Chimeric DNA polymerases
The present invention provides, among other things, chimeric DNA polymerases containing heterologous domains having sequences derived from at least two DNA polymerases that have at least one distinct functional characteristics (e.g., elongation rate, processivity, error rate or fidelity, salt tolerance or resistance) and methods of making and using the same. In some embodiments, the present invention can combine desired functional characteristics (e.g., high processivity; high elongation rate; thermostability; resistance to salt, PCR additives (e.g., PCR enhancers) and other impurities; and high fidelity) of different DNA polymerases in a chimeric polymerase.
1. A DNA polymerase of whose amino acid sequence shows at least 95% overall identity with SEQ ID NO:16, the DNA polymerase characterized in that:
a) it shows a higher fidelity than a wildtype KOD polymerase; and
b) it shows higher salt tolerance or thermostability than a wildtype Pfu polymerase.
2. A DNA polymerase of claim 1 whose amino acid sequence comprises:
a first consensus sequence selected from the group consisting of
(1)
XXLXXXXXXXEGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXV LIXXXXXNXXXAXXKXXCXXXXXNFALXXXXXXXXXXXXIXXMXXRFXXXX XXXXXXXXXPXXRXXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTTXXXT (SEQ ID NO:30), wherein X is any amino acid or a peptide bond;
(2)
XXEXXXXYXXXXEXXFXXXXKXXXAXXXXXXXXAXXXXTVXTVKRXXXXQ XXXXXRXVEXXXXFTXXXXXXAXXDXIXXXXX (SEQ ID NO:31), wherein X is any amino acid or a peptide bond;
(3)
XXXXXXXXXXXXXXXXALXXDXXXXKXXXXXXXXTEXXSKXXVXXXXXVX HXXXXXDXKDXXXTXXXXXXXXRXXXRXXXXRXXTXXSXXXXKXSXRXGD XXXPFDXFXXTXXXXXXXXXXXXXXXXXXEXXXRAXX (SEQ ID NO:32), wherein X is any amino acid or a peptide bond;
(4)
NGX 1 FKIEX 2 DRTFX 3 PYX 4 YALLX 5 DDSX 6 IEEVKKITX 7 ERHGX 8 X 9 VX 10 X 11 X 12 X 13 VEKVX 14 KKFLGX 15 PX 16 X 17 VWKLYX 18 X 19 HPQDVPX 20 IRX 21 KX 22 REHPA (SEQ ID NO:33), wherein X 1 is not K; X 2 is not H; X 3 is not R; X 4 is not I; X 5 is not R; X 6 is not K; X 7 is not G; X 8 is not K; X 9 is not I; X 10 is not R; X 11 is not I; X 12 is not V; X 13 is not D; X 14 is not E; X 15 is not K; X 16 is not I; X 17 is not T; X 18 is not L; X 19 is not E; X 20 is not T; X 21 is not E; and X 22 is not V;
(5)
PIX 1 MISYADEX 2 X 3 AX 4 VITWKNX 5 DLPYVX 6 VVSX 7 EREMIKRFLRX 8 X 9 X 10 EKDPDX 11 X 12 X 13 TYNGDX 14 FDFX 15 YLX 16 KRX 17 EKLGIX 18 X 19 X 20 X 21 GRDGSEPKX 22 QRX 23 GDX 24 X 25 AVEVKGRIHFDLYX 26 VIX 27 RTINLPTYTLEAVYEAX 28 FGX 29 PKEKVYAX 30 EIX 31 X 32 AWEX 33 (SEQ ID NO:34), wherein X 1 is not I; X 2 is not N; X 3 is not E; X 4 is not K; X 5 is not I; X 6 is not E; X 7 is not S; X 8 is not I; X 9 is not I; X 10 is not R; X 11 is not I; X 12 is not I; X 13 is not V; X 14 is not S; X 15 is not P; X 16 is not A; X 17 is not A; X 18 is not K; X 19 is not L; X 20 is not T; X 21 is not I; X 22 is not M; X 23 is not I; X 24 is not M; X 25 is not T; X 26 is not H; X 27 is not T; X 28 is not I; X 29 is not K; X 30 is not D; X 31 is not A; X 32 is not K; and X 33 is not S; and
