PHI29 DNA polymerase

• US 5,198,543 A
• Filed: 03/13/1991
• Issued: 03/30/1993
• Est. Priority Date: 03/24/1989
• Status: Expired due to Fees

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Consejo Superior Investigaciones Cientificas
• Inventors: Bernad, Antonio, Salas, Margarita, Blanco, Luis
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/668,945
• Time in Patent Office: 748 Days
• Field of Search: 435/194, 536/26, 536/27, 536/28, 536/29
• US Class Current: 536/23.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

An improved method for determining the nucleotide base sequence of a DNA molecule. The method includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each the agent terminates DNA synthesis at a different nucleotide base; and separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA can be determined. The improvement is provision of a DNA polymerase which is a φ29-type DNA polymerase.

Claims

1. A non-naturally occurring DNA fragment consisting essentially of DNA encoding a modified φ

29-type DNA polymerase, wherein said polymerase comprises sufficient DNA polymerase activity for use in DNA sequencing, and an exonuclease activity which is less than 10% the activity of the corresponding naturally occurring φ

29-type DNA polymerase.

2. The fragment of claim 1 wherein said DNA fragment encodes a DNA polymerase having substantially no exonuclease activity.

3. The DNA fragment of claim 1 wherein said DNA fragment comprises a DNA sequence encoding a φ

29 DNA polymerase in which the amino acid moiety at position 12, 14, or 66 of the polymerase is replaced by an alanine moiety.