Claims
- 1. A method of introducing a change in a target DNA sequence to form a different, desired sequence in a recombination/repair competent prokaryotic cell comprising:
(A) transforming the prokaryotic cell with a duplex mutational vector comprising:
(i) a first oligonucleobase strand of at least 12 linked nucleobases and not more than 75 linked nucleobases, that has a first and a second terminal nucleobase; (ii) a second oligonucleobase strand having a 3′ most and a 5′ most nucleobase and having a number of nucleobases equal to the first oligonucleobase strand, which second oligonucleobase strand is optionally divided into a first chain and a second chain; and (iii) a single 3′ end nucleobase and a single 5′ end nucleobase; in which
(a) the 3′ most and 5′ most nucleobases of the second oligonucleobase strand are Watson-Crick base paired to the first terminal and the second terminal nucleobase of the first oligonucleobase strand, respectively, (b) said 3′ most nucleobase and said second terminal nucleobase are protected from 3′ exonuclease attack; and (c) the second oligonucleobase strand contains at least two non-overlapping regions of at least 5 contiguous nucleobases that are Watson-Crick base paired to nucleobases of the first oligonucleobase strand, provided that for not more than two contiguous nucleobases of a strand is a deoxy-type nucleobase Watson-Crick paired with a ribo-type nucleobase or a ribo-type nucleobase Watson-Crick paired with a deoxy-type nucleobase; and wherein the sequence of at least one oligonucleobase strand comprises the different, desired sequence; and (B) detecting the presence of the different desired sequence from or in the prokaryotic cell or progeny thereof.
- 2. The method of claim 1, further comprising the step of transiently producing functional RecA in the prokaryotic cell.
- 3. The method of claim 1, wherein the prokaryotic cell contains a RecA 35 gene which is operably linked to an inducible promoter.
- 4. The method of claim 1, wherein the DNA target sequence is a sequence of a bacterial artificial chromosome or a plasmid.
- 5. The method of claim 1, wherein the first oligonucleobase strand comprises not more than 60 nucleobases.
- 6. The method of claim 1, wherein the first oligonucleobase strand and the second oligonucleobase strand contain not more than 2 contiguous ribo-type nucleobases.
- 7. The method of claim 1, wherein a terminal nucleobase is linked by a nuclease resistant linker to the 3′ most or the 5′ most nucleobase, whereby a nucleobase is protected from 3′ exonuclease attack.
- 8. The method of claim 7, wherein the nuclease resistant linker comprises a moiety selected from the group consisting of 2′-methoxy-uridine, 2′-allyloxy-uridine, 2′-fluoro-uridine, 2′-methoxy-thymidine, 2′-allyloxy-thymidine, 2′-fluoro-thymidine, polyethylene glycol and trans-4,4′-stilbenecarboxamide.
- 9. The method of claim 7, wherein the first terminal and the second terminal nucleobase are linked to the 3′ most and 5′ most nucleobase by nuclease resistant linkers.
- 10. The method of claim 9, wherein each nuclease resistant linker comprises a moiety selected from the group consisting of 2′-methoxy-uridine, 2′-allyloxy-uridine, 2′-fluoro-uridine, 2′-methoxy-thymidine, 2′-allyloxy-thymidine, 2′-fluoro-thymidine, polyethylene glycol and trans-4,4′-stilbenecarboxamide.
- 11. The method of claim 1, wherein the second oligonucleobase strand or the first chain contain no RNA-type nucleobases.
- 12. The method of claim 1, wherein the 3′ end nucleobase is protected from 3′ exonuclease activity by a blocking group.
- 13. A method of introducing a change in a target DNA sequence to form a different, desired sequence in a recombination/repair competent prokaryotic cell comprising:
(A) transforming the prokaryotic cell with a heteroduplex mutational vector comprising:
(i) a first oligonucleobase strand of at least 12 linked nucleobases and not more than 75 linked nucleobases, which first oligonucleobase strand has a first and a second terminal nucleobase; (ii) a second oligonucleobase strand having a 3′ most and a 5′ most nucleobase and having a number of nucleobases equal to the first oligonucleobase strand, which second oligonucleobase strand is optionally divided into a first chain and a second chain; and (iii) a single 3′ end nucleobase and a single 5′ end nucleobase; in which
(a) the 3′ most and 5′ most nucleobases of the second oligonucleobase strand are Watson-Crick base paired to the first terminal and the second terminal nucleobase of the first oligonucleobase strand, respectively, (b) the 3′ most nucleobase of the second oligonucleobase strand and the second terminal nucleobase of the first oligonucleobase strand are protected from 3′ exonuclease attack, and (c) the second oligonucleobase strand contains at least two non-overlapping regions of at least 5 contiguous nucleobases that are Watson-Crick base paired to nucleobases of the first oligonucleobase strand, provided that at least one nucleobase of the first oligonucleobase strand is paired with a non-complementary base of the second oligonucleobase strand; and wherein the sequence of at least one oligonucleobase strand comprises the different, desired sequence; and (B) detecting the presence of the different desired sequence from or in the prokaryotic cell or progeny thereof.
