Claims for Patent: 6,248,569

Summary for Patent: 6,248,569

Title:	Method for introducing unidirectional nested deletions
Abstract:	Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment in the context of a cloning vector which contains an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment. Also disclosed is a method for producing single-stranded DNA probes utilizing the same cloning vector. An optimal vector, PZIP is described. Methods for introducing unidirectional deletions into a terminal location of a cloned DNA sequence which is inserted into the vector of the present invention are also disclosed. These methods are useful for introducing deletions into either or both ends of a cloned DNA insert, for high throughput sequencing of any DNA of interest.
Inventor(s):	Dunn; John J. (Bellport, NY), Quesada; Mark A. (Horseheads, NY), Randesi; Matthew (New York, NY)
Assignee:	Brookhaven Science Associates (Upton, NY)
Application Number:	09/342,353
Patent Claims:	1. A DNA cloning vector for generating unidirectional deletions in a cloned insert, comprising: a) a cloning region for insertion of a DNA sequence having a first and second terminus; b) recognition sequences for a first and a second Exo III resistance cutter adjacently located at discrete positions on a first side of the cloning region, the first Exo III resistance cutter recognition sequence being located between the cloning region and the second Exo III resistance cutter recognition sequence; c) recognition sequences for a set of Exo III sensitizing cutters located between the first Exo III resistance cutter recognition sequence and the cloning region; and d) an f1 endonuclease recognition sequence adjacently located on the second side of the cloning region. 2. The DNA cloning vector of claim 1 wherein the first and second Exo III resistance cutters are intron encoded endonucleases. 3. The DNA cloning vector of claim 2 wherein the intron encoded endonucleases are selected from the group consisting of I-CeuI, PI-PspI, and I-SceI. 4. The DNA cloning vector of claim 1 wherein one or more of the set of Exo III sensitizing cutters is an endonuclease which has an 8-base recognition sequence. 5. The DNA cloning vector of claim 4 wherein the endonuclease with an 8-base recognition sequence is selected from the group consisting of SrfI, PmeI, and SwaI. 6. The DNA cloning vector of claim 1 which further comprises recognition sequences for a second set of Exo III sensitizing cutters located between the f1 endonuclease recognition sequence and the cloning region. 7. The DNA cloning vector of claim 6 wherein one or more of the second set of Exo III sensitizing cutters is an endonuclease which has an 8-base recognition sequence. 8. The DNA cloning vector of claim 7 wherein the endonuclease with a 8-base recognition sequence of the second set of Exo III sensitizing cutters is selected from the group consisting of NotI, PacI, and AscI. 9. The DNA cloning vector of claim 6 further comprising a recognition sequence for a third Exo III resistance cutter located between the f1 endonuclease recognition sequence and the recognition sequences for the second set of Exo III sensitizing cutters. 10. The DNA cloning vector of claim 1 wherein the cloning region is a multiple cloning region which contains unique restriction sites for shotgun and directional cloning. 11. The DNA cloning vector of claim 10 wherein the cloning region and flanking recognition sequences for Exo III resistance cutters, Exo III sensitizing cutters and f1 comprise the sequence listed in SEQ ID NO: 3. 12. The DNA cloning vector of claim 1 which further comprises one or more sequencing primer binding sites. 13. The DNA cloning vector of claim 1 which is a single-copy vector for generating normalized full-length cDNA libraries. 14. The DNA cloning vector of claim 13 wherein the vector contains a P1 lytic replicon which is under the control of an inducible promoter. 15. The DNA cloning vector of claim 14 wherein the inducible promoter is a Lac promoter which is inducible with IPTG. 16. The DNA cloning vector of claim 13 which is capable of stably propagating a DNA sequence of up to 15 kb in length which is inserted at the cloning region. 17. The DNA cloning vector of claim 16 which is the pZIP vector, the sequence of which is listed in SEQ ID NO: 2. 18. A method for introducing a unidirectional deletion into both termini of a cloned DNA sequence, comprising the steps: a) providing a recombinant DNA construct comprising a cloning vector for generating nested deletions in a cloned insert, the cloning vector comprising: i) a cloning vector for insertion of a cloned DNA sequence, the cloning region having a first and second side; ii) recognition sequences for a first and a second Exo III resistance cutter adjacently located at discrete positions on a first side of the cloning region, the first Exo III resistance cutter recognition sequence being located between the cloning region and the second Exo III resistance cutter recognition sequence; iii) recognition sequences for a set of Exo III sensitizing cutters located between the first Exo III resistance cutter recognition sequence and the cloning region; and iv) an f1 endonuclease recognition sequence adjacently located on the second side of the cloning region, the recombinant DNA construct further comprising a cloned DNA sequence which is inserted into the cloning region of the cloning vector such that the cloned DNA sequence has a first terminus located directly adjacent the first side of the cloning region, and a second terminus located directly adjacent