Thermostable DNA polymerases having reduced discrimination against ribo-NTPs

• US 5,939,292 A
• Filed: 08/05/1997
• Issued: 08/17/1999
• Est. Priority Date: 08/06/1996
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Reichert, Fred Lawrence, Gelfand, David Harrow, Kalman, Lisa Vivian
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Stole, Einar
• Application Number:
  US08/906,484
• Time in Patent Office: 742 Days
• Field of Search: 435/194, 435/91.2, 536/23.2
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase

Abstract

Modified thermostable DNA polymerases having enhanced efficiency for incorporating unconventional nucleotides, such as ribonucleotides, into DNA products, are advantageous in many in vitro synthesis applications. Such enzymes are particularly useful for use in nucleic acid sequencing protocols and provide novel means for DNA sequence analysis. Genes encoding the modified enzymes and methods for their production and use offer cost and efficiency advantages for DNA sequencing.

Claims

1. A recombinant thermostable DNA polymerase that is a mutant form of a naturally occurring thermostable DNA polymerase, wherein said naturally occurring thermostable DNA polymerase has an amino acid sequence comprising amino acid sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO:

2), wherein "Xaa" at position 7 of said sequence motif is a valine residue (Val) or an isoleucine residue (Ile);

wherein said mutant form has been modified to contain an amino acid other than glutamic acid (Glu) at position 4 of said sequence motif; and

wherein said mutant form possesses reduced discrimination against incorporation of an unconventional nucleotide in comparison to said naturally occurring thermostable DNA polymerase.

3. The recombinant thermostable DNA polymerase of claim 1 further characterized in that the ability of said polymerase to incorporate an unconventional nucleotide, relative to the ability of said corresponding native form of polymerase to incorporate said unconventional nucleotide, is increased by at least 20 fold.

4. The recombinant thermostable DNA polymerase of claim 3 further characterized in that said polymerase has sufficient activity for use in a DNA sequencing reaction that comprises an unconventional nucleotide and a corresponding conventional nucleotide in a ratio of 1:

1 or less.

5. The recombinant thermostable DNA polymerase of claim 4, wherein said unconventional nucleotide is a ribonucleoside triphosphate.

6. The recombinant thermostable DNA polymerase of claim 5 wherein said ribonucleoside triphosphate is present at a concentration of less than about 100 μ

M and said corresponding conventional nucleotide is present at a concentration of more than about 100 μ

M.

7. The recombinant thermostable DNA polymerase of claim 6 wherein said naturally occurring DNA polymerase is selected from the group consisting of Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus oshimai, Thermus ruber, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus DNA polymerases.

8. The recombinant thermostable DNA polymerase of claim 6 wherein said naturally occurring DNA polymerase is a thermostable Thermus species DNA polymerase.

9. The recombinant thermostable DNA polymerase of claim 6 wherein said naturally occurring DNA polymerase is a thermostable Thermotoga species DNA polymerase.

10. The recombinant thermostable DNA polymerase of claim 6 wherein said naturally occurring DNA polymerase comprises the amino acid sequence LeuAspTyrSerGlnIleGluLeuArgValLeuAlaHisLeuSer (SEQ ID NO:

5).

18. A composition for use in a DNA sequencing reaction that comprises;

a nucleic acid template;

an oligonucleotide primer complementary to said template;

a recombinant thermostable DNA polymerase of claim 5, and a mixture of conventional dNTPs and at least one unconventional nucleotide, wherein said unconventional nucleotide is a ribonucleotide and wherein the ratio of said unconventional nucleotide to said corresponding conventional nucleotide is 1;

1 or less.

19. The composition of claim 18 wherein said ribonucleoside is present at a concentration of less than about 100 μ

M and the corresponding conventional nucleotide is present at a concentration of more than about 100 μ

M.

