Nucleic acid for a cloning vector

• US 20040241852A1
• Filed: 03/15/2004
• Published: 12/02/2004
• Est. Priority Date: 09/16/2001
• Status: Abandoned Application

• Current Assignee: Gene Architects AG
• Original Assignee: Gene Architects AG
• Inventors: Heiermann, Reinhard
• Application Number:
  US10/489,749
• Publication Number:
  US 20040241852A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• 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 invention concerns a nucleic acid forming part of a cloning vector or designed to be inserted in a cloning vector. Said nucleic acid comprises at least a functional nucleotide sequence, which is needed for cloning and/or expression and is coupled with at least one sequence for identifying type II restriction endo nucleases (REII), so that the cleavage site of the REII concerned is inside said functional nucleotide sequence. The invention also relates to a method using said nucleic acid.

Claims

1. Nucleic acid in a cloning vector or suitable for incorporation into a cloning vector with at least one functional nucleotide sequence necessary for cloning and/or expression thereof, characterized in that the nucleic acid or at least its functional nucleotide sequence is coupled with at least one REIIs-recognition sequence or “

homing nuclease”

recognition sequence, so that the cleavage site of the respective REIIs or the “

homing nuclease”

is located within the functional nucleotide sequence.

2. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least its functional nucleotide sequence is the recognition sequence and/or cleavage site of a restriction endonuclease Type II (REII).

3. Nucleic acid according to claim 1, characterized in that it has several REIIs-coupled REII cleavage sites.

4. Nucleic acid according to claim 1, characterized in that it has a REIIs coupled with different REII cleavage sites, or several different REIIs coupled with one REII cleavage site.

5. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is the recognition sequence of a REIIs or a “

homing nuclease”

6. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least the functional nucleotide is the promoter sequence of an RNA polymerase.

7. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is the recognition sequence of a DNA binding protein or RNP particle.

8. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is an enhancer sequence.

9. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is a silencer sequence.

10. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is a termination sequence.

11. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is a polyadenylization sequence.

12. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is a gene or an open reading frame.

13. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is another regulatory sequence.

14. Nucleic acid according to claim 1, characterized in that the nucleic acid or at least a functional nucleotide sequence thereof is a sequence which facilitates the purification the protein expressed from the cloned fragment.

15. Nucleic acid according to claim 1, characterized in that it is a component of a cloning vector.

16. Nucleic acid according to claim 1, characterized in that it is coupled to a carrier material.

17. Nucleic acid according to claim 16, wherein the carrier material is selected from the group consisting of agarose, silica compounds, polystyrene compounds, teflon-acrylamid, polypropylene, nylon, sepharcryl, latex, paramagnetic particles, nanoparticles and cellulose derivatives.

18. Method for serial and linear linkage of nucleic acids fragments by means of the nucleic acid according to the invention, the method comprising the following steps:

(i) providing a nucleic acid according to claim 1, wherein the cutting site of REIIs or the “

homing nuclease”

is located within the REII recognition- and cutting sequence and optionally is identical with the REII cutting site,

(ii) opening the nucleic acid according to the invention with the respective REIIs or REII,

(iii) inserting of a first nucleic acid fragment which has, at the 5′

end as well as also at the 3′

end, an end compatible with the REII of the nucleic acid according to the invention,

(iv) opening of the nucleic acid according to the invention with the respective REIIs (not REII), and

(v) inserting a second nucleic acid fragment, which has, at the 5′

end as well as also at the 3′

end, an end compatible with the REII of the nucleic acid according to the invention,

wherein the steps (iv) to (v) for inserting further nucleic acid fragments an optionally be repeated as desired.

19. Method for the selective inactivation of promoter sequences, the method comprising the following steps:

(i) providing a nucleic acid according to claim 6, wherein

a) the cleavage site of REIIs or the “

homing nuclease”

is located within the promoter sequence,

b) the promoter sequence is provided two times in opposite direction, wherein the two promoter sequences can be identical or different, and

c) both promoter sequences coupled with a least one of each REIIs (REIIs-A, REIIs-B) where REIIs-A is different from REIIs-B, and

(ii) opening the nucleic acid with optionally REIIs-A and/or REIIs-B.

20. Cloning vector, which contains a nucleic acid according to claim 1.

21. Cloning vector according to claim 20, characterized in that it contains no further recognition—

or cleavage sites for the REIIs or “

homing nuclease”

occurring in the said nucleic acid.

22. Cells containing a cloning vector, or a nucleic acid according to claim 1.

23. Kit, comprising at least a nucleic acid according to claim 1, at least a suitable REIIs, optionally in combination with one or more REII, a ligase, as well as optionally additional suitable enzymes and suitable reaction buffers.