the second side of the cloning region; b) generating a unidirectional deletion in the first terminus of the cloned DNA sequence by: i) linearizing the recombinant DNA construct at a site in the vector located directly adjacent the first terminus of the cloned DNA sequence by digesting the recombinant DNA construct of step a) with endonucleases which generate one Exo III sensitive end, corresponding to the end directly adjacent the first terminus of the cloned DNA sequence, and one Exo III insensitive end; ii) digesting the linearized recombinant DNA construct generated in step b)i) with E. coli Exonuclease III, thereby digesting 3' to 5' one strand of the Exo III sensitive end, thereby generating a linearized recombinant DNA construct having a single-stranded deletion in the First terminus of the cloned DNA sequence; iii) contacting the linearized recombinant DNA cost ruct generated in step b)ii) with a single-strand-specific endonuclease, thereby generating a DNA molecule containing a double-stranded deletion in the first terminus of the cloned DNA sequence, the deletion corresponding in size to the single-stranded deletion of step b)ii); and iv) ligating the DNA molecule generated in step b)iii with DNA ligase, thereby re-circularizing the molecule; and c) generating a unidirectional deletion in the second terminus of the cloned DNA sequence by: i) contacting either the recombinant DNA construct of step a) or the recombinant DNA construct generated in step b)iv) with protein gpII encoded by gene II of phage f1 thereby generating a recombinant DNA construct having a single-stranded nick; ii) digesting the recombinant DNA having a single-stranded nick with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap, thereby generating a recombinant DNA construct having a single-stranded gap; iii) contacting the recombinant DNA construct generated by step c)ii) with a single-strand-specific endonuclease, thereby producing a linearized DNA molecule containing a double-stranded deletion in the cloned DNA of the second terminus, the deletion corresponding in size to the single-stranded gap of step c)ii); and iv) ligating the linearized DNA molecule generated by step c)iii) with DNA ligase, thereby recircularizing the molecule. 19. The method of claim 18 wherein the cloning vector of step a) further comprises recognition sequences for a second set of Exo III sensitizing cutters located between the f1 endonuclease recognition sequence and the cloning region, and a recognition sequence for a third Exo III resistance cutter located between the f1 endonuclease recognition sequence and the recognition sequences for the second set of Exo III sensitizing cutters. 20. The method of claim 19 further comprising digesting the recombinant DNA construct generated in step b) with the third Exo III resistance cutter, thereby reducing background from undigested parent DNA constructs. 21. The method of claim 18 wherein the Exo III sensitive end and the Exo III insensitive end of step b)i) are generated by digesting the recombinant DNA construct of step a) with the second Exo III resistance cutter and a member of the first set of Exo III sensitizing cutters. 22. The method of claim 18 wherein the Exo III sensitive end and the Exo III insensitive end of step b)i) are generated by digesting the recombinant DNA construct of step a) with the first Exo III resistance cutter and then blunting the resulting ends thereby generating the Exo III sensitive end of step b)i) and an intermediate Exo III sensitive end, and then further digesting with the second Exo III resistance cutter, thereby converting the intermediate Exo III sensitive end into the Exo III resistant end of step b)i). 23. The method of claim 18 wherein the Exonuclease III digestion of step c)ii) is timed to produce a single-stranded gap having a specific length, the time of digestion required for said specific length being determined by empirical experimentation. 24. The method of claim 18 wherein the Exonuclease III digestion of step b)ii) is timed to produce a single-stranded deletion having a specific length, the time of digestion required for said specific length being determined by empirical experimentation. 25. The method of claim 18 wherein the cloning vector is a single copy cloning vector for generating normalized full-length cDNA libraries. 26. The method of claim 18 wherein the single-strand-specific endonuclease is selected from the group consisting of S1 endonuclease and mung bean endonuclease. 27. The method of claim 26 wherein the single-strand-specific endonuclease is S1 nuclease. 28. The method of claim 18 wherein step c)i) is carried out in a buffer containing the divalent cation Mn.sup.2+. 29. The method of claim 18 wherein the cloning vector further comprises a sequencing primer binding site. 30. A method for introducing a unidirectional deletion at a terminal location of a cloned DNA sequence, comprising the steps: a) providing a recombinant DNA construct comprising a cloning vector for generating nested deletions in a cloned insert, the cloning vector comprising: i) a cloning vector for insertion of a cloned DNA sequence, the cloning region having a first and second side; ii) recognition sequences for a first and a second Exo III resistance cutter adjacently located at discrete positions on a first side of the cloning region, the first Exo III resistance cutter recognition sequence being locate( between the cloning region and the second Exo III resistance cutter recognition sequence; iii) recognition sequences for a set of Exo III sensitizing cutters located