Cloning vectors and their preparation and use for mRNA expression pattern analysis

• US 6,303,308 B1
• Filed: 05/10/1999
• Issued: 10/16/2001
• Est. Priority Date: 05/18/1998
• Status: Expired due to Fees

• Current Assignee: Switch Biotech AG
• Original Assignee: Switch Biotech AG
• Inventors: Goppelt, Andreas, Halle, Jörn-Peter, Regenbogen, Johannes
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/307,924
• Time in Patent Office: 890 Days
• Field of Search: 435/6, 435/91.1, 435/91.3, 536/24.33
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection

Abstract

Disclosed herein are cloning vectors which include:

(a) a cloning site which permits the cloning of a nucleic acid in defined orientation;

(b) at least one cleavage site adjacent to the cloning site, the cleavage site being rarely-occurring in nucleic acids; and

(c) a long region which is located on the side of the cloning site opposite to the cleavage site (b), wherein the long region and the region between the cloning site and the cleavage site (b) contain neither the cloning site nor at least two frequently-occurring cleavage sites.

Claims

1. A cloning vector comprising:

(a) a cloning site which permits the cloning of a nucleic acid in a defined orientation;

(b) at least one cleavage site located adjacent to said cloning site, said cleavage site being rarely-occurring in nucleic acids, wherein said cleavage site is recognized by a restriction endonuclease selected from AscI, BaeI, FseI, NotI, PmeI, PpuMI, RsrII, SanDI, SapI, SexAI, SfiI, SgfI, SgrAI, SrfI, Sse8387I, SwaI, I-CeuI, PI-PspI, I-PpoI, PI-TliI, and PI-Scel, or is recognized by a restriction endonuclease with a recognition sequence of not less than 8 nucleotides that includes a CG combination;

(c) a short region having several different cleavage sites which are frequently-occurring in nucleic acids, said short region being positioned on the side of said cleavage site (b) opposite to said cloning site, wherein said short region is shorter than about 100 nucleotides; and

(d) a long region located on the side of said cloning site opposite to said cleavage site (b), wherein said long region and the region between said cloning site and said cleavage site (b) contain neither said cloning site nor at least three of said frequently-occurring cleavage sites, wherein said long region is longer than at least 500 nucleotides.

2. The cloning vector according to claim 1, characterized in that said cloning site contains two different cleavage sites.

3. The cloning vector according to claim 1, characterized in that said frequently-occurring cleavage sites, independently of one another, are recognized by restriction endonucleases having recognition sequences of not more than 4 nucleotides.

4. The cloning vector according to claim 3, characterized in that each of said frequently-occurring cleavage sites is recognized independently by a restriction endonuclease selected from AciI, AluI, BfaI, BsaJI, Bs1I, BscFI, BssKI, BstUI, Cac8I, CfoI, Csp6I, CviJI, DdeI, DpnI, DpnII, FmuI, Fnu4HI, HaeIII, HhaI, HinfI, HinPI, HpaII, MaeII, MaeIII, MboI, MnlI, MseI, MspI, MwoI, NlaIII, NlaIV, RsaI, Sau3AI, Sau96I, ScrFI, TaiI, TaqI, Tsp4CI, or Tsp509I.

5. The cloning vector according to claim 1, characterized in that said cloning site is selected from a cleavage site which is recognized by restriction endonucleases with a recognition sequence of not less than 5 nucleotides.

6. The cloning vector according to claim 1, characterized in that said cloning site is selected from a cleavage site which is recognized by restriction endonucleases with a recognition sequence of not less than 6 nucleotides.

7. The cloning vector according to claim 1, characterized in that said cloning site is selected from a cleavage site which is recognized by restriction endonucleases with a recognition sequence of not less than 8 nucleotides.

8. The cloning vector according to claim 6, characterized in that said cloning site is recognized by a restriction endonuclease selected from AClWI, Alw26I, AlwI, AsuHPI, AvaII, BbvI, BccI, BcefI, BinI, BsbI, BscGI, Bse1I, BseNI, BsmAI, BsmFI, BspLU11III, BsrI, BsrSI, Bst71I, BstF5I, BstNI, CjeI, CjePI, EcoRII, FauI, FinI, FokI, HgaI, HphI, MboII, NciI, PleI, SfaNI, SimI, TauI, TfiI, TseI, Tsp45I, TspRI, or Vpa11AI.

