Modified Proteins and Methods of Making and Using Same
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.
1 - 15 . (canceled)
16 . A method for sequencing a nucleic acid, 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 a modified polymerase including one or more amino acid substitutions that substantially reduce its buffering capacity within the range of about pH 7 to about pH 9 relative to the unmodified 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 such nucleotide incorporation; and
(d) detecting the incorporation of the one or more nucleotides by detecting the generation of the one or more hydrogen ions using the FET.
17 . The method of claim 16 , 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.
18 . The method of claim 17 , wherein the other amino acid residue is selected from the group consisting of: Ala Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).
19 . The method of claim 15 , wherein at least one of the one or more amino acid substitutions 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.
20 . The method of claim 16 , wherein the at least one of the one or more amino acid substitutions is a conservative amino acid substitution.
21 . The method of claim 16 , 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.
22 . The method of claim 16 , 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.
23 . The method of claim 16 , wherein the modified polymerase includes a modified Bst DNA polymerase.
24 . The method of claim 16 , 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.
25 . The method of claim 16 , wherein the one or more amino acid substitutions in the modified Bst DNA polymerase are 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.
26 . The method of claim 16 , wherein at least one of the one or more amino acid substitutions includes a substitution of an N terminal amino acid residue of the polymerase with any other different amino acid residue.
27 . The method of claim 16 , wherein the modified polymerase includes the amino acid sequence of SEQ ID NO: 2 except for the N-terminal methionine residue of SEQ ID NO: 2, which is not included in the modified polymerase.