Thermostable DNA polymerases incorporating nucleoside triphosphates labeled with fluorescein family dyes

• US 6,346,379 B1
• Filed: 09/03/1998
• Issued: 02/12/2002
• Est. Priority Date: 09/11/1997
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: F Hoffmann-La Roche AG (Roche Holding AG)
• Inventors: Reichert, Fred Lawrence, Gelfand, David Harrow, Sigua, Christopher Lim, Kalman, Lisa Vivian, Myers, Thomas W.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US09/146,631
• Time in Patent Office: 1,258 Days
• Field of Search: 435/6, 435/91.1, 435/975, 530/350, 530/300, 530/327, 536/23.1, 536/23.2
• US Class Current: 435/6.11
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

Modified thermostable DNA polymerases having enhanced efficiency for incorporating unconventional nucleotides such as those labeled with fluorescein family dyes are advantageous in many in vitro DNA synthesis applications. Such enzymes are particularly useful for use in chain termination nucleic acid sequencing protocols, as are native forms of such enzymes. 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 which is characterized in that

a) in its native form said polymerase comprises the amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:

1), whereby “

Xaa”

at positions 3, 4, 6, 9, and 10 of said sequence are any amino acid residue, and “

Xaa”

at position 7 of said sequence is Val or Ile;

b) said “

Xaa”

at position 4 is mutated in comparison to the native sequence to Lys; and

c) said thermostable DNA polymerase has a level of discrimination against incorporation of nucleotides labeled with fluorescein family dyes which is reduced in comparison to the native form of the polymerase.

2. The recombinant thermostable DNA polymerase of claim 1 wherein said nucleotide is a dideoxynucleotide and said level of discrimination is at least 3-fold lower than that of said native form of said polymerase, wherein said level of discrimination is measured by determining the concentration of a dideoxynucleotide labeled with a fluorescein dye that is required for 50% inhibition of DNA synthesis.

3. The thermostable DNA polymerase of claim 2 wherein said polymerase is from a Thermus species.

4. The recombinant thermostable DNA polymerase of claim 3 wherein said polymerase comprises the amino acid sequence LeuSerXaaXaaLeuXaaIleProTyrGluGlu (SEQ ID NO:

2), whereby “

Xaa”

at position 3 is Gln or Gly, and “

Xaa”

at position 6 is Ser or Ala.

5. The recombinant thermostable DNA polymerase of claim 4 wherein said polymerase comprises the amino acid sequence LeuSerGlnXaaLeuAlalleProTyrGluGlu (SEQ ID NO:

3).

6. The thermostable DNA polymerase of claim 2 wherein said polymerase is from a Thermotoga species.

7. The recombinant thermostable DNA polymerase of claim 6 wherein said polymerase comprises the amino acid sequence LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO:

4), whereby “

Xaa”

at position 7 is Val or Ile.

8. A nucleic acid sequence encoding a recombinant thermostable DNA polymerase which is characterized in that

a) in its native form said polymerase comprises the amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:

1), whereby “

Xaa”

at positions 3, 4, 6, 9, and 10 of said sequence are any amino acid residue, and “

Xaa”

at position 7 of said sequence is Val or Ile;

b) said “

Xaa”

at position 4 is mutated in comparison to the native sequence to Lys; and

c) said thermostable DNA polymerase has a level of discrimination against incorporation of nucleotides labeled with fluorescein family dyes which is reduced in comparison to the native form of said polymerase.

9. The nucleic acid sequence of claim 8 wherein said nucleotide is a dideoxynucleotide and said level of discrimination is at least 3-fold lower than that of said native form of said polymerase, wherein said level of discrimination is measured by determining the concentration of a dideoxynucleotide labeled with a fluorescein dye that is required for 50% inhibition of DNA synthesis.

