Recombinant polymerases with increased phototolerance
First Claim
1. A composition comprising a ϕ
- 29-type (phi29-type) recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO;
2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position D17, an amino acid substitution at position P305, a Q96W substitution, a K128S substitution, an R258Q substitution, an K303Q substitution, a C452A substitution, an E463K substitution, an E463R substitution, and an E505Q substitution, wherein identification of positions is relative to SEQ ID NO;
2, and wherein said polymerase exhibits polymerase activity.
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Abstract
Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties include increased resistance to photodamage, and can also include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.
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Citations
19 Claims
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1. A composition comprising a ϕ
- 29-type (phi29-type) recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO;
2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position D17, an amino acid substitution at position P305, a Q96W substitution, a K128S substitution, an R258Q substitution, an K303Q substitution, a C452A substitution, an E463K substitution, an E463R substitution, and an E505Q substitution, wherein identification of positions is relative to SEQ ID NO;
2, and wherein said polymerase exhibits polymerase activity. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
- 29-type (phi29-type) recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO;
Specification