Purified thermostable DNA polymerase obtainable from thermococcus litoralis

• US 5,210,036 A
• Filed: 04/26/1990
• Issued: 05/11/1993
• Est. Priority Date: 04/26/1990
• Status: Expired due to Term

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Kucera, Rebecca B., Comb, Donald G.
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): Weber, Jon P.
• Application Number:
  US07/513,994
• Time in Patent Office: 1,111 Days
• Field of Search: 435/193, 435/194
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

There is provided an extremely thermostable enzyme obtainable from Thermococcus litoralis. The thermostable enzyme has a molecular weight of about 90,000-95,000 daltons, a half-life of about 60 minutes at 100° C. in the absence of stabilizer, and a half-life of about 95 minutes at 100° C. in the presence of stabilizer, such as octoxynol (TRITON X-100) or bovine serum albumin. The thermostable enzyme possesses a 3'"'"'-5'"'"' proofreading exonuclease activity. The thermostable enzyme may be native or recombinant and may be used for second-strand cDNA synthesis in cDNA cloning, DNA sequencing, and DNA amplification.

Claims

1. A purified thermostable enzyme obtainable from Thermococcus litoralis which catalyzes the polymerization of DNA, wherein said enzyme has a molecular weight of 90,000 to 95,000 daltons, a 3'"'"'-5'"'"' exonuclease activity and is substantially free of Thermococcus litoralis nucleic acid.

2. The thermostable enzyme of claim 1, having a half life of about 60 minutes at 100°

C. in the absence of a stabilizer.

3. The thermostable enzyme of claim 1, having a half life of about 95 minutes at 100°

C. in the presence of a stabilizer.

4. The thermostable enzyme of claim 3, wherein said stabilizer is a nonionic-detergent.

5. The thermostable enzyme of claim 4, wherein said nonionic-detergent is selected from the group consisting of octoxynol, polyoxyethylated sorbitan monolaurate and ethoxylated nonyl phenol.

6. The thermostable enzyme of claim 3, wherein the stabilizer is a protein selected from the group consisting of Bovine Serum Albumin and gelatin.

7. The polymerase of claim 1, wherein said polymerase is isolated from a recombinant organism transformed with a vector that codes for the expression of Thermococcus litoralis DNA polymerase.