Method for the directional cloning of DNA

• US 5,523,221 A
• Filed: 06/16/1993
• Issued: 06/04/1996
• Est. Priority Date: 06/16/1993
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Weiner, Michael P.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): Gurian-Sherman, Douglas
• Application Number:
  US08/078,662
• Time in Patent Office: 1,084 Days
• Field of Search: 435/172.3, 435/320.1, 435/91.1, 435/91.4, 435/91.42, 536/27.11
• US Class Current: 435/91.2
• 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 directionally cloning an insert DNA fragment into a target sequence using differential phosphorylation is disclosed, Monophosphorylated PCR fragments are directionally cloned into a monophosphorylated plasmid, Methods for directionally cloning non-PCR fragments into target DNA sequences are also discussed.

Claims

1. A method for directionally cloning an insert DNA sequence into a target DNA sequence comprising:

generating a monophosphorylated target DNA sequence;

generating a monophosphorylated insert DNA sequence; and

combining said insert DNA sequence with said target sequence, wherein said insert sequence can ligate in only one orientation with respect to said target sequence.

2. The method of claim 1 wherein said target DNA sequence is a plasmid.

3. The method of claim 2 wherein said plasmid is an expression plasmid.

4. The method of claim 1 wherein said target DNA is lambda phage.

5. The method of claim 1 wherein said insert DNA is a PCR fragment.

6. The method of claim 1 wherein said combining step is a ligation with DNA ligase.

7. The method of claim 1 wherein said target sequence is produced using calf intestinal alkaline phosphatase (CIAP).

8. A method for directionally cloning a PCR generated DNA fragment into a target DNA comprising:

generating a PCR fragment having only one phosphorylated 5'"'"' end;

digesting a target DNA sequence with a blunt-end restriction enzyme;

dephosphorylating the 5'"'"' ends of said target sequence;

cleaving said target sequence to expose one 5'"'"' phosphate; and

ligating said PCR generated DNA fragment with said target sequence, wherein said PCR generated DNA sequence can ligate in only one orientation with respect to said target sequence.

9. The method of claim 8 wherein said target DNA sequence is a plasmid.

10. The method of claim 9 wherein said plasmid is an expression plasmid.

11. The method of claim 8 wherein said target DNA is lambda phage.

12. The method of claim 8 wherein said dephosphorylating step uses calf intestinal alkaline phosphatase (CIAP).

13. The method of claim 8 wherein said blunt-end restriction enzyme is SrfI.

14. The method of claim 8 wherein said cleaving step uses Sma I.

15. The method of claim 8 wherein the generating step comprises PCR amplification of the insert DNA sequence by using one 5-phosphorylated primer and one unphosphorylated primer.

16. The method of claim 8 wherein the generating step comprises PCR amplification of the insert DNA sequence by using one DNA kinase-treated primer and one unphosphorylated primer.