Purified thermostable enzyme

• US 5,352,600 A
• Filed: 11/05/1992
• Issued: 10/04/1994
• Est. Priority Date: 08/22/1986
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Gelfand, David H., Stoffel, Susanne
• Primary Examiner(s): Fleisher, Mindy B.
• Application Number:
  US07/971,798
• Time in Patent Office: 698 Days
• Field of Search: 435/183, 435/194, 435/172.3, 935/14
• US Class Current: 435/194
• 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

Abstract

A purified thermostable enzyme is obtained that has unique characteristics. Preferably the enzyme is isolated from the Thermus aquaticus species and has a molecular weight of about 86,000-95,000 daltons. The thermostable enzyme may be native or recombinant and may be used in a temperature-cycling chain reaction wherein at least one nucleic acid sequence is amplified in quantity from an existing sequence with the aid of selected primers and nucleotide triphosphates. The enzyme is preferably stored in a buffer containing non-ionic detergents that lends stability to the enzyme.

Claims

1. A purified thermostable template-dependent DNA polymerase from the species Thermus aquaticus, wherein said polymerase is substantially free of contaminating deoxyribonucleases and retains activity at 60°

C. to 80°

C. for after exposure to a temperature of 90°

C. for up to four minutes and has a specific activity of about 200,000 units/mg, wherein one unit corresponds to 10 nmoles of product synthesized on activated salmon sperm DNA in 30 minutes.

2. The polymerase of claim 1 that is produced in a recombinant host cell.

3. A purified thermostable template-dependent DNA polymerase from the species Thermus aquaticus, wherein said polymerase is substantially free of contaminating deoxyribonucleases and retains activity at 60°

C. to 80°

C. after exposure to a temperature of 90°

C. for up to four minutes and has a specific activity of about 20 units/pmole enzyme, wherein one unit corresponds to 10 nmoles of product synthesized on activated salmon sperm DNA in 30 minutes.

4. The polymerase of claim 3 that is produced in a recombinant host cell.