Mutant DNA Polymerases with Improved Pyrophasphorolysis Activated Polymerization (PAP) Ability
AssigneeRoche Molecular Systems, Inc.
InventorsKeith A. Bauer, David Harrow Gelfand
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Abstract
Disclosed are mutant DNA polymerases having improved extension rates relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.
Claims38
A DNA polymerase, comprising R-X1-X2-X3-K-L-X4-X5-X6-Y-X7-X8-X9-X10-X11, wherein
- X1, X3, X7, and X9 are any amino acid,
- X2 is L, I, Y,
- X4 is K, R, Q,
- X5 is N, S, G,
- X6 is not T,
- X8 is D or E,
- X10 is L or I,
- X11 is P or L (SEQ ID NO:36);
- wherein the polymerase has improved pyrophosphorolysis activated polymerization (PAP) relative to an otherwise identical polymerase where X6 is T.
A mutant DNA polymerase characterized in that
- (i) in its unmodified form, the polymerase has pyrophosphorolysis activated polymerization activity (PAP) and comprises an amino acid sequence having the following consensus sequence:
- R-X1-X2-X3-K-L-X4-X5-X6-Y-X7-X8-X9-X10-X11 (SEQ ID NO:24), wherein
- X1, X3, X7, and X9 are any amino acid,
- X2 is L, I, Y,
- X4 is K, R, Q,
- X5 is N, S, G,
- X6 is T,
- X8 is D or E,
- X10 is L or I,
- X11 is P or L;
- (ii) the mutant polymerase has an amino acid substitution, relative to its unmodified form, at position X6 to an amino acid other than T and wherein;
- (iii) the polymerase has improved PAP relative to the unmodified form where X6 is T.
The mutant DNA polymerase of 12, wherein the unmodified for of the polymerase is selected from the group consisting of:
- (a) CS5 DNA polymerase
- (b) Thermus thermophilus
- (c) Thermus caldophilus
- (d) Thermus sp. Z05
- (e) Thermus aquaticus
- (f) Thermus flavus
- (g) Thermus filiformis
- (h) Thermus sp. sps17
- (i) Deinococcus radiodurans
- (j) Bacillus stearothermophilus
- (k) Bacillus caldotenax
- (l) Escheria coli
- (m) Thermotoga maritima
- (n) Thermosipho africanus
- (o) Thermotoga neapolitana
- (p) Hot Spring family A
- (q) a DNA polymerase having at least 90% sequence identity to any one of (a)-(p).
Dependencies are inferred from the claim text.