- 14. The method of claim 13, further comprising the step of transiently producing functional RecA in the prokaryotic cell.
- 15. The method of claim 13, wherein the prokaryotic cell contains a RecA gene which is operably linked to an inducible promoter.
- 16. The method of claim 13, wherein the DNA target sequence is a sequence of a bacterial artificial chromosome or a plasmid.
- 17. The method of claim 13, wherein the first oligonucleobase strand comprises not more than 50 nucleobases.
- 18. The method of claim 13, wherein not more than 3 nucleobases of the first oligonucleobase strand are paired with non-complementary nucleobases of the second oligonucleobase strand.
- 19. The method of claim 18, wherein the non-complementary nucleobases are deoxyribo-type nucleobases.
- 20. The method of claim 18, wherein not more than one nucleobase of the first oligonucleobase strand is paired with a non-complementary nucleobase of the second oligonucleobase strand.
- 21. The method of claim 18, wherein the second oligonucleobase strand is comprised of a first chain and a second chain and the first chain contains a mismatched nucleobase.
- 22. The method of claim 21, wherein the first chain contains no ribo-type nucleobases.
- 23. The method of claim 21, wherein the first chain contains the 5′ end nucleobase.
- 24. The method of claim 18, wherein the first oligonucleobase strand contains at least 10 ribo-type oligonucleobases.
- 25. The method of claim 24, wherein the first oligonucleobase strand contains no deoxyribo-type oligonucleobases.
- 26. The method of claim 13, wherein 5 most nucleobase is linked by a nuclease resistant linker to the second terminal nucleobase, whereby said second terminal nucleobase is protected from 3′ exonuclease attack.
- 27. The method of claim 13, wherein the 3′ most nucleobase is linked by a nuclease resistant linker to the first terminal nucleobase, whereby said 3′ most nucleobase is protected from 3′ exonuclease attack.
- 28. The method of claim 27, wherein the 5′ most nucleobase is linked by a nuclease resistant linker to the second terminal nucleobase, whereby said second terminal nucleobase is protected from 3′ exonuclease attack.
- 29. The method of claim 27, wherein the linker comprises a moiety selected from the group consisting of 2′-methoxy-uridine, 2′-allyloxy-uridine, 2′-fluoro-uridine, 2′-methoxy-thymidine, 2′-allyloxy-thymidine, 2′-fluoro-thymidine, polyethylene glycol and trans-4,4′-stilbenecarboxamide.
- 30. The method of claim 13, wherein the first chain contains no ribo-type nucleobases.
- 31. The method of claim 13, wherein the 3′ end nucleobase is protected from 3′ exonuclease activity by a blocking group.
- 32. A method of introducing a change in a target DNA sequence to form a different, desired sequence in a recombination/repair competent prokaryotic cell comprising:
(A) transforming the prokaryotic cell with a chimeric duplex mutational vector having no intervening segment comprising:
(i) a first oligonucleobase strand of at least 12 linked nucleobases and not more than 75 linked nucleobases, which first oligonucleobase strand has a first terminal and a second terminal nucleobase; (ii) a second oligonucleobase strand having a 3′ most nucleobase and a 5′ most nucleobase and having a number of nucleobases equal to the first strand, which second oligonucleobase strand is divided into a first chain and a second chain; and (iii) a 3′ end nucleobase and a 5′ end nucleobase; in which
(a) the 3′ most and 5′ most nucleobases of the second oligonucleobase strand are Watson-Crick base paired to the first terminal and the second terminal nucleobase of the first oligonucleobase strand, respectively, (b) the nucleobases of the first chain are deoxy-type nucleobases and nucleobases of the first oligonucleobase strand paired therewith are nuclease resistant ribo-type nucleobases, (c) the second oligonucleobase strand contains at least two non-overlapping regions of at least 5 contiguous nucleobases that are Watson-Crick base paired to nucleobases of the first oligonucleobase strand, provided that the first oligonucleobase strand does not contain an intervening segment of deoxy-type nucleobases between two segments of ribo-type nucleobases; and wherein the sequence of at least one oligonucleobase strand comprises the different, desired sequence; and (B) detecting the presence of the different desired sequence from or in the prokaryotic cell or progeny thereof.
- 33. The method of claim 32, further comprising the step of transiently producing functional RecA in the prokaryotic cell.
- 34. The method of claim 32, wherein the prokaryotic cell contains a RecA gene which is operably linked to an inducible promoter.
- 35. The method of claim 32, wherein the DNA target sequence is a sequence of a bacterial artificial chromosome or a plasmid.
