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 exo-sample nucleotide residues selected from the group consisting of deoxyuridine, bromodeoxyuridine, and a ribonucleotide;
- (B) treating said first region of pre-selected sequence under conditions sufficient to result in the removal of at least one of said exo-sample nucleotide 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 exo-sample nucleotide-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 exo-sample nucleotide-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 said exo-sample nucleotide residues.
- 9. The method of claim 1 wherein in step (B), said exo-sample nucleotide residues are treated under conditions sufficient to remove at least one of said 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 strand 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 strand 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 strand 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 exo-sample nucleotide residue selected from the group consisting of deoxyuridine, bromodeoxyuridine, and a ribonucleotide;
- (ii) a second region of pre-selected sequence at a 5' terminus of a second strand, said sequence containing at least one exo-sample nucleotide residue selected from the group consisting of deoxyuridine, bromodeoxyuridine, and a ribonucleotide; and
- (II) treating said first and second regions of pre-selected sequence under conditions sufficient to result in the removal of at least one of said exo-sample nucleotide residues, to thereby form said modified vector having said protruding 3' termini.
Parent Case Info
This application is a continuation of U.S. patent application Ser. No. 07/715,623 (filed Jun. 14, 1991) now U.S. Pat. No. 5,137,814.
US Referenced Citations (3)
Number |
Name |
Date |
Kind |
4873192 |
Kunkel |
Oct 1989 |
|
5035996 |
Hartley |
Jul 1991 |
|
5137814 |
Rashtchian et al. |
Aug 1992 |
|
Foreign Referenced Citations (1)
Number |
Date |
Country |
329822 |
Aug 1989 |
EPX |
Non-Patent Literature Citations (4)
Entry |
Longo, M. C. et al., Gene 93:125-128 (1990). |
Frohman, M. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:8998-9002 (1988). |
Ohara, O. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:5673-5677 (1989). |
Duncan, B., The Enzymes XIV:565-586 (1981). |
Continuations (1)
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Number |
Date |
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Parent |
715623 |
Jun 1991 |
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