10. The nucleic acid sequence of claim 9 wherein said polymerase is from a Thermus species.

11. The nucleic acid sequence of claim 10 wherein said nucleic acid sequence encodes a polymerase that comprises the amino acid sequence LeuSerXaaXaaLeuXaaIleProTyrGluGlu (SEQ ID NO:

2) whereby “

Xaa”

at position 3 is Gln or Gly, and “

Xaa”

at position 6 is Ser or Ala.

12. The nucleic acid sequence of claim 10 wherein said nucleic acid sequence encodes a polymerase that comprises the amino acid sequence LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:

3).

13. The nucleic acid sequence of claim 9 wherein said polymerase is from a Thermotoga species.

14. The nucleic acid sequence of claim 10 wherein said nucleic acid sequence encodes a polymerase that comprises the amino acid sequence LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO:

4), whereby “

Xaa”

at position 7 is Val or Ile.

15. A method of DNA sequencing which comprises

a) providing a thermostable DNA polymerase characterized in that

i) said polymerase comprises the amino acid, sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:

1), whereby “

Xaa”

at positions 3, 6, 9, and 10 of this sequence are any amino acid residue, and “

Xaa”

at position 4 is Lys, and “

Xaa”

at position 7 of this sequence is Val or Ile

ii) said polymerase has a reduced level of discrimination against incorporation of nucleotides labeled with fluorescein family dyes,

b) providing a dye-terminator labeled with a negatively charged fluorescent dye, and

c) performing a dye-terminator sequencing reaction.

16. The method of claim 15 wherein said nucleotide is a dideoxynucleotide and said level of discrimination is measured by determining the ratio of the concentration of a dideoxynucleotide labeled with a fluorescein dye required for 50% inhibition of DNA synthesis versus the concentration of an unlabeled dideoxynucleotide required for 50% inhibition.

17. The method of claim 16 wherein said ratio is 4 or less.

18. The method of claim 16 wherein said polymerase is from a thermophilic species selected from the group consisting of Thermosipho africanus, Bacillus caldotenax, and Bacillus stearothermophilus.

19. The method of claim 17 wherein said thermostable DNA polymerase is from a Thermus species.

20. The method of claim 19 wherein said amino acid sequence comprises:

LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO;

3), whereby “

Xaa”

at position 4 is Lys.

21. The method of claim 16 wherein said polymerase is from a Thermotoga species.

22. The method of claim 21 wherein said amino acid sequence comprises:

LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO;

     4), whereby “

Xaa”

at position 4 is Lys and “

Xaa”

at position 7 is Val or Ile.

23. A method of producing labeled DNA which comprises:

a) providing a thermostable DNA polymerase characterized in that

i) said polymerase comprises the amino acid sequence LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO;

     4), whereby “

Xaa”

at position 4 is Lys, and “

Xaa”

at position 7 of this sequence is Val or Ile;

ii) said polymerase has a reduced level of discrimination against incorporation of nucleotides labeled with fluorescein family dyes,

b) providing a nucleotide labeled with a fluorescein family dye, and

c) performing a DNA synthesis reaction.

24. A method of producing labeled primer extension products which comprises:

a) providing a thermostable DNA polymerase characterized in that

i) said polymerase comprises the amino acid sequence LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO;

     4), whereby “

Xaa”

at position 4 is Lys, and “

Xaa”

at position 7 of this sequence is Val or Ile;

ii) said polymerase has a reduced level of discrimination against incorporation of nucleotides labeled with fluorescein family dyes;

iii) said polymerase also comprises the-second amino acid sequence SQIXLR(V/I) (SEQ ID NO;

     18) where “

X”

is any amino acid except E;

iv) said polymerase has reduced discrimination against incorporation of ribonucleotides labeled with fluorescein family dyes;

b) providing a ribonucleotide labeled with a fluorescein family dye, and

c) performing a primer extension reaction.

