High fidelity BbsI
View Patent ↗A composition comprising a variant BbsI restriction endonuclease having reduced star activity is provided, as well as kits and methods employing the same.
1. A composition comprising a variant BbsI restriction endonuclease comprising a subunit A and a subunit B, wherein:
the subunit A comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1, and
the subunit B comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2, and wherein the amino acid at the position corresponding to amino acid 442 of SEQ ID NO:2 in the variant BbsI restriction endonuclease is not a Pro.
2. The composition of claim 1 , wherein the amino acid at the position corresponding to amino acid 442 of SEQ ID NO:2 in the variant BbsI restriction endonuclease is an Ala.
3. The composition of claim 1 , wherein the composition comprises a non-naturally occurring buffering agent.
4. The composition of claim 1 , wherein the composition comprises glycerol.
5. The composition of claim 1 , wherein the composition comprises a fragment of DNA comprising one or more recognition sequences for the variant BbsI restriction endonuclease.
6. The composition of claim 1 , wherein the composition comprises multiple fragments of DNA that each comprises at least one recognition sequence for the variant BbsI restriction endonuclease.
7. The composition of claim 6 , wherein the multiple fragments have compatible overhangs after they are cleavage by the BbsI variant.
8. The composition of claim 1 , wherein the composition further comprises a ligase.
9. A kit comprising:
(a) the composition of claim 1 ; and
(b) a concentrated buffer comprising a non-naturally occurring buffering agent.
10. The kit of claim 9 , wherein the amino acid at the position corresponding to amino acid 442 of SEQ ID NO:2 in the variant BbsI restriction endonuclease is an Ala.
11. The kit of claim 9 , further comprising:
(c) a ligase.
12. The kit of claim 11 , wherein the ligase is T4 DNA ligase.
13. A method comprising:
combining the composition of claim 1 with a fragment of DNA comprising a recognition sequence for the variant BbsI restriction endonuclease and a buffer to produce a reaction mix; and
incubating the reaction mix to cleave the fragment.
14. The method of claim 13 , wherein the amino acid at the position corresponding to amino acid 442 of SEQ ID NO:2 in the variant BbsI restriction endonuclease is an Ala.
15. The method of claim 13 , wherein the reaction mix comprises multiple fragments of DNA that each comprises at least one recognition sequence for the variant BbsI restriction endonuclease.
16. The method of claim 14 , wherein at least two of the cleavage products have compatible overhangs.
17. The method of claim 15 , wherein the reaction mix further comprises a ligase, wherein the ligase ligates the compatible overhangs together.
18. The composition of claim 1 , wherein:
subunit A of the variant BbsI restriction endonuclease comprises an amino acid sequence that is identical to SEQ ID NO:1, and
subunit B of the variant BbsI restriction endonuclease comprises an amino acid sequence that is identical to SEQ ID NO:2 with the exception that the amino acid at the position corresponding to amino acid 442 of SEQ ID NO:2 is not a Pro.
19. The composition of claim 18 , wherein the amino acid at the position corresponding to amino acid 442 of SEQ ID NO:2 in subunit B is an Ala.