Claims
- 1. A method for the recombination of nucleic acid templates of interest comprising:
a) providing at least one double-stranded first nucleic acid template and at least one second double-stranded nucleic acid template, wherein the 5′ region of the sense strand of the first nucleic acid template is of different sequence than the 5′ region of the sense strand of the second nucleic acid template and the 3′ region of the sense strand of the first nucleic acid template is of different sequence than the 3′ region of the sense strand of the second nucleic acid template; b) providing at least one forward primer that will only anneal to the 3′ region of the antisense strand of the first nucleic acid template; c) providing at least one reverse primer that will only anneal to the 3′ region of the sense strand of the second nucleic acid template; d) contacting the first and second nucleic acid templates of (a) with the primers of (b) and (c) in a replication composition whereby primer directed extension of the primers takes place; e) terminating extension of the primers of (d) after the addition of no more than about 1000 nucleotides; f) separating the extended forward and reverse primers from the first and second templates; g) re-annealing the extended forward and reverse primers with the first and second templates whereby the extended primers anneal to either the first or second template; and h) repeating steps (d)-(g) until at least one full-length extension product is generated comprising the recombination of the first and second double stranded templates.
- 2. A method for the recombination of nucleic acid templates of interest comprising:
a) providing at least one first antisense single-stranded nucleic acid template and at least one second sense single-stranded nucleic acid template; b) providing at least one forward primer that will only anneal to the 3′ region of the first nucleic acid template; c) providing at least one reverse primer that will only anneal to the 3′ region of the second nucleic acid template; d) contacting the first and second nucleic acid templates of (a) with the primers of (b) and (c) in a replication composition whereby primer directed extension of the primers takes place; e) terminating extension of the primers of (d) after the addition of no more than about 1000 nucleotides; f) separating the extended forward and reverse primers from the first and second templates; g) re-annealing the extended forward and reverse primers with the first and second templates whereby the extended primers anneal to either the first or second template; and h) repeating steps (d)-(g) until at least one full-length extension product is generated comprising the recombination of the first and second templates.
- 3. A method according to claim 1 wherein said first and second templates have at least 50% identity to each other.
- 4. A method according to claim 1 wherein the first and second templates have at least 70% identity to each other.
- 5. A method according to claim 1 wherein the first and second templates have at least 90% identity to each other.
- 6. A method according to either of claims 1 or 2 wherein the length of the templates is from about 0.03 kB to about 20 kB.
- 7. A method according to either of claims 1 or 2 wherein the primer extension is terminated after the addition of no more than about 500 nucleotides.
- 8. A method according to either of claims 1 or 2 wherein the primer extension is terminated after the addition of no more than about 300 nucleotides
- 9. A method according to either of claims 1 or 2 wherein the primer extension is terminated after the addition of no more than about 20-50 nucleotides.
- 10. A method according to either of claims 1 or 2 wherein the primer extension is terminated by a process step selected from the group consisting of: altering the annealing temperature, altering the pH, and adding a polymerase inhibitor.
- 11. A method according to either of claims 1 or 2 wherein the 3′ region of either the first or second template is from about 6 to about 200 bases in length.
- 12. A method according to claim 1 wherein the first template is an antisense single-stranded template, and the forward primer anneals only to the 3′ region of the first template.
- 13. A method according claim 1 wherein the second template is a sense single-stranded template and the reverse primer anneals only to the 3′ region of the second template.
- 14. A method according to either of claims 1 or 2 wherein the 3′ region of the antisense strand is an engineered flanking region.
- 15. A method according to either of claims 1 or 2 wherein the 3′ region of the sense strand is an engineered flanking region.
- 16. A method according to either of claims 1 or 2 wherein the full-length extension product is optionally purified from the first and second templates.
- 17. A method according to either of claims 1 or 2 wherein the full-length extension product is optionally amplified.
- 18. A method according to either of claims 1 or 2 wherein the forward and reverse primers anneal under optimal conditions.
- 19. A method according to either of claims 1 or 2, wherein the forward and reverse primers are from about 6 to about 200 nucleotides in length.
