Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof

• US 6,306,588 B1
• Filed: 02/06/1998
• Issued: 10/23/2001
• Est. Priority Date: 02/07/1997
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Solus, Joseph, Chatterjee, Deb K., Yang, Shuwei
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/019,160
• Time in Patent Office: 1,355 Days
• Field of Search: 435/6, 435/91.2, 435/174, 530/350, 530/820, 530/388.21
• US Class Current: 435/5
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present invention provides methods for use in identifying, analyzing and typing polymorphic DNA fragments, particularly minisatellite, microsatellite or STR DNA fragments. In particular, the invention provides methods using DNA polymerases, more particularly thermostable DNA polymerases, and most particularly Thermotoga polymerases or mutants or derivatives thereof, whereby minisatellite, microsatellite or STR DNA molecules maybe amplified and analyzed for polymorphisms. The invention also relates to polymerases having reduced, substantially reduced or eliminated ability to add non-template 3′ nucleotides to a synthesized nucleic acid molecule. In accordance with the invention, such reduction or elimination may be accomplished by modifying or mutating the desired polymerase.

Claims

1. A polymerase which comprises one or more modifications or mutations in the O-helix of said polymerase that reduce, substantially reduce or eliminate the ability of the polymerase to add non-templated 3′

nucleotides to a synthesized nucleic acid molecule, wherein said polymerase is not a Thermotoga polymerase.

2. The polymerase of claim 1, wherein said polymerase is selected from the group consisting of Taq DNA polymerase, Tth DNA polymerase, Tli DNA polymerase, VENT®

DNA polymerase, Pfu DNA polymerase, DEEPVENT®

DNA polymerase, Pwo DNA polymerase, Bst DNA polymerase, Bca DNA polymerase, and Tfl DNA polymerase.

3. The polymerase of claim 1, wherein said O-helix is defined as RXXXKXXXFXXXYX (SEQ ID NO:

11), wherein X is any amino acid.

4. The polymerase of claim 3, wherein said mutation or modification is at one or more positions or combinations of positions of said O-helix selected from the group consisting of position R (Arg), position F (Phe), position K (Lys), positions R and F, positions R and K, positions F and K, and positions R, F and K.

5. The polymerase of claim 3, wherein said mutation or modification is an amino acid substitution at one or more positions or combinations of positions of said O-helix selected from the group consisting of position R, position F, position K, positions R and F, positions R and K, positions F and K, and positions R, F and K.

6. The polymerase of claim 5, wherein K (Lys) is substituted with Arg or His.

7. A mutant Tne DNA polymerase protein selected from the group consisting of:

Tne N′

Δ

283, D323A;

Tne N′

Δ

284, D323A;

Tne N′

Δ

193, D323A;

Tne G195D, D323A;

Tne G37D, D323A;

Tne N′

Δ

283;

Tne D137A, D323A, R722K;

Tne D137A, D323A, R722Y;

Tne D137A, D323A, R722L;

Tne D137A, D323A, R722H;

Tne D137A, D323A, R722Q;

Tne D137A, D323A, K726R;

Tne D137A, D323A, K726H;

Tne D137A, D323A, R722K, F730Y;

Tne D137A, D323A, R722K, K726R;

Tne D137A, D323A, R722K, K726H;

Tne D137A, D323A, R722H, F730Y;

Tne D137A, D323A, R722H, K726R;

Tne D137A, D323A, R722H, K726H;

Tne D137A, D323A, R722Q, F730Y;

Tne D137A, D323A, R722Q, K726R;

Tne D137A, D323A, R722Q, K726H;

Tne D137A, D323A, R722N, F730Y;

Tne D137A, D323A, R722N, K726R;

Tne D137A, D323A, R722N, K726H;

Tne D137A, D323A, F730S;

Tne N′

Δ

283, D323A, R722K/H/Q/N/Y/L;

Tne N′

Δ

219, D323A, R722K;

Tne N′

Δ

219, D323A, K726R;

Tne N′

Δ

219, D323A, K726H;

Tne D137A, D323A, F730S, R722K/Y/Q/N/H/L, K726R/H;

Tne D137A, D323A, F730T, R722K/Y/Q/N/H/L, K726R/H;

Tne D137A, D323A, F730T; and

Tne D137A, D323A, R722N.

8. A polymerase which comprises one or more modifications or mutations in the O-helix of said polymerase that reduce, substantially reduce or eliminate the ability of the polymerase to add non-templated 3′

nucleotides to a synthesized nucleic acid molecule, wherein said mutation or modification is at one or more positions or combinations of positions of said O-helix selected from the group consisting of position R (Arg), position F (Phe), position K (Lys), positions R and F, positions R and K, positions F and K, and positions R, F and K, and wherein said polymerase is not a Thermotoga polymerase.

9. A polymerase which comprises one or more modifications or mutations in the O-helix of said polymerase that reduce, substantially reduce or eliminate the ability of the polymerase to add non-templated 3′

nucleotides to a synthesized nucleic acid molecule, wherein said mutation or modification is an amino acid substitution at one or more positions or combinations of positions of said O-helix selected from the group consisting of position R, position F, position K, positions R and F, positions R and K, positions F and K, and positions R, F and K, and wherein said polymerase is not a Thermotoga polymerase.