.phi.29 DNA polymerase

• US 5,576,204 A
• Filed: 02/11/1993
• Issued: 11/19/1996
• Est. Priority Date: 03/24/1989
• Status: Expired due to Term

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Consejo Superior Investigaciones Cientificas
• Inventors: Salas, Margarita, Blanco, Luis, Bernad, Antonio
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/010,568
• Time in Patent Office: 1,377 Days
• Field of Search: 435/193
• US Class Current: 435/193
• 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 employs a φ-29 type DNA polymerase modified to have reduced or no exonuclease activity. 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 Φ

29-type DNA polymerase produced from a non-naturally occurring DNA fragment consisting 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 φ

29-type DNA polymerase of claim 1, wherein said DNA encoding a modified φ

29-type DNA polymerase encodes a DNA polymerase having no exonuclease activity.

3. The φ

29-type DNA polymerase of claim 1, wherein said φ

29-type DNA polymerase is that polymerase present in cells infected with the phage φ

29.

4. The φ

29-type DNA polymerase of claim 2, wherein said φ

29-type DNA polymerase is that polymerase present in cells infected with the phage φ

29.

5. The φ

29-type DNA polymerase of claim 1, wherein said phage is selected from one of the group consisting of φ

29, Cp-1, PRD1, φ

15, φ

21, PZE, PZA, Nf, M2Y, B103, SF5, GA-1, Cp-5, Cp-7, PR4, PR5, PR722, and L17.

6. The φ

29-type DNA polymerase of claim 2, wherein said phage is selected from one of the group consisting of φ

29, Cp-1, PRD1, φ

15, φ

21, PZE, PZA, Nf, M2Y, B103, SF5, GA-1, Cp-5, Cp-7, PR4, PR5, PR722, and L17.