- 20. A method according to either of claims 1 or 2 wherein the primer directed extension is accomplished using a technique selected from the group consisting of polymerase chain reaction, ligase chain reaction and strand displacement amplification.
- 21. A method according to either of claims 1 or 2 wherein the first and second templates are of unknown sequence.
- 22. A method according to claim 1 wherein the first and second nucleic acid templates are contacted with an enzyme under conditions whereby the template is nicked.
- 23. A method according to claim 22 wherein the nucleic acid templates are RNA and the enzyme is an RNase.
- 24. A method according to claim 22 wherein the nucleic acid templates are DNA and the enzyme is selected from the group consisting of DNAse, DNA gyrase and recA.
- 25. A method according to either of claims 1 or 2 wherein the first and second nucleic acid templates are linear.
- 26. A method according to claim 1 wherein either one or both of the first and second nucleic acid templates are comprised on a plasmid.
- 27. A method according to either of claims 1 or 2 wherein either one or both of the first and second nucleic acid template are allelic variants.
- 28. A method according to either of claims 1 or 2 wherein the full-length extension product of (h) comprising the recombined first and second templates is further subjected to steps (c)-(g).
- 29. A method according to either of claims 1 or 2 wherein the forward and reverse primers are of different sequence.
- 30. A method according to either of claims 1 or 2 wherein the forward and reverse primers have the same sequence.
- 31. A method according to either of claims 1 or 2 wherein:
a) said 3′ region of the antisense strand of the first template comprises a first unique restriction site, and wherein said 3′ region of the sense strand of the second template comprises a second unique restriction site wherein said first unique restriction site is not present in the second template and wherein the second unique restriction site is not present in said first template; and b) wherein the full-length extension product comprises the first and second unique restriction sites.
- 32. A method according to claim 31 further comprising:
a) restricting the full-length extension product with either one or both of a first restriction enzyme which cuts at said first unique restriction site and a second restriction enzyme which cuts at said second unique restriction site to generate a restricted full-length extension product; and b) ligating said restricted full-length extension product into a vector.
- 33. A method for the generation of a recombined polypeptide having altered properties comprising:
a) providing at least one double-stranded first nucleic acid template and at least one second double-stranded nucleic acid template, wherein the 5′ region of the sense strand of the first nucleic acid template is of different sequence than the 5′ region of the sense strand of the second nucleic acid template and the 3′ region of the sense strand of the first nucleic acid template is of different sequence than the 3′ region of the sense strand of the second nucleic acid template; b) providing at least one forward primer that will only anneal to the 3′ region of the antisense strand of the first nucleic acid template; c) providing at least one reverse primer that will only anneal to the 3′ region of the sense strand of the second nucleic acid template; d) contacting the first and second nucleic acid templates of (a) with the primers of (b) and (c) in a replication composition whereby primer directed extension of the primers takes place; e) terminating extension of the primers of (d) after the addition of no more than about 1000 nucleotides; f) separating the extended forward and reverse primers from the first and second templates; g) re-annealing the extended forward and reverse primers with the first and second templates whereby the extended primers anneal to either the first or second template; h) repeating steps (d)-(g) until at least one full-length extension product is generated comprising the recombination of the first and second templates; i) expressing the full-length extension product of (h) to generate a recombined polypeptide; and j) screening the recombined polypeptide of (i) for altered properties as compared with the polypeptide expressed from either the first or second double-stranded nucleic acid template.
- 34. A method for the generation of a recombined polypeptide having altered properties comprising:
a) providing at least one first antisense single-stranded nucleic acid template and at least one second sense single-stranded nucleic acid template; b) providing at least one forward primer that will only anneal to the 3′ region of the first nucleic acid template; c) providing at least one reverse primer that will only anneal to the 3′ region of the second nucleic acid template; d) contacting the first and second nucleic acid templates of (a) with the primers of (b) and (c) in a replication composition whereby primer directed extension of the primers takes place; e) terminating extension of the primers of (d) after the addition of no more than about 1000 nucleotides; f) separating the extended forward and reverse primers from the first and second templates; g) re-annealing the extended forward and reverse primers with the first and second templates whereby the extended primers anneal to either the first or second template; h) repeating steps (d)-(g) until at least one full-length extension product is generated comprising the recombination of the first and second templates; i) expressing the full-length extension product of (h) to generate a recombined polypeptide; and j) screening the recombined polypeptide of (i) for altered properties as compared with the polypeptide expressed from either the first or second double-stranded nucleic acid template.