between the first Exo III resistance cutter recognition sequence and the cloning region; and iv) an f1 endonuclease recognition sequence adjacently located on the second side of the cloning region, the recombinant DNA construct further comprising a cloned DNA sequence which is inserted into the cloning region of the cloning vector such that the cloned DNA sequence has a first terminus located directly adjacent the first side of the cloning region and a second terminus located directly adjacent the second side of the cloning region; and b) generating a unidirectional deletion in the first terminus of the cloned DNA sequence by: i) linearizing the recombinant DNA construct at a sate in the vector located directly adjacent the first terminus of the cloned DNA sequence, by digesting the recombinant DNA construct of step a) with endonucleases which generate one Exo III sensitive end, corresponding to the end directly adjacent the first terminus of the cloned DNA sequence, and one Exo III insensitive end; ii) digesting the linearized recombinant DNA construct generated in step b)i) with E. coli Exonuclease III, thereby digesting 3' to 5' one strand of the Exo III sensitive end, thereby generating a linearized recombinant DNA construct having a single-stranded deletion in the first terminus of the cloned DNA sequence; iii) contacting the linearized recombinant DNA construct generated in step b)ii) with a single-strand-specific endonuclease, thereby generating a DNA molecule containing a double-stranded deletion in the first terminus of the cloned DNA sequence, the deletion corresponding in size to the single-stranded deletion of step b)ii); and iv) ligating the DNA molecule generated in step b) iii) with DNA ligase, thereby re-circularizing the molecule. 31. The method claim 30 wherein the Exo III sensitive end and the Exo III insensitive end of step b)i) are generated by digesting the recombinant DNA construct of step a) with the second Exo III resistance cutter and a member of the first set of Exo III sensitizing cutters. 32. The method of claim 30 wherein the Exo III sensitive end and the Exo III insensitive end of step b)i) are generated by digesting the recombinant DNA construct of step a with the first Exo III resistance cutter and then blunting the resulting ends thereby generating the Exo III sensitive end of step b)i) and an intermediate Exo III sensitive end, and then further digesting with the second Exo III resistance cutter, thereby converting the intermediate Exo III sensitive end into the Exo III resistant end of step b)i). 33. A method for introducing a unidirectional deletion at a terminal location of a cloned DNA sequence, comprising the steps: a) providing a recombinant DNA construct comprising a cloning vector for generating nested deletions in a cloned Insert, The cloning vector comprising: i) a cloning vector for insertion of a cloned DNA sequence, the cloning region having a first and second side; ii) recognition sequences for a first and a second Exo III resistance cutter adjacently located at discrete positions on a first side of the cloning region, the first Exo III resistance cutter recognition sequence being located between the cloning region and the second Exo III resistance cutter recognition sequence; iii) recognition sequences for a first set of Exo III sensitizing cutters located between the first Exo III resistance cutter recognition sequence and the cloning region; iv) an f1 endonuclease recognition sequence adjacently located on the second side of the cloning region; v) recognition sequences for a second set of Exo III sensitizing cutters located between the f1 endonuclease recognition sequence and the cloning region; and vi) a recognition sequence for a third Exo III resistance cutter located between the f1 endonuclease recognition sequence and the recognition sequences for the second set of Exo III sensitizing cutters, the recombinant DNA construct further comprising a cloned DNA sequence which is inserted into the cloning region of the cloning vector such that the cloned DNA sequence has a first terminus located directly adjacent the first side of the cloning region and a second terminus located directly adjacent the second side of the cloning region; and b) generating a unidirectional deletion in the second terminus of the cloned DNA sequence by: i) digesting the recombinant DNA construct with the third Exo III resistance cutter and with an Exo III sonsitizing cutter from the second set of Exo III sensitizing cutters, thereby generating an Exo III sensitive end and an Exo III resistant end; ii) digesting the linearized recombinant DNA construct generated by step b)i) with E. coli Exonuclease III, thereby digesting 3' to 5' one strand of the Exo III sensitive end, thereby generating a linearized recombinant UNA construct having a single-stranded deletion in the second terminus of the cloned DNA sequence; iii) contacting the linearized recombinant DNA construct generated in step b)ii) with a single-strand-specific endonuclease, thereby generating a DNA molecule containing a double-stranded deletion in the second terminus of the cloned DNA sequence, the double-stranded deletion corresponding in size to the single-stranded deletion of step b)ii); and iv) ligating the DNA molecule generated in step b)iii) with DNA ligase, thereby re-circularizing the molecule.

Details for Patent 6,248,569

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2017-11-10
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2017-11-10
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2017-11-10