9. The cloning vector according to claim 6, characterized in that said cloning site is recognized by a restriction endonuclease selected from AccI, AflIII, ApoI, AvaI, AvaII, BanI, BanII, BmgI, BsaI, BsaHI, BsaWI, BsiEI, BsiHKAI, BsoBI, Bsp1286I, BsrFI, BstYI, DsaI, EaeI, EcoO109I, GdiII, HaeI, HaeII, Hin4I, HincII, MmeI, Ms1I, MspA1I, NspI, SfcI, StyI, TatI, Tth111II, AatI, Acc131I, Acc65I, AcINI, AfIII, Alw44I, ApaI, ApaLI, AseI, Asp718I, AvrII, Ba1I, BamHI, BbuI, BbsI, Bc1I, BfrI, Bg1I, Bg1II, BlnI, BpiI, BpmI, BsaI, BsaMI, BseRI, BsmBI, BsmI, Bsp120I, Bsp1407I, Bsp191I, BspHI, BspLU11I, BspMI, BspTI, BsrGI, Bst1107I, Bst98I, DraI, Eam1104I, EarI, Ecl136II, Eco147I, Eco255I, Eco57I, EcoNI, EcoRI, EcoRV, EcoT22I, HindIII, HpaI, KpnI, MfeI, MscI, NcoI, NdeI, NheI, NsiI, PstI, PvuII, SacI, ScaI, SpeI, SphI, SspI, SstI, StuI, or XbaI.

10. The cloning vector according to claim 6, characterized in that said cloning site is recognized by a restriction endonuclease with a recognition sequence which contains a rare nucleotide combination.

11. The cloning vector according to claim 10, characterized in that said rare nucleotide combination is CG.

12. The cloning vector according to claim 10, characterized in that said cloning site is recognized by one of the restriction endonuclease selected from AatII, BbeI, BsiI, BsiWI, BsmBI, BspDI, BsrBI, BssHII, Bst2BI, BstBI, ClaI, EagI, EciI, Eco47III, EheI, Esp3I, FspI, KasI, MluI, NarI, NruI, Pfl1108I, PmlI, Psp1406I, PvuI, SacII, SalI, SnaBI, or XhoI.

13. The cloning vector according to claim 6, characterized in that said cloning site is recognized by a restriction endonuclease selected from AscI, BaeI, FseI, NotI, PacI, PmeI, PpuMI, RsrII, SanDI, SapI, SexAI, SfiI, SgfI, SgrAI, SrfI, Sse8387I, SwaI, I-CeuI, PI-PspI, I-PpoI, PI-TliI, or PI-SceI.

14. The cloning vector according to claim 1, characterized in that said long region is longer than the fragments obtainable by cutting with restriction endonucleases which recognize frequently-occurring cleavage sites.

15. The cloning vector according to claim 1, wherein said long region is longer than about 1000 nucleotides.

16. The cloning vector according to claim 1, characterized in that said short region is smaller than the length of the nucleic acid which extends from cleavage site (b) to the first possible cleavage site in the nucleic acid to be inserted into said cloning vector.

17. The cloning vector according to claim 16, characterized in that said frequently-occurring cleavage site occurs at the start of the poly(A) tail of a cDNA to be inserted into said cloning site of said cloning vector.

18. The cloning vector according to claim 1, wherein said short region is shorter than about 30 nucleotides.

19. A method for the preparation of a cloning vector according to claim 1, said method comprising combining the individual components of the vector.

20. A cloning vector comprising:

(a) a cloning site which permits the cloning of a nucleic acid in a defined orientation;

(b) at least one cleavage site located adjacent to said cloning site, said cleavage site being rarely-occurring in nucleic acids, wherein said cleavage site is recognized by a restriction endonuclease selected from SfiI, Bg1I, BbsI, BsaI, and BspMI; and

(c) a long region located on the side of said cloning site opposite to said cleavage site (b), wherein said long region and the region between said cloning site and said cleavage site (b) contain neither said cloning site nor at least three different frequently-occurring cleavage sites, said frequently-occurring cleavage sites comprising DdeI, DpnI, and RsaI, and wherein said long region is longer than at least 500 nucleotides.

21. The cloning vector of claim 20, wherein said cloning vector further comprises a short region, said short region having several different cleavage sites which are frequently-occurring in nucleic acids but which do not occur in said long region, said short region being positioned on the side of said cleavage site (b) opposite to said cloning site, wherein said short region is shorter than about 100 nucleotides.