- 35. A method according to claim 33 wherein said first and second templates have at least 50% identity to each other.
- 36. A method according to claim 33 wherein the first and second templates have at least 70% identity to each other.
- 37. A method according to claim 33 wherein the first and second templates have at least 90% identity to each other.
- 38. A method according to either of claims 33 or 34 wherein the primer extension is terminated after the addition of no more than about 500 nucleotides.
- 39. A method according to either of claims 33 or 34 wherein the primer extension is terminated after the addition of no more than about 300 nucleotides.
- 40. A method according to either of claims 33 or 34 wherein the primer extension is terminated after the addition of no more than about 20-50 nucleotides.
- 41. A method according to either of claims 33 or 34 wherein the primer extension is terminated by a process step selected from the group consisting of: altering the annealing temperature, altering the pH, and adding a polymerase inhibitor.
- 42. A method according to either of claims 33 or 34 wherein the 3′ region of either the first or second template is from about 6 to about 200 bases in length.
- 43. A method according to either of claims 33 or 34 wherein the 3′ region of the antisense strand is an engineered flanking region.
- 44. A method according to either of claims 33 or 34 wherein the 3′ region of the sense strand is an engineered flanking region.
- 45. A method according to either of claims 33 or 34 wherein the forward and reverse primers anneal under stringent conditions.
- 46. A method according to either of claims 33 or 34 wherein the forward and reverse primers are about 6-200 nucleotides in length.
- 47. A method according to either of claims 33 or 34 wherein the primer directed extension is accomplished using a technique selected from the group consisting of polymerase chain reaction, ligase chain reaction and strand displacement amplification.
- 48. A method according to either of claims 33 or 34 wherein the recombined polypeptide is an enzyme.
- 49. A method according to claim 48 wherein the enzyme is selected from the group consisting of: a protease, esterase, pectinase, xylanase, amylase, cellulase, levanase, lipase, RNase, nucleotidase, transfructosylase, lactase, desaturases, dehydrogenase, oxidases, transferases, isomerases, dehydratases, desulfurases, hydratases, phosphatases, kinases, glucose isomerase phosphatase, phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, o-methyltransferase, cinnamate 4-hydroxylase, 4-coumarate-CoA ligase, cinnamoyl CoA reductase, and acetolactate synthase.
- 50. A method according to claim 49 wherein the enzyme is glycerol dehydratase.
- 51. A method according to claim 48 wherein the properties of the enzyme are altered with respect to properties selected from the group consisting of: enzyme activity, substrate specificity, stability against inhibitors, thermal stability, protease stability, solvent stability, detergent stability, and folding properties.
- 52. A method according to either of claims 33 or 34 wherein the recombined polypeptide is not an enzyme.
- 53. A method according to claim 52 wherein the recombined polypeptide is selected from the group consisting of cytokines, glycoproteins, growth factors, biotin binding proteins, immunoglobin binding proteins, structural proteins, viral proteins, envelope proteins and microbial antigens.
- 54. A method according to claim 52 wherein the properties of the polypeptide are altered with respect to properties selected from the group consisting of: thermal stability, protease stability, solvent stability, detergent stability, folding properties, structural properties.
- 55. A polypeptide having altered properties made by the method of either of claims 33 or 34.
- 56. A polypeptide according to claim 55 having the amino acid sequence selected from the group consisting of SEQ ID NO:16, 18, 20, 22, and 24.
- 57. A polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:12 and 14.
Parent Case Info
[0001] This application claims the benefit of U.S. Provisional Application No. 60/360,279, filed Feb. 26, 2002.
Provisional Applications (1)
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Number |
Date |
Country |
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60360279 |
Feb 2002 |
US |