: 11, amino acid residues 22 to 308 of SEQ ID NO: 11, SEQ ID NO: 17, amino acid residues 1 to 320 of SEQ ID NO: 17, and amino acid residues 22 to 331 of SEQ ID NO: 17.
10. A recombinant nucleic acid of claim 1 comprised on an expression construct.
11. The recombinant nucleic acid of claim 3 wherein the transcriptional control element is a promoter.
12. The recombinant nucleic acid molecule of claim 1 , comprising:
a) a nucleic acid sequence exhibiting 90% or greater sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 09, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 32, a complement thereof or a fragment of either; or
b) a nucleic acid sequence encoding a polypeptide exhibiting 90% or greater sequence identity to an amino acid 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.
13. The recombinant nucleic acid molecule of claim 12 comprising a nucleic acid sequence encoding a polypeptide exhibiting 90% or greater identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 16.
14. The recombinant nucleic acid molecule of claim 12 comprising a nucleic acid sequence encoding a polypeptide exhibiting 95% or greater identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 16.
15. A polypeptide sequence encoded by the nucleic acid molecule of claim 13 .
16. A polypeptide sequence encoded by the nucleic acid molecule of claim 1 .