Method of nucleic acid sequence determination
Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.
1. A mutant DNA polymerase comprising a polypeptide sequence, said polypeptide sequence comprising SEQ ID NO:12,
wherein said mutant DNA polymerase forms ternary complexes with primed template nucleic acid molecules and cognate nucleotides, and
wherein said mutant DNA polymerase is incapable of magnesium-catalyzed phosphodiester bond formation.
2. The mutant DNA polymerase of claim 1 , wherein the mutant DNA polymerase catalyzes formation of phosphodiester bonds in the presence of manganese ions.
3. The mutant DNA polymerase of claim 1 , wherein the polypeptide sequence of the mutant DNA polymerase comprises SEQ ID NO:1 with the exception of comprising SEQ ID NO:12 and having glutamate at amino acid position 381.
4. The mutant DNA polymerase of claim 1 , wherein the polypeptide sequence of the mutant DNA polymerase comprises SEQ ID NO:2 with the exception of comprising SEQ ID NO:12 and having glutamate at amino acid position 364.
5. The mutant DNA polymerase of claim 1 , wherein the polypeptide sequence of the mutant DNA polymerase comprises SEQ ID NO:3 with the exception of comprising SEQ ID NO:12 and having glutamate at amino acid position 355.
6. The mutant DNA polymerase of claim 1 , further comprising an exogenous label.
7. A ternary complex, comprising the mutant DNA polymerase of claim 1 , a primed template nucleic acid and a next correct nucleotide for the primed template nucleic acid.
8. The ternary complex of claim 7 , wherein the next correct nucleotide comprises an exogenous label.
9. The ternary complex of claim 7 , wherein the mutant DNA polymerase comprises an exogenous label.