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AssigneeRoche Sequencing Solutions, Inc.
InventorsCleoma Arnold, Aruna Ayer, David Beyer, Neha Jain, Naga Kishore Kakani, Yarrow Madrona, Ashtamurthy Pawate, David Penkler, Christopher Witbooi
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Described herein is a variant pol6 polymerase having improved thermostability. The disclosed polymerases each contain one or more substitutions relative to SEQ ID NO: 1 selected from the group consisting of G12W/Y/F, K114W/F/I, L117L/F/P, N194F/W/V, M232W/G/R, G313E/Q/L, A451F/W/Y, K490W/F/Y, Q565Y/I/V, Q590P/V/Y, and D681G/N/H. Additionally, the present specification discloses substitutions that can reduce stuttering in such polymerases, the substitutions selected from the group consisting of N298L, L538R, P542A, I570H/T/W/R/N/G, N574L, E633W/F, S636F, E639K, and K655G.
An isolated polypeptide having a DNA polymerase activity, the isolated polypeptide comprising an amino acid sequence having at least 75% sequence identity to amino acids 11-739 of SEQ ID NO:
The isolated polypeptide of any one of the preceding claims, wherein the at least one substitution is selected from the group consisting of G12W, G12Y, G12F, A451F, A451W, N194W, E5651, E590Y, and D681H.
The isolated polypeptide of any one of the preceding claims, wherein the at least one substitution is selected from the group consisting of A451F, N194W, D681H.
The isolated polypeptide of any of any one of the preceding claims, wherein said isolated polypeptide has an improved thermostability relative to Pol6-1743 at a temperature in the range of 37° C. to 47° C.
The isolated polypeptide of any one of the preceding claims, wherein the isolated polypeptide has at least 1.5-fold greater thermostability of DNA polymerase activity relative to Pol6-1743 at 36° C., 38° C., 40° C., and/or 46° C.
The isolated polypeptide of any of any one of the preceding claims, wherein said amino acid sequence further comprises at least one substitution selected from the group consisting of N298L, L538R, P542A, I570H/T/W/R/N/G, N574L, E633 W/F, S636F, E639K, and K655G.
The isolated polypeptide of any one of the preceding claims, wherein the substitutions are selected from the group consisting of I570H, N574L, S636F, L538R, and E633F.
The isolated polypeptide of any one of the preceding claims, wherein said polypeptide comprises at least one substitution selected from the group consisting of G12W/Y/F, K114W/F/I, L117L/F/P, N194F/W/V, M232W/G/R, G313E/Q/L, A451F/W/Y, K490W/F/Y, Q565Y/I/V, Q590P/V/Y, and D681G/N/H;
The isolated polypeptide of any one of the preceding claims, wherein at least one substitution is selected from the group consisting of G12W/Y/F, K114W/F/I, L117L/F/P, N194F/W/V, M232W/G/R, G313E/Q/L, A451F/W/Y, K490W/F/Y, Q565Y/I/V, Q590P/V/Y, and D681G/N/H;
The isolated polypeptide of any one of the preceding claims, wherein at least one substitution is selected from the group consisting of G12W, G12Y, G12F, A451F, A451W, N194W, E5651, E590Y, and D681H;
The isolated polypeptide of any one of the preceding claims, wherein at least one substitution is selected from the group consisting of G12W, G12Y, G12F, A451F, A451 W, N194 W, E5651, E590Y, and D681H;
The isolated polypeptide of any one of the preceding claims, wherein at least one substitution is selected from the group consisting of A451F, N194W, and D681H;
The isolated polypeptide of any one of the preceding claims, wherein at least one substitution is selected from the group consisting of A451F, N194W, and D681H;
A composition comprising a DNA polymerase attached to nanopore, wherein said DNA polymerase comprises the isolated polypeptide of any one of the preceding claims.
Dependencies are inferred from the claim text.