- 36. The method of claim 32, wherein the first chain comprises the 5′ end nucleobase.
- 37. The method of claim 36, wherein not more than one nucleobase of the first chain is paired with a non-complementary nucleobase of the first oligonucleobase strand.
- 38. The method of claim 32, wherein the 3′ most nucleobase and the first terminal nucleobase are linked by a linker comprising a moiety selected from the group consisting of 2′-methoxy-uridine, 2′-allyloxy-uridine, 2′-fluoro-uridine, 2′-methoxy-thymidine, 2′-allyloxy-thymidine, 2′-fluoro-thymidine, polyethylene glycol and trans-4,4′-stilbenecarboxamide.
- 39. The method of claim 32, wherein the 5′ most nucleobase and the second terminal nucleobase are linked by a linker comprising a moiety selected from the group consisting of 2′-methoxy-uridine, 2′-allyloxy-uridine, 2′-fluoro-uridine, 2′-methoxy-thymidine, 2′-allyloxy-thymidine, 2′-fluoro-thymidine, polyethylene glycol and trans-4,4′-stilbenecarboxamide.
- 40. A method of introducing a change in a target DNA sequence to form a different, desired sequence in a recombination/repair competent prokaryotic cell comprising:
(A) transforming the prokaryotic cell with an overhang containing chimeric duplex mutational vector having no intervening segment comprising:
(i) an oligonucleobase strand of at least 12 linked nucleobases and not more than 75 linked nucleobases, which oligonucleobase strand has a first terminal and a second terminal nucleobase; and (ii) an oligonucleobase chain having a 3′ most nucleobase and a 5′ end nucleobase; and (iii) a 3′ overhang attached to the second terminal nucleobase; in which
(a) the 3′ most and 5′ end nucleobases of the oligonucleobase chain are Watson-Crick base paired to the first terminal and the second terminal nucleobase of the oligonucleobase strand, respectively, (b) the nucleobases of the oligonucleobase chain are deoxy-type nucleobases and nucleobases of the oligonucleobase strand paired therewith are nuclease resistant ribo-type nucleobases; and (c) the oligonucleobase chain contains at least two non-overlapping regions of at least 5 contiguous nucleobases that are Watson-Crick base paired to nucleobases of the oligonucleobase strand; and (B) detecting the presence of the different desired sequence from or in the prokaryotic cell or progeny thereof.
- 41. The method of claim 40, further comprising the step of transiently producing functional RecA in the prokaryotic cell.
- 42. The method of claim 40, wherein the prokaryotic cell contains a RecA gene which is operably linked to an inducible promoter.
- 43. The method of claim 40, wherein the DNA target sequence is a sequence of a bacterial artificial chromosome or a plasmid.
- 44. The method of claim 40, wherein at least one nucleobase of the oligonucleobase chain is paired with a non-complementary nucleobase of the oligonucleobase strand.
- 45. The method of claim 44, wherein not more than one nucleobase of the oligonucleobase chain is paired with a non-complementary nucleobase of the oligonucleobase strand.
- 46. The method of claim 40, wherein the 3′ most nucleobase and the first terminal nucleobase are linked by a linker comprising a moiety selected from the group consisting of 2′-methoxy-uridine, 2′-allyloxy-uridine, 2′-fluoro-uridine, 2′-methoxy-thymidine, 2′-allyloxy-thymidine, 2′-fluoro-thymidine, polyethylene glycol and trans-4,4′-stilbenecarboxamide.
- 47. The method of claim 1, 13, 32 or 40, wherein the vector is transformed into the prokaryotic cell by electroporation.
- 48. The method of claim 1, 13, 32 or 40, wherein the vector is preincubated at room temperature with spermidine prior to mixing with the prokaryotic cell.
- 49. The method of claim 48, wherein the amount of the vector is 1-2 mg/ml and the amount of spermidine is 3-200 nM.
- 50. The method of claim 1, 13, 32 or 40, wherein the vector is preincubated at room temperature with spermine prior to mixing with the prokaryotic cell.
- 51. A nucleic acid comprising a nucleotide sequence encoding RecA operable linker to an inducible promoter.
- 52. A prokaryotic cell comprising the nucleic acid of claim 51 and the vector of claim 1, 13, 32 or 40.
Parent Case Info
[0001] This is a division of copending application Ser. No. 09/429,292, filed Oct. 28, 1999, which is a continuation of application Ser. No. 09/078,063, filed May 12, 1998.
Divisions (1)
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Number |
Date |
Country |
Parent |
09429292 |
Oct 1999 |
US |
Child |
09825124 |
Apr 2001 |
US |
Continuations (1)
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Number |
Date |
Country |
Parent |
09078063 |
May 1998 |
US |
Child |
09429292 |
Oct 1999 |
US |