DNA POLYMERASES FOR EFFICIENT AND EFFECTIVE INCORPORATION OF METHYLATED-DNTPS
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
C12N-009/12
C12P-019/34
C12Q-001/6806
출원번호
16981397
(2019-03-21)
공개번호
20210032609
(2021-02-04)
국제출원번호
PCT/EP2019/057080
(2019-03-21)
발명자
/ 주소
BAUER, Keith A.
ECKERT, Barbara
출원인 / 주소
BAUER, Keith A.
인용정보
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0인용 특허 :
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초록▼
Disclosed are DNA polymerases having improved ability to incorporate methylated-dNTPs, relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vect
Disclosed are DNA polymerases having improved ability to incorporate methylated-dNTPs, relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases. Further disclosed are kits and reaction mixtures comprising the improved DNA polymerases as well as methods of primer extension using the improved DNA polymerases.
대표청구항▼
1. A modified DNA polymerase having increased efficiency in incorporating methylated deoxynucleotide triphosphates (dNTPs) as compared with a control DNA polymerase, wherein, the modified DNA polymerase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ
1. A modified DNA polymerase having increased efficiency in incorporating methylated deoxynucleotide triphosphates (dNTPs) as compared with a control DNA polymerase, wherein, the modified DNA polymerase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2,wherein the amino acid sequence of the modified DNA polymerase and the amino acid sequence of the control DNA polymerase differ only at a position corresponding to 640 of SEQ ID NO:2 and/or at a position corresponding to position 705 of SEQ ID NO:2,wherein the amino acid of the modified DNA polymerase at the position corresponding to position 640 of SEQ ID NO:2 is any amino acid other than I, and/or wherein the amino acid of the modified DNA polymerase at the position corresponding to position 705 of SEQ ID NO:2 is any amino acid other than V, andwherein the amino acid of the control DNA polymerase at the position corresponding to position 640 of SEQ ID NO:2 is I, and/or wherein the amino acid of the control DNA polymerase at the position corresponding to position 705 of SEQ ID NO:2 is V. 2. The modified DNA polymerase of claim 1, wherein the amino acid of the control DNA polymerase at the position corresponding to position 640 of SEQ ID NO:2 is I, and wherein the amino acid of the control DNA polymerase at the position corresponding to position 705 of SEQ ID NO:2 is V. 3. The modified DNA polymerase of claim 2, wherein the control DNA polymerase comprises the amino acid sequence of SEQ ID NO:2. 4. The modified DNA polymerase of claim 1, wherein the amino acid of the modified DNA polymerase corresponding to position 640 of SEQ ID NO:2 is M. 5. The modified DNA polymerase of claim 4, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:4. 6. The modified DNA polymerase of claim 4, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 7. The modified DNA polymerase of claim 1, wherein the amino acid of the modified DNA polymerase corresponding to position 705 of SEQ ID NO:2 is L. 8. The modified DNA polymerase of claim 7, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:5. 9. The modified DNA polymerase of claim 7, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 10. The modified DNA polymerase of claim 1, wherein the amino acid of the modified DNA polymerase corresponding to position 640 of SEQ ID NO:2 is M, and wherein the amino acid of the modified DNA polymerase corresponding to position 705 of SEQ ID NO:2 is L. 11. The modified DNA polymerase of claim 10, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 12. The modified DNA polymerase of claim 1, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:4. 13. The modified DNA polymerase of claim 1, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:5. 14. The modified DNA polymerase of claim 1, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 15. A recombinant nucleic acid encoding the modified DNA polymerase according to claim 1. 16. An expression vector comprising the recombinant nucleic acid of claim 15. 17. A kit for producing an extended primer comprising at least one container providing a modified DNA polymerase as in claim 1. 18. The kit of claim 17, further comprising one or more additional containers selected from the group consisting of: (a) a container providing a primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template;(b) a container providing dNTPs; and(c) a container providing a buffer suitable for primer extension. 19. The kit of claim 18, wherein the dNTPs are methylated dNTP. 20. A reaction mixture comprising the modified DNA polymerase of claim 1, at least one primer, a polynucleotide template, and dNTPs. 21. The reaction mixture of claim 20, wherein the polynucleotide template is RNA. 22. The reaction mixture of claim 20, wherein the polynucleotide template is DNA. 23. The reaction mixture of claim 20, further comprising Mg2+. 24. The reaction mixture of claim 20, further comprising a second thermostable DNA polymerase. 25. A method for conducting extension of one or more primers, the method comprising: contacting a modified DNA polymerase with the one or more primers, a polynucleotide template, and deoxynucleotide triphosphates (dNTPs), under conditions suitable for extension of the one or more primers, thereby extending the one or more primers, wherein, the modified DNA polymerase has increased efficiency in incorporating methylated dNTPs as compared with a control DNA polymerase,wherein, the modified DNA polymerase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2,wherein the amino acid sequence of the modified DNA polymerase and the amino acid sequence of the control DNA polymerase differ only at a position corresponding to 640 of SEQ ID NO:2 and/or at a position corresponding to position 705 of SEQ ID NO:2,wherein the amino acid of the modified DNA polymerase at the position corresponding to position 640 of SEQ ID NO:2 is any amino acid other than I, and/or wherein the amino acid of the modified DNA polymerase at the position corresponding to position 705 of SEQ ID NO:2 is any amino acid other than V, andwherein the amino acid of the control DNA polymerase at the position corresponding to position 640 of SEQ ID NO:2 is I, and/or wherein the amino acid of the control DNA polymerase at the position corresponding to position 705 of SEQ ID NO:2 is V. 26. The method of claim 25, wherein the amino acid of the control DNA polymerase at the position corresponding to position 640 of SEQ ID NO:2 is I, and wherein the amino acid of the control DNA polymerase at the position corresponding to position 705 of SEQ ID NO:2 is V. 27. The method of claim 26, wherein the control DNA polymerase comprises the amino acid sequence of SEQ ID NO:2. 28. The method of claim 25, wherein the amino acid of the modified DNA polymerase corresponding to position 640 of SEQ ID NO:2 is M. 29. The method of claim 28, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:4. 30. The method of claim 28, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 31. The method of claim 25, wherein the amino acid of the modified DNA polymerase corresponding to position 705 of SEQ ID NO:2 is L. 32. The method of claim 31, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:5. 33. The method of claim 31, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 34. The method of claim 25, wherein the amino acid of the modified DNA polymerase corresponding to position 640 of SEQ ID NO:2 is M, and wherein the amino acid of the modified DNA polymerase corresponding to position 705 of SEQ ID NO:2 is L. 35. The method of claim 34, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 36. The method of claim 25, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:4. 37. The method of claim 25, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:5. 38. The method of claim 25, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:3. 39. A DNA polymerase comprising the amino acid sequence of SEQ ID NO:4. 40. A DNA polymerase comprising the amino acid sequence of SEQ ID NO:5. 41. A DNA polymerase comprising the amino acid sequence of SEQ ID NO:3.
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