Purified thermostable enzyme

• US 5,079,352 A
• Filed: 05/15/1990
• Issued: 01/07/1992
• Est. Priority Date: 08/22/1986
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Cetus Corporation (Novartis Ag)
• Inventors: Gelfand, David H., Saiki, Randall K., Stoffel, Susanne, Lawyer, Frances C.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US07/523,394
• Time in Patent Office: 602 Days
• Field of Search: 435/183, 435/194, 435/320.1, 435/172.3, 435/68, 435/70, 536/27, 935/12, 935/29
• US Class Current: 536/23.2
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS

Abstract

Recombinant DNA vectors that encode a thermostable DNA polymerase are useful in the recombinant production of thermostable DNA polymerase. The recombinant thermostable polymerase is preferred for use in the production of DNA in a polymerase chain reaction. Especially useful vectors encode the ˜94,000 dalton thermostable DNA polymerase from thermus aquaticus.

Claims

1. A recombinant DNA sequence that encodes the thermostable DNA polymerase activity of Thermus aquaticus.

2. The recombinant DNA sequence of claim 1 shown in FIG. 1.

5. A recombinant DNA vector that comprises the DNA sequence of claim 1 and can be used to drive expression of the thermostable DNA polymerase activity of Thermus aquaticus in a host cell transformed with the vector.

6. The recombinant vector of claim 5 that is plasmid pLSG1.

7. The recombinant vector of claim 5 that is plasmid pLSG2.

8. The recombinant vector of claim 5 that is plasmid pLSG5.

9. The recombinant vector of claim 5 that is plasmid pLSG6.

10. The recombinant vector of claim 5 that is plasmid pLSG10.

18. A host cell transformed with a vector of claim 5.

19. The host cell of claim 18 that is E. coli/pLSG1.

20. The host cell of claim 18 that is E. coli/pLSG2.

21. The host cell of claim 18 that is E. coli/pLSG5.

22. The host cell of claim 18 that is E. coli/pLSG6.

23. The host cell of claim 18 that is E. coli/pLSG7.

24. The host cell of claim 18 that is E. coli/pLSG8.

25. The host cell of claim 18 that is E. coli/pLSG10.

26. The host cell of claim 18 that is E. coli/pFC82.

27. The host cell of claim 18 that is E. coli/pFC85.

3. A recombinant DNA sequence that encodes amino acid residues 290 through 832 shown in FIG. 1.

4. The recombinant DNA sequence of claim 3 that encodes amino acid residues 4 through 832 shown in FIG. 1.

11. A recombinant DNA vector that comprises the DNA sequence of claim 3 and can be used to drive expression of a protein that has thermostable DNA polymerase activity in a host cell transformed with the vector.

12. The recombinant vector of claim 11 that is plasmid pLSG8.

13. The recombinant vector of claim 11 that is plasmid pFC82.

14. The recombinant vector of claim 11 that is plasmid pFC85.

15. A recombinant DNA vector that comprises the DNA sequence of claim 4 and can be used to drive expression of a protein that has thermostable DNA polymerase activity in a host cell transformed with the vector.

16. The recombinant vector of claim 15 that is plasmid pLSG7.

17. A recombinant vector selected from the group consisting of plasmid pFC83, phage CH35:

Taq #4-2, and plasmid pSYC1578.