(6)
RDWSEIAKETQARVLEX 1 X 2 LKX 3 GDVEX 4 AVRIVKEVX 5 X 6 KLX 7 X 8 YEX 9 PPEKLX 10 IX 11 EQITRX 12 LX 13 X 14 YKAX 15 GPHVAVAKX 16 LAAX 17 GVKIX 18 PGX 19 VIX 20 YIVLX 21 GX 22 GX 23 IX 24 X 25 RAIX 26 X 27 X 28 EX 29 DPX 30 KHKYDAEYYIENQVLPAVX 31 RILX 32 X 33 FG (SEQ ID NO:35), wherein X 1 is not T; X 2 is not I; X 3 is not H; X 4 is not E; X 5 is not I; X 6 is not Q; X 7 is not A; X 8 is not N; X 9 is not I; X 10 is not A; X 11 is not Y; X 12 is not P; X 13 is not H; X 14 is not E; X 15 is not I; X 16 is not K; X 17 is not K; X 18 is not K; X 19 is not M; X 20 is not G; X 21 is not R; X 22 is not D; X 23 is not P; X 24 is not S; X 25 is not N; X 26 is not L; X 27 is not A; X 28 is not E; X 29 is not Y; X 30 is not K; X 31 is not L; X 32 is not E; and X 33 is not G;
and a second consensus sequence selected from the group consisting of
(1)
XKXXXXXXXXXXXXAXXXXXXXXXXXXXXXXXLXXXXNXXIXXXXXXKXX XXIXXXXXXXXXHXXXXXXXXXTXXXEXQXXXXKIXXXXXXKXXXLXXXXF XXXXXXXKXXXXXXXXXXXXXXXXXKXXELVWXXLXXXFXXXXLXIXXXXL YXXXXXGESXEIXXXXLX (SEQ ID NO:36), wherein X is any amino acid or a peptide bond;
(2)
EX 1 GLWENIVYLDFRX 2 LYPSIIITHNVSPDTLNX 3 EGCKX 4 YDX 5 APQVGHX 6 FCKDX 7 PGFIPSLLGX 8 LLEERQKIKX 0 KMKX 10 TX 11 DPIEX 12 X 13 LLDYRQX 14 AIKX 15 LANSX 16 YGYYGYAX 17 ARWYCKECAESVTAWGRX 18 YIX 19 X 20 X 21 X 22 KEX 23 EEKX 24 GFKVX 25 YX 26 DTDGX 27 X 28 ATIPGX 29 X 30 X 31 EX 32 X 33 KKKAX 34 E (SEQ ID NO:37), wherein X 1 is not R; X 2 is not S; X 3 is not R; X 4 is not E; X 5 is not V; X 6 is not R; X 7 is not F; X 8 is not D; X 9 is not K; X 10 is not A; X 11 is not I; X 12 is not R; X 13 is not K; X 14 is not R; X 15 is not I; X 16 is not Y; X 17 is not R; X 18 is not E; X 19 is not T; X 20 is not M; X 21 is not T; X 22 is not I; X 23 is not I; X 24 is not Y; X 25 is not I; X 26 is not S; X 27 is not F; X 28 is not F; X 29 is not A; X 30 is not D; X 31 is not A; X 32 is not T; X 33 is not V; X 34 is not M.
3. The DNA polymerase of claim 1 , wherein the amino acid sequence is identical to SEQ ID NO:16.
4. The DNA polymerase of claim 1 , whose amino acid sequence comprises:
XXXXTXXXXXDXXXXXXIXXXXXXEXXXXYXXXXEXXFXXXXFXXXAXXXXXXXX AXXXXTVXTVKRXXXXQXXXXXRXVEXXXXXFTXXXXXXAXXDXIXXXXXXIXXYX XXXXXXXXXXXXXXXVXXXXDXXXXMXXXXXXXXXXXXXXXAEXXXLXXXXXXX EGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXVLIXXXXXNXXXAXXK XXCXXXXXNFALXXXXXXXXXXIXXMXXRFXXXXXXXXXXXXXPXXRXXXXXXXX XXXXXXXXVXXQXXXXXXXEXXTTXXXTXXXXXXXXRXXXXXXXVXXXXXXXXX XXXAXXXXXVXXPXXXXXXXXXXXXXXXXXXXXXXXXXXVXXXXXSXEXYQXXX XEXXTXXFXXXXXKXXXXXXXXXXXXAXXXXXXXXXXXXXXXXXLXXXXNXXIXX XXXXKXXXXIXXXXXXXXXHXXXXXXXXXTXXXEXQXXXXKIXXXXXXKXXXLXX XXFXXXXXXXKXXXXXXXXXXXXXXXXXKXXELVWXXLXXXFXXXXLXIXXXXLY XXXXXGESXEIXXXXLXXLXXXXAXXXXAXXXXXXXXXXXXXXXXXXKXXXXXXX XXITXXXXXXXXXXXXXXXXXXXXXXXALXXDXXXXKXXXXXXXXTEXXSKXXV XXXXXVXHXXXXXDXKDXXXTXXXXXXXXRXXXRXXXXRXXTXXSXXXXKXSXRX GDXXXPFDXFXXTXXXXXXXXXXXXXXXXXXEXXXRAXXXXXXXXXXXXXXXXXX SAXXKPXGT (SEQ ID NO:38)
wherein X is any amino acid or a peptide bond.
5. A kit comprising a DNA polymerase of claim 1 .