20. The composition of claim 19 further characterized in that said unconventional nucleotide is unlabeled.

2. A nucleic acid sequence encoding a recombinant thermostable DNA polymerase that is a mutant form of a naturally occurring thermostable DNA polymerase, wherein said naturally occurring thermostable DNA polymerase has an amino acid sequence comprising amino acid sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO:

2), wherein "Xaa" at position 7 of said sequence motif is a valine residue (Val) or an isoleucine residue (Ile);

wherein said mutant form has been modified to contain an amino acid other than glutamic acid (Glu) at position 4 of said sequence motif; and

wherein said mutant form possesses reduced discrimination against incorporation of an unconventional nucleotide in comparison to said naturally occurring thermostable DNA polymerase.

11. The nucleic acid sequence of claim 2 wherein said recombinant thermostable DNA polymerase has sufficient activity for use in a DNA sequencing reaction.

12. The nucleic acid sequence of claim 11 wherein said DNA sequencing reaction comprises an unconventional nucleotide and the corresponding conventional nucleotides in a ratio of 1:

1 or less.

13. The nucleic acid sequence of claim 12 wherein said unconventional nucleotide is a ribonucleoside triphosphate.

14. The nucleic acid sequence of claim 13 wherein said naturally occurring thermostable DNA polymerase is selected from the group consisting of Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus oshimai, Thermus ruber, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus DNA polymerases.

15. The nucleic acid sequence of claim 13 wherein said naturally occurring thermostable DNA polymerase is a thermostable Thermus species DNA polymerase.

16. The nucleic acid sequence of claim 13 wherein said naturally occurring thermostable DNA polymerase is a thermostable Thermotoga species DNA polymerase.

17. The nucleic acid sequence of claim 12 wherein said naturally occurring DNA polymerase comprises the amino acid sequence LeuAspTyrSerGlnIleGluLeuArgValLeuAlaHisLeuSer (SEQ ID NO:

5).

21. A method for sequencing a nucleic acid target which method comprises the steps of:

(a) providing an unconventional nucleotide and a corresponding conventional nucleotide in a DNA sequencing reaction, wherein said unconventional and corresponding conventional nucleotides are present in a ratio of less than about 1;

1, wherein said DNA sequencing reaction contains a recombinant thermostable DNA polymerase that is a mutant form of a naturally occurring thermostable DNA polymerase, wherein said naturally occurring thermostable DNA polymerase has an amino acid sequence comprising amino acid sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO;

     2), wherein "Xaa" at position 7 of said sequence motif is a valine residue (Val) or an isoleucine residue (Ile);

wherein said mutant form has been modified to contain an amino acid other than glutamic acid (Glu) at position 4 of said sequence motif; and

wherein said mutant form possesses reduced discrimination against incorporation of an unconventional nucleotide in comparison to said naturally occurring thermostable DNA polymerase;

(b) treating the reaction of step (a) under conditions for primer extension to provide primer extension products comprising said unconventional nucleotide;

(c) treating the primer extension products of step (b) under conditions for hydrolyzing said primer extension products;

(d) resolving reaction products from step (c); and

(e) determining the sequence of the nucleic acid target.

22. The method for sequencing of claim 21 wherein said unconventional nucleotide is a ribonucleotide.

23. The method for sequencing of claim 22 wherein said ribonucleotide is present at a concentration of about 0.1 μ

M-100 μ

M.

24. The method for sequencing of claim 23 wherein said corresponding conventional nucleotide is present at a concentration of about 50 μ

M-500 μ

M.

25. A kit for sequencing a nucleic acid comprising a thermostable DNA polymerase enzyme that comprises the amino acid sequence SerGlnIleXaaLeuArgXaa (SEQ ID NO:

1), wherein "Xaa" at position 4 of this sequence is any amino acid residue but not a glutamic acid residue (Glu) and "Xaa" at position 7 of this sequence is a valine residue (Val) or an isoleucine residue (Ile), and a mixture of conventional dNTPs and at least one unconventional nucleotide, wherein the ratio of said unconventional nucleotide to said corresponding conventional nucleotide is less than one.