Methods for identifying and isolating variant T4 DNA polymerases

• US 5,660,980 A
• Filed: 06/06/1995
• Issued: 08/26/1997
• Est. Priority Date: 08/02/1993
• Status: Expired due to Fees

• Current Assignee: University of Alberta, University of Southern California
• Original Assignee: University of Alberta, University of Southern California
• Inventors: Goodman, Myron F., Reha-Krantz, Linda J.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/465,995
• Time in Patent Office: 812 Days
• Field of Search: 435/91.2, 435/5, 435/183, 435/235.1, 435/69.1, 530/358, 935/82, 935/79
• US Class Current: 435/5
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Method for identifying and isolating variant T4 DNA polymerase by isolating and selecting for T4 strains having variant DNA polymerase defective in DNA replication, and at least one additional mutation which corrects or compensates for said defect in DNA replication; identifying the additional mutation(s) and introducing the mutation(s) into T4 phage or T4 DNA polymerase expression vectors.

Claims

1. A method of identifying and isolating variant T4 DNA polymerases comprising the steps of:

identifying T4 strains having variant T4 DNA polymerases defective in some aspect of DNA replication;

isolating further mutated forms of said variant T4 DNA polymerases by selection in an E. coli optA1 host;

isolating T4 strains which contain variant T4 DNA polymerases having at least one additional mutation which corrects or compensates said defect in DNA replication;

identifying additional correcting/compensating mutation(s) in said variant T4 DNA polymerases; and

introducing said identified correcting/compensating mutation(s) T4 DNA polymerases into T4 phage or T4 DNA polymerase expression vectors.