Modified proteins and methods of making and using same

• US 10,059,987 B2
• Filed: 06/16/2017
• Issued: 08/28/2018
• Est. Priority Date: 02/26/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Davidson, John, Hinz, Wolfgang, Rothberg, Jonathan, Whitaker, Richard
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US15/625,800
• Publication Number:
  US 20170292156A1
• Time in Patent Office: 438 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Abstract

Methods, compositions, systems, apparatuses and kits comprising modified proteins, particularly modified nucleic acid-binding proteins with altered buffering properties are provided. For example, in some embodiments, methods of forming modified proteins including one or more amino acid modifications to achieve desired pKa values are described. Furthermore, the invention provides methods for using such modified proteins in ion-producing reactions, such as ion-based nucleic acid sequencing reactions.

Claims

1. A modified Bst DNA polymerase comprising three or more amino acid substitutions selected from the group consisting of H46R, H273R, H281R, E446Q, H473R, Y477F, H528R, and H572R, the numbering of amino acid residues being in accordance with that of SEQ ID NO:

1, wherein the modified polymerase has reduced buffering capacity compared to wild type Bst DNA polymerase, retains the ability to catalyze nucleotide polymerization, and comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO;

1.

2. The modified Bst DNA polymerase of claim 1, wherein the amino acid substitutions comprise H46R, E446Q, and H572R.

3. The modified Bst DNA polymerase of claim 2, further comprising one or more amino acid substitutions of one or more amino acid residues shown in Table 2 or 3.

4. The modified Bst DNA polymerase of claim 1, wherein the modified polymerase has reduced buffering capacity compared to wild type Bst DNA polymerase within a range of about pH 4 to about pH 10.

5. The modified Bst DNA polymerase of claim 1, wherein the modified polymerase has reduced buffering capacity compared to wild type Bst DNA polymerase within a range of about pH 7 to about pH 9.

6. The modified Bst DNA polymerase of claim 1, wherein polymerase activity of the modified Bst DNA polymerase is at least 90% compared to wild type Bst DNA polymerase.

7. An aqueous solution comprising the modified Bst DNA polymerase of claim 1, wherein said aqueous solution further comprises a template nucleic acid and nucleotides.

8. A method of detecting a nucleotide incorporation, comprising:

(a) performing a nucleotide incorporation by contacting a template nucleic acid with the modified Bst DNA polymerase of claim 1 in the presence of nucleotides, wherein one or more hydrogen ions are generated as a by-product of the nucleotide incorporation; and

(b) detecting the one or more hydrogen ions generated as a by-product of the nucleotide incorporation, thereby detecting the nucleotide incorporation.

9. The method of claim 8, wherein the amino acid substitutions comprise H46R, E446Q, and H572R.

10. The method of claim 9, wherein the modified Bst DNA polymerase further comprises one or more amino acid substitutions of one or more amino acid residues shown in Table 2 or 3.

11. The method of claim 8, wherein the modified Bst DNA polymerase has reduced buffering capacity compared to wild type Bst DNA polymerase within a range of about pH 4 to about pH 10.

12. The method of claim 8, wherein the modified Bst DNA polymerase has reduced buffering capacity compared to wild type Bst DNA polymerase within a range of about pH 7 to about pH 9.

13. The method of claim 8, wherein the buffering capacity of the modified Bst DNA polymerase between pH 7 and pH 9 has been removed.

14. A method of sequencing a nucleic acid, comprising:

(a) contacting a template nucleic acid with the modified Bst DNA polymerase of claim 1 and nucleotides;

(b) synthesizing a new nucleic acid strand by sequentially incorporating one or more of the nucleotides into a nucleic acid molecule; and

(c) detecting such incorporation by detecting one or more hydrogen ion byproducts generated when the nucleotide is complementary to a corresponding nucleotide in the template nucleic acid.

15. The method of claim 14, wherein the amino acid substitutions comprise H46R, E446Q, and H572R.

16. The method of claim 15, wherein the modified Bst DNA polymerase further comprises one or more amino acid substitutions of one or more amino acid residues shown in Table 2 or 3.

17. The method of claim 14, wherein the modified Bst DNA polymerase has reduced buffering capacity compared to wild type Bst DNA polymerase within a range of about pH 4 to about pH 10.

18. The method of claim 14, wherein the modified Bst DNA polymerase has reduced buffering capacity compared to wild type Bst DNA polymerase within a range of about pH 7 to about pH 9.

19. The method of claim 14, wherein the buffering capacity of the modified Bst DNA polymerase between pH 7 and pH 9 has been removed.

20. The method of claim 14, wherein the template nucleic acid is hybridized to a sequencing primer when contacting the modified Bst DNA polymerase and synthesizing the new nucleic acid strand by incorporating the one or more nucleotides at the 3′

end of the sequencing primer.