Thermostable blunt-end ligase and methods of use
Fusion proteins having thermostable blunt-end ligase activity are provided. Blunt-end ligases are useful for DNA amplification, sequencing, production of recombinant DNA and recombinant fusion proteins, and other purposes. These thermostable blunt-end DNA ligases are useful in ligation schemes which include, e.g., an incubation at about 60-65° C. or higher, or as high as about 94° C., or at other temperatures. The ligases disclosed herein may enable high temperature blunt-end ligation without need for molecular crowding agents, and so may be useful for many nucleic acid ligation-amplification schemes, e.g., ones which operate at a uniform temperature (e.g., at about 60° C. or higher), including ones which require temperature cycling, e.g., from about 94° C. to about 60° C. (or higher) for one, two, three, or more cycles. The thermostable blunt-end DNA ligases disclosed herein enable higher specificity target amplification, for example, by permitting temperature denaturation of double-stranded DNA templates as well as specific primer binding.
1. A method of performing a blunt-end DNA ligase reaction, comprising:
Combining DNA molecules to be ligated and a thermostable blunt-end DNA ligase in a solution, wherein said thermostable blunt-end DNA ligase comprises the amino acid sequence: mghhhhhhhhhhssghiegrasadgpylqileqpkqrgfrfryvcegpshgglpgasseknkksypqvkicnyvgpakvivqlvtn gknihlhahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdr ekelirqaalqqtkemdlsvvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddiqir fyeeeenggvwegfgdfsptdvhrqfaivfkpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsg ggsgggmtleearkrvnelrdliryhnyryyvladpeisdaeydrllrelkeleerfpelkspdsptlqvgarpleatfrpvrhptrmysld nafnldelkafeerieralgrkgpfaytvehkvdglsvnlyyeegylvygatrgdgevgeevtqnlltiptiprrlkgvperlevrgevym pieaflrineeleergerifknprnaaagslrqkdpritakrglratfyalglgleeveregvatqfallhwlkekgfpvehgyaravgaegv eavyqdwlkkrralpfeadgvvvkldelalwrelgytaraprfaiaykfpaeeketrlldvvfqvgrtgrvtpvgilepvflegsevsrvtl hnesyieeldirigdwvlvhkaggvipevlrvlkerrtgeerpirwpetcpecghrllkegkvhrcpnplcpakrfeairhfasrkamdi qglgeklierllekglvkdvadlyrlrkedlvglermgeksaqnllrqieeskkrglerllyalglpgvgevlarnlaarfgnmdrlleasle elleveevgeltarailetlkdpafrdlvrrlkeagvemeakekggealkgltfvitgelsrpreevkallrrlgakvtdsysrktsylvvgen pgsklekaralgvptlteeelyffleartgkkaeelv (SEQ ID NO: 8); and
performing a blunt-ended DNA ligation reaction at a temperature of about 60° C. or higher.
2. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction is performed at a temperature of about 60° C. or higher is performed at a uniform temperature.
3. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction is performed at a temperature of about 60° C. to about 65° C.
4. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction is performed at a temperature of about 75° C.
5. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction comprises temperature cycling.
6. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction comprises temperature cycling from about 94° C. to about 60° C.
7. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction comprises two or more cycles of temperature cycling.
8. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction comprises two or more cycles of temperature cycling from about 94° C. to about 60° C.
9. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction comprises three or more cycles of temperature cycling.
10. The method of claim 1 , wherein said thermostable blunt-end DNA ligase reaction comprises three or more cycles of temperature cycling from about 94° C. to about 60° C.
11. The method of claim 1 , wherein said thermostable blunt-end DNA ligase comprises a fusion protein comprising a polymer.
12. The method of claim 1 , wherein said thermostable blunt-end DNA ligase comprises a fusion protein comprising a sugar.
13. The method of claim 1 , wherein said thermostable blunt-end DNA ligase comprises a fusion protein comprising a sugar and comprising a polymer.
14. The method of claim 1 , wherein said thermostable blunt-end DNA ligase comprises a fusion protein comprising a component selected from a peptide linker, an N-terminal addition, a C-terminal addition, and a peptide mimetic.
15. The method of claim 14 , wherein said method is performed at about 60° C. to about 65° C.
16. The method of claim 14 , wherein said thermostable blunt-end DNA ligase comprises a fusion protein comprising a sugar and comprising a polymer.
17. The method of claim 14 , wherein said thermostable blunt-end DNA ligase comprises an affinity tag comprising polyhistidine.
18. The method of claim 1 , comprising multiple ligation events effective to provide concatamers.
19. The method of claim 18 , wherein said multiple ligation events effective to provide concatamers are performed at about 75° C.
20. The method of claim 18 , wherein said thermostable blunt-end DNA ligase comprises a fusion protein comprising a component selected from a peptide linker, an N-terminal addition, a C-terminal addition, and a peptide mimetic.