Degenerate oligonucleotide gene shuffling
View Patent ↗A method for gene shuffling to form a mutant or chimeric gene, the method comprising: (a) assigning one or more segments of one or more genes based on regions of encoded amino acid sequence; (b) amplifying the one or more assigned segments of the gene using primers specific for each segment; and (c) causing recombination of the one or more amplified segments to form a mutant or chimeric gene. An oligonucleotide primer suitable for use in gene shuffling, the primer having a non-degenerate core based on a segment or template of a gene to be amplified, and the core being flanked by both 5′ and 3′ degenerate ends.
1. A method for gene shuffling to form at least one mutant or chimeric gene, the method comprising:
amplifying at least a first segment of a first parental gene and a second segment of a second parental gene, wherein at least one first segment is amplified using a first primer comprising a non-degenerate core flanked by 5′ and 3′ degenerate ends, and wherein at least one second segment is amplified using a second primer comprising a non-degenerate core flanked by 5′ and 3′ degenerate ends, wherein the non-degenerate core of the first primer is complementary to the non-degenerate core of the second primer; and
causing recombination between at least one amplified first segment and at least one amplified second segment to form at least one mutant or chimeric gene.
2. The method according to claim 1 wherein the recombination forms two or more mutant or chimeric genes.
3. The method according to claim 1 or 2 wherein the parental genes belong to the same gene family encoding proteins having the same function.
4. The method according to claim 1 or 2 wherein the parental genes belong to different gene families encoding proteins with different functions.
5. The method according to claim 1 or 2 wherein the gene segments are selected based on regions of conserved amino acid sequence of the respective gene products encoded by the parental genes.
6. The method according to claim 1 or 2 wherein the amplification is by polymerase chain reaction (PCR).
7. The method according to claim 1 or 2 wherein the amplified segments are mixed in substantially equal ratios for the recombination.
8. The method according to claim 1 or 2 wherein at least one mutant or chimeric gene is produced by overlap extension of the combined amplified segments and multiple copies of the mutant or chimeric gene are produced by PCR amplification.
9. The method according to claim 1 or 2 wherein at least one mutant or chimeric gene is placed in suitable expression vector and the gene product is produced from the mutant or chimeric gene.
10. The method according to claim 1 or 2 wherein at least one mutant or chimeric gene encodes a protein selected from the group consisting of enzyme, growth factor, inhibitor, antibody, antigen, structural protein, transport protein, toxin, and combination thereof.
11. The method according to claim 1 or 2 wherein at least one mutant or chimeric gene is further mutated or altered.
12. The method according to claim 1 or 2 , wherein the non-degenerate cores flanked by both 5′ and 3′ degenerate ends of the primers act as a stabilizing clamp.
13. The method according to claim 1 or 2 wherein the 3′ degenerate ends of the primers give the primers template-binding specificity.
14. The method according to claim 1 or 2 wherein subsequent rounds of amplification are performed, wherein the non-degenerate cores of the primers act as a stabilizing clamp.
15. The method according to claim 1 or 2 wherein the amplified gene segments are subjected to overlap-extension amplification, wherein the portions of amplified gene segments derived from the 5′ degenerate ends of the primers allow for efficient binding and overlap-extension.
16. The method according to claim 1 or 2 wherein the non-degenerate cores of the primers are based upon the corresponding coding sequence of at least one parental gene.
17. The method according to claim 1 or 2 wherein the non-degenerate core of the primers has 11 nucleotides.
18. The method according to claim 1 or 2 wherein the non-degenerate core of the primers has from 5 to 16 nucleotides.
19. The method according to claim 1 or 2 wherein the non-degenerate core of the primers has 16 nucleotides.
20. The method according to claim 1 or 2 wherein the non-degenerate core of the primers has 5 nucleotides.
21. The method according to claim 1 or 2 wherein multiple mutant or chimeric genes are produced and wherein the multiple mutant or chimeric genes are further mutated or altered.