25. A kit for DNA sequencing which comprises

a) an isolated thermostable DNA polymerase characterized in that

i) said polymerase comprises the amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:

1), whereby “

Xaa”

at positions 3, 6, 9, and 10 of this sequence are any amino acid residue, and “

Xaa”

at position 4 is Lys, and “

Xaa”

at position 7 of this sequence is Val or Ile

ii) said polymerase has reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes, and

b) a terminator labeled with negatively-charged fluorescent dye.

26. The kit of claim 25 wherein said reduced level of discrimination is measured by determining the ratio of the concentration of ddNTP labeled with a fluorescein family dye required for 50% inhibition of DNA synthesis compared to that for an unlabeled ddNTP and said ratio is 4 or less.

27. The kit of claim 26 wherein said amino acid sequence comprises:

LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO;

3), whereby “

Xaa”

at position 4 is Lys.

28. The kit of claim 26 wherein said amino acid sequence comprises:

LeuSerValXaaLeuGLyXaaProValLysGlu (SEQ ID NO;

     4), whereby “

Xaa”

at position 4 is Lys except Glu and “

Xaa”

at position 7 is Val or Ile.

29. A kit for DNA sequencing which comprises

a) an isolated mutant thermostable DNA polymerase characterized in that

i) in its native form said polymerase comprises the amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:

1), whereby “

Xaa”

at positions 3, 4, 6, 9, and 10 of this sequence are any amino acid residue, and “

Xaa”

at position 7 of this sequence is Val or Ile;

ii) said “

Xaa”

at position 4 is mutated in comparison to the native sequence to Lys; and

iii) said thermostable DNA polymerase has a level of discrimination against incorporation of nucleotides labeled with fluorescein family dyes which is reduced in comparison to the native form of said polymerase.

30. The kit of claim 29 wherein said level of discrimination is at least 5-fold lower than that of said native form of said polymerase.

31. The kit of claim 30 wherein said mutant thermostable DNA polymerase its native form said polymerase comprises the amino acid sequence LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:

3).

32. The kit of claim 30 wherein said mutant thermostable DNA polymerase comprises the amino acid sequence LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO:

4), whereby “

Xaa”

at position 7 is Val or Ile.

33. A kit for producing labeled DNA which comprises

a) an isolated thermostable DNA polymerase characterized in that

i) said polymerase comprises the amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:

7), whereby “

Xaa”

at positions 3, 6, 9, and 10 of this sequence are any amino acid residue, and “

Xaa”

at position 4 is Lys, and “

Xaa”

at position 7 of this sequence is Val or Ile

ii) said polymerase has reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes, and

b) a nucleotide labeled with a negatively-charged fluorescent dye.

34. The kit of claim 33 wherein said amino acid sequence comprises:

LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO;

     14), whereby “

Xaa”

at position 4 is Lys.

35. The kit of claim 33 wherein said amino acid sequence comprises:

LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO;

     15), whereby “

Xaa”

at position 4 is Lys and “

Xaa”

at position 7 is Val or Ile.

36. A kit for producing labeled primer extension products which comprises

a) an isolated thermostable DNA polymerase which is characterized in that

i) the polymerase comprises a first amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:

1), whereby “

Xaa”

at positions 3, 6, 9, and 10 of this sequence are any amino acid residue, and “

Xaa”

at position 4 is Lys, and “

Xaa”

at position 7 of this sequence is Val or Ile;

ii) the polymerase has reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes;

iii) the polymerase also comprises a second amino acid sequence SQIXLR(V/I) (SEQ ID NO;

     18), where “

X”

is any amino acid except E;

iv) the polymerase has reduced discrimination against incorporation of ribonucleotides labeled with fluorescein family dyes; and

b) a ribonucleotide labeled with a fluorescein family dye.

37. The kit of claim 36 wherein said first amino acid sequence comprises:

LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO;

3), whereby “

Xaa”

at position 4 is Lys.

38. The kit of claim 36 wherein said first amino acid sequence comprises:

LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO;

     4), whereby “

Xaa”

at position 4 is Lys and “

Xaa”

at position 7 is Val or Ile.