Use of exo-sample nucleotides in gene cloning

• US 5,137,814 A
• Filed: 06/14/1991
• Issued: 08/11/1992
• Est. Priority Date: 06/14/1991
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Rashtchian, Ayoub, Berninger, Mark S.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US07/715,623
• Time in Patent Office: 424 Days
• Field of Search: 435/6, 435/91, 435/172.3, 435/320.1, 536/27
• US Class Current: 435/91.41
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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

The present invention provides improved methods for manipulating recombinant DNA in gene cloning and expression. More specifically, the invention provides methods capable of altering a nucleic acid sequence present at the termini of a target sequence.

Claims

1. A method for incorporating a double-stranded linear desired nucleic acid molecule having a first and a second strand into a double-stranded vector, comprising the steps of:

(A) forming a modified desired nucleic acid molecule characterized in possessing a first region of pre-selected sequence at at least one terminus of a first strand, said sequence being of a length of from about 10 to about 20 nucleotides, and being composed of at least about one third dU residues;

(B) treating said first region of pre-selected sequence under conditions sufficient to result in the removal of the uracil base of at least one of said dU residues, to thereby form a protruding terminus capable of hydrogen bonding to a complementary sequence on at least one strand of said modified desired molecule;

(C) incubating said treated modified molecule of step (B) in the presence of a modified vector having at least one protruding single-stranded terminus, and being capable of hydrogen bonding to at least one of said protruding terminus of said modified desired DNA molecule, to thereby incorporate said double-stranded linear desired nucleic acid molecule into said double-stranded vector.

2. The method of claim 1 wherein only one terminus of said modified desired molecule contains said dU-containing sequence.

3. The method of claim 2 wherein said terminus is a 3'"'"' terminus of said modified desired molecule.

4. The method of claim 2 wherein said terminus is a 5'"'"' terminus of said modified desired molecule.

5. The method of claim 1 wherein said desired nucleic acid molecule is characterized in possessing a first region of pre-selected sequence at two termini, and wherein said two termini of said modified desired molecule contain said du-containing sequence.

6. The method of claim 5 wherein both of said termini are 3'"'"' termini of said modified desired molecule.

7. The method of claim 5 wherein both of said termini are 5'"'"' termini of said modified desired molecule.

8. The method of claim 1 wherein said termini of said first and second strands of said desired DNA molecule contain a plurality of dU residues.

9. The method of claim 1 wherein in step (B), said dU residues are treated with UDG under conditions sufficient to remove the uracil base of at least one of said dU residues, to thereby form an abasic site.

10. The method of claim 9 wherein step (B) additionally comprises treating said abasic site with Endonuclease IV under conditions sufficient to cleave the modified desired molecule at said abasic site.

11. The method of claim 1 wherein said regions of pre-selected sequence of said modified desired DNA molecule are identical.

12. The method of claim 1 wherein in step (C), said modified vector has two protruding single-stranded termini, wherein said termini are produced through the action of a restriction endonuclease.

13. The method of claim 1 wherein in step (C), said modified vector has two protruding single-stranded termini, wherein said termini are produced through the ligation of an oligonucleotide to said vector.

14. The method of claim 1 wherein in step (C), said modified vector has two protruding single-stranded termini, wherein said termini are produced by

(I) adding to said vector:

(i) a first region of pre-selected sequence at a 5'"'"' terminus of a first strand, said sequence containing at least one dU residue;

(ii) a second region of pre-selected sequence at a 5'"'"' terminus of a second strand, said sequence containing at least one dU residue; and

(II) treating said first and second regions of pre-selected sequence under conditions sufficient to result in the removal of the uracil base of at least one of said dU residues, to thereby form said modified vector having said protruding 3'"'"' termini.