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OwnerAlexei Slesarev, Andrey Pavlo +2inventors
InventorsAlexei Slesarev, Andrey Pavlo, Sergei Kozyavkin, Nadezhda Pavlova
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The utility of Topo V's HhH motifs for modulating DNA binding properties of a DNA processing enzyme, such as Taq DNA polymerase or PfuDNA polymerase, is demonstrated. In one embodiment, an amino acid residue comprising one or more HhH domains derived from Topoisomerase V, linked to either the NH2-terminus or COOH-terminus of a Taq polymerase fragment significantly broadens the processivity and/or salt concentration range of polymerase activity. The specific activities of the chimeric polymerases are not affected by added HhH motifs. Depending on the type of the construct, the thermal stability of chimeric polymerases increases or remains the same as that of Taq DNA polymerase or its Stoffel fragment. This invention further provides a method of increasing the salt tolerance of Taq polymerases. The methods of this invention may be applied to all pol A-type DNA polymerases, as well as to other DNA processing enzymes.
A chimeric DNA processing enzyme comprising a DNA processing enzyme or a fragment thereof linked to an amino acid sequence comprising one or more helix-hairpin-helix (HhH) motifs not naturally associated with said DNA processing enzyme or said fragment, wherein said amino acid sequence is derived from Topoisomerase V.
A method of increasing the processivity of a DNA processing enzyme or a fragment thereof, comprising linking to said DNA processing enzyme or said fragment to an amino acid sequence comprising one or more helix-hairpin-helix (HhH) motifs not naturally associated with said DNA processing enzyme or said fragment, wherein said amino acid sequence is derived from Topoisomerase V.
A method of increasing the salt tolerance of a DNA processing enzyme or a fragment thereof in an amplification reaction, comprising linking to said DNA processing enzyme or said fragment to an amino acid sequence comprising one or more helix-hairpin-helix (HhH) motifs not naturally associated with said DNA processing enzyme or said fragment, wherein said amino acid sequence is derived from Topoisomerase V.
A method of increasing the thermal stability of a DNA processing enzyme or a fragment thereof, comprising linking to said DNA processing enzyme or said fragment to an amino acid sequence comprising one or more helix-hairpin-helix (HhH) motifs not naturally associated with said DNA processing enzyme or said fragment, wherein said amino acid sequence is derived from Topoisomerase V.
A method of amplifying a nucleic acid, comprising combining said nucleic acid with a chimeric DNA processing enzyme having a DNA processing enzyme or a fragment thereof linked to an amino acid sequence is derived from Topoisomerase V and contains one or more helix-hairpin-helix (HhH) motifs, wherein said nucleic acid and said chimeric DNA processing enzyme are combined in an amplification reaction mixture under conditions that allow amplification of said nucleic acid.
A chimeric DNA processing enzyme comprising a DNA processing enzyme or a fragment thereof linked to an amino acid sequence comprising one or more helix-hairpin-helix (HhH) motifs not naturally associated with said DNA processing enzyme or said fragment.
Dependencies are inferred from the claim text.