Modified cloning vectors for restriction mapping, their preparation and use

• US 5,196,328 A
• Filed: 05/21/1987
• Issued: 03/23/1993
• Est. Priority Date: 05/21/1987
• Status: Expired due to Fees

• Current Assignee: The Institute for Cancer Research (Fox Chase Cancer Center)
• Original Assignee: The Institute for Cancer Research (Fox Chase Cancer Center)
• Inventors: Tartof, Kenneth D.
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/052,799
• Time in Patent Office: 2,133 Days
• Field of Search: 435/5, 435/6, 435/29, 435/172.3, 435/320.1, 536/27, 935/26, 935/29, 935/31, 935/78
• US Class Current: 435/91.41
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

Modified cloning vectors having general utility for easily obtaining unambiguous restriction maps of recombinant DNA molecules and the methods of preparation and use of such vectors.

Claims

1. A modified cloning vector for facilitating mapping of a DNA molecule present in said modified cloning vector, said vector having a multiple cloning site including said DNA molecule and a DNA construct, said construct being a linear, synthetic oligonucleotide consisting essentially of an infrequent cutter segment providing a recognition sequence for a site-specific restriction enzyme, said recognition sequence being at lest eight base pairs in length, and a discrete, hybridizable linker segment, said construct being positioned in said vector with said linker segment adjacent to said DNA molecule.

2. A modified cloning vector as claimed in claim 1, wherein said recognition sequence is GCGGCCGC.

3. A modified cloning vector as claimed in claim 1, wherein said vector is selected from the group consisting of a plasmid, a lambda phage and a cosmid.

4. A modified cloning vector as claimed in claim 3, wherein said vector comprises two DNA constructs, said recognition sequence of each construct being the same and said linker segment of each construct being different with respect to nucleotide sequence, and wherein said multiple cloning site including the DNA molecule to be mapped is located intermediate the two DNA constructs, adjacent the linker terminus of each construct.

5. A modified cloning vector as claimed in claim 3, wherein comprises a plasmid selected from the group consisting of pUC 18 and pUC 19.

6. A modified cloning vector as claimed in claim 3, which comprises the lambda phage EMBL 4.

7. A modified cloning vector as claimed in claim 3, wherein comprises the cosmid cos4.

8. A modified cloning vector as claimed in claim 7, wherein said cos4 vector comprises two DNA constructs, each construct comprising a linear, synthetic oligonucleotide including an infrequent cutter segment and a linker segment, the infrequent cutter segment of each construct being the same and the linker segment of each construct being different with respect to nucleotide sequence and wherein the multiple cloning site of said vector is located intermediate said DNA constructs adjacent the linker terminus of each construct.

9. A vector as claimed in claim 8, wherein said multiple cloning site is a plasmid-derived oligonucleotide.

12. A host cell transformed in vitro with the vector of claim 1, and cells grown from said host cell, said cells containing said vector.

10. A method of preparing a modified cloning vector useful for obtaining a restriction map of a DNA molecule present in said vector, said method comprising inserting into a cloning vector a DNA construct, said construct being a linear, synthetic oligonucleotide consisting essentially of an infrequent cutter segment providing a recognition sequence for a site-specific restriction enzyme, said restriction sequence being at least eight base pairs in length, and a discrete, hybridizable linker segment, said construct being positioned in said vector with said linker segment adjacent to said DNA molecule and wherein said vector comprises a multiple cloning site including said DNA molecule and said DNA construct is positioned adjacent to said multiple cloning site.

11. A method as claimed in claim 10, wherein said construct is inserted into a vector selected from the group consisting of a plasmid, a lambda phage or a cosmid.

13. A DNA construct for facilitating restriction mapping of a DNA molecule, said construct being a linear, synthetic oligonucleotide consisting essentially of an infrequent cutter segment providing a recognition sequence for a sitespecific restriction enzyme, said recognition sequence being at least eight base pairs in length, and a discrete, hybridizable linker segment, said construct being adapted for insertion into a vector to provide a modified vector, each terminus of said linear oligonucleotide comprising a recognition sequence for a sitespecific restriction enzyme, whereby said construct is insertable into said vector, said linker segment being positioned intermediate said frequency cutter segment and said DNA molecule to be mapped and wherein the recognition sequence of each terminus is the same.