Six base oligonucleotide linkers and methods for their use

• US 4,719,179 A
• Filed: 11/30/1984
• Issued: 01/12/1988
• Est. Priority Date: 11/30/1984
• Status: Expired due to Fees

• Current Assignee: Pharmacia P-L Biochemicals Incorporated
• Original Assignee: Pharmacia P-L Biochemicals Incorporated
• Inventors: Barany, Francis
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Mays, Thomas D.
• Application Number:
  US06/676,935
• Time in Patent Office: 1,138 Days
• Field of Search: 435/172.3, 935/6, 935/10, 536/27
• 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

A method for inserting a selected restriction site into a double stranded genetic sequence at a selected tab site, and a linker for use therewith are disclosed. The method involves treating the genetic sequence with an opening agent (such as a restriction enzyme) so as to open both strands of the tab site. One then exposes the opened genetic sequence to two hexameric single stranded oligonucleotide linkers in the presence of a ligating enzyme. The two linkers have the same nitrogenous base sequence, a sequence which is partially palindromic complementary to itself at least one end of the nitrogenous sequence, but is not completely palindromic complementary to itself. Through use of this method, the two linkers are inserted in the opening with partial complementary overlap, and at least one of the linkers is affixed to each strand. Subsequently, the site is reclosed at the point of the insertion so as to contain a six base insertion.

Claims

1. A method for inserting a selected restriction site into a double stranded genetic sequence at a selected tab site comprising:

treating a double stranded DNA genetic sequence with a type II restriction enzyme that cleaves both strands at a selected tab site and produces complementary single stranded termini on each strand of said genetic sequence;

exposing said cleaved genetic sequence to two hexameric single stranded DNA oligonucleotide linkers in the presence of a ligating enzyme, wherein said linkers have the same nitrogenous base sequence, and wherein said linker sequence is palindromic complementary to both ends of said linker sequence with respect to only either two or four bases per end and wherein said linker sequence is a recognition site for a restriction enzyme; and

selecting said genetic sequence, wherein said linker sequence has been inserted and ligated complementary to said single stranded termini.

2. The method of claim 1, wherein the selected tab site is a pre-existing restriction enzyme site.

3. The method of claim 2, wherein the pre-existing restriction enzyme site, when cleaved, has a four base-5'"'"' overhang.

4. The method of claim 2, wherein the pre-existing restriction enzyme site, when cleaved, has a two base-5'"'"' overhang.

5. The method of claim 2, wherein the pre-existing restriction enzyme site, when cleaved, has a two base-3'"'"' overhang.

6. The method of claim 2, wherein the pre-existing restriction enzyme site, when cleaved, as a four base-3'"'"' overhang.