Modified Proteins and Methods of Making and Using Same

• US 20110262903A1
• Filed: 02/25/2011
• Published: 10/27/2011
• Est. Priority Date: 02/26/2010
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: DAVIDSON, John, Rothberg, Jonathan M., Hinz, Wolfgang
• Application Number:
  US13/035,081
• Publication Number:
  US 20110262903A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

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 method of detecting a nucleotide incorporation, comprising:

(a) performing a nucleotide incorporation using a modified polymerase and generating one or more hydrogen ions as a by-product of the nucleotide incorporation, where the modified polymerase includes one or more amino acid substitutions that reduce the buffering capacity of the modified polymerase relative to an unmodified polymerase; and

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

2. The method of claim 1, wherein the modified polymerase includes one or more amino acid substitutions that reduce the buffering capacity of the modified polymerase within the pH range of about pH 7 to about pH 9 relative to the unmodified polymerase.

3. The method of claim 1, wherein the polymerase comprises one or more substitutions of an amino acid residue having a pKa within the range of about 7 to about 9 with another amino acid residue.

4. The method of claim 3, wherein the other amino acid residue is selected from the group consisting of Ala Arg, Asp, Gln, Iy, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

5. The method of claim 3, wherein at least one of the one or more amino acid modifications includes a substitution of an amino acid residue having a pKa of between about 7 and about 9 with an amino acid residue having a pKa that is less than about 7 or greater than about 9.

6. The method of claim 5, wherein the at least one of the one or more amino acid substitutions is a conservative amino acid substitution.

7. The method of claim 1, wherein at least one of the one or more amino acid substitutions is selected from the group consisting of histidine to arginine, glutamic acid to glutamine, aspartic acid to asparagine, lysine to arginine, and tyrosine to phenylalanine.

8. The method of claim 1, wherein at least one of the one or more amino acid substitutions is selected from the group consisting of:

substitution of a surface histidine with an arginine, substitution of a surface glutamic acid with a glutamine, and substitution of a surface lysine with an arginine.

9. The method of claim 1, wherein the modified polymerase is a modified. Phi29 DNA polymerase.

10. The method of claim 1, wherein the modified polymerase is a modified Bst DNA polymerase.

11. The method of claim 10, wherein the one or more amino acid substitutions in the modified Bst DNA polymerase are of one or more amino acid residues shown in Table 1.

12. The method of claim 10, wherein the one or more conservative amino acid substitutions in the modified Bst DNA polymerase are selected from the group consisting of H46R, H273R, H428R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acid residues being in accordance with that of SEQ ID NO:

1.

13. The method of claim 1, wherein at least one of the one or more amino acid substitutions includes a substitution of an amino acid residue of the polymerase with an alanine residue.

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

providing template nucleic acid hybridized to a sequencing primer and bound to a bufferless polymerase;

synthesizing a new nucleic acid strand by incorporating one or more known nucleoside triphosphates sequentially at the 3′

end of the sequencing primer; and

detecting such incorporation at the 3′

end of the primer by measuring a concentration of a hydrogen ion byproduct generated if the known nucleoside triphosphate is complementary to corresponding nucleotides in the template nucleic acid.

15. The method of claim 14, wherein the bufferless polymerase is a polymerase including the amino acid sequence of SEQ ID NO:

2.