Modified proteins and methods of making and using same

• US 9,714,915 B2
• Filed: 05/28/2015
• Issued: 07/25/2017
• 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: Whitaker, Richard, Davidson, John, Hinz, Wolfgang, Rothberg, Jonathan
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US14/724,354
• Publication Number:
  US 20150260680A1
• Time in Patent Office: 789 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 system for nucleic acid sequencing, comprising:

(a) a field effect transistor (FET); and

(b) a modified Bst DNA polymerase comprising three or more amino acid substitutions selected from the group consisting of H46R, H273R, H281R, E446Q, H473R, H528R, H572R and Y477F, 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, is configured 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; and

wherein the FET comprises an ion sensitive FET (ISFET) configured to detect the release of hydrogen ions as a chemical byproduct of nucleotide incorporation catalyzed by the modified polymerase.

2. The system of claim 1, wherein the amino acid substitutions are H46R, E446Q and H572R.

3. The system of claim 2, 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.

4. The system of claim 2, wherein the modified Bst DNA polymerase comprises an amino acid sequence at least 95% identical to SEQ ID NO. 2.

5. The system of claim 1, 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.

6. The system of claim 1, wherein the reduced buffering capacity can be observed within the range of from pH 4 to pH 10.

7. The system of claim 1, wherein the reduced buffering capacity can be observed within the range of from pH 7 to pH 9.

8. The system of claim 1, wherein the buffering capacity of the modified Bst DNA polymerase between pH 7 and pH 9 has been removed.

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

10. A method of sequencing a nucleic acid using a system of claim 1, comprising:

(a) disposing a plurality of template nucleic acids into a plurality of reaction chambers, wherein one or more of the reaction chambers are in contact with a field effect transistor (FET);

(b) contacting at least one of the template nucleic acids with the modified Bst DNA polymerase;

(c) synthesizing a new nucleic acid strand by sequentially incorporating one or more nucleotides into a nucleic acid molecule and generating one or more hydrogen ions as a byproduct of the nucleotide incorporation; and

(d) detecting the incorporation of the one or more nucleotides by detecting the generation of the one or more hydrogen ions use the FET.

11. The method of claim 10, wherein the amino acid substitutions are H46R, E446Q and H572R.

12. The method of claim 11, 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.

13. The method of claim 10, wherein the modified Bst DNA polymerase comprises an amino acid sequence at least 95% identical to SEQ ID NO. 2.

14. The method of claim 10, wherein the reduced buffering capacity can be observed within the range of from pH 4 to pH 10.

15. The method of claim 10, wherein the reduced buffering capacity can be observed within the range of from pH 7 to pH 9.

16. The method of claim 10, wherein polymerase activity of the modified Bst polymerase is at least 90% compared to wild type Bst DNA polymerase.