Exonuclease deficient polymerases

Patent metadata

Field	Value
Publication number	US-10947516-B2
Application number	US-201916280890-A
Country	US
Kind code	B2
Filing date	Feb 20, 2019
Priority date	Feb 29, 2016
Publication date	Mar 16, 2021
Grant date	Mar 16, 2021

Abstract

Described herein are polymerase variants that are exonuclease deficient. Some variants retain the strand displacement capability comparable to the wild-type or parental polymerase. Some variants have a strand displacement capability that is improved relative to the wild-type or parental polymerase. The variants may have an extension rate that is greater than the wild-type or parental polymerase. The variants may have a waiting time that is less than the wild-type or parental polymerase.

First claim

What is claimed is:

1. A modified DNA polymerase, derived from a parental DNA polymerase polypeptide having a DNA polymerase activity comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, comprising a substitution at a position corresponding to a position selected from the group consisting of T45, H123, L125, W127, D128, V179, S211, Y212, I215, T216, E219, Q221, E239, D241, Y259, Q260, A292, S293, and S294 of SEQ ID NO: 2, wherein the modified polymerase is exonuclease deficient.

2. The modified DNA polymerase according to claim 1 , wherein said modified polymerase retains a strand displacement capability.

3. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution selected from L125K, D128H, V179Y, V179R, S211F, Y212K, I215L, T216K, Q221R, Y259G/K/Q, A292K, S293G, and S294T.

4. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution corresponding to L125K.

5. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution corresponding to D128H.

6. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution corresponding to V179Y or V179R.

7. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution selected from the group consisting of S211F, Y212K, I215L, T216K, and Q221R.

8. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution corresponding to Y259G.

9. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution corresponding to Y259K.

10. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution corresponding to Y259Q.

11. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a substitution selected from the group consisting of A292K, S293G, and S294T.

12. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

13. The modified DNA polymerase according to claim 1 , wherein said modified polymerase comprises a set of substitutions selected from the group consisting of:
a. T529M+S366A+A547F+Y259G/K/Q,
b. T529M+S366A+A547F+D128H,
c. T529M+S366A+A547F+Y212G, and
d. T529M+S366A+A547F+Y212K.

14. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has an extension rate that is greater than the parental polymerase.

15. The modified DNA polymerase of claim 14 , wherein the extension rate is at least 1.5 times greater than the parental polymerase.

16. The modified DNA polymerase according to claim 1 , wherein said modified polymerase has a median waiting time that is less than the wild-type or parental polymerase.

17. The modified DNA polymerase according to claim 16 , wherein said modified polymerase has a median waiting time that is less than the waiting time of the polymerase according to SEQ ID NO: 11.

18. The modified DNA polymerase according to claim 16 , wherein said modified polymerase has a median waiting time that is less than 3 seconds.

19. An exonuclease-deficient DNA polymerase having a strand displacement activity, wherein said exonuclease-deficient DNA polymerase comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 and comprises a substitution at a position corresponding to a position selected from the group consisting of T45, H123, L125, W127, D128, V179, S211, Y212, I215, T216, E219, Q221, E239, D241, Y259, Q260, A292, S293, and S294 of SEQ ID NO: 2.

20. The exonuclease-deficient DNA polymerase of claim 19 , wherein said exonuclease-deficient DNA polymerase has a shorter wait time than a DNA polymerase according to SEQ ID NO: 1.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• AYER Aruna
• Bibillo Arkadiusz
• Sarvabhowman Preethi
• Dalal Dhruti Vasudev
• Lederman Ilya
• Thai Eileen

Classifications

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title