Mutant reverse transcriptase and methods of use

Patent metadata

Field	Value
Publication number	US-9783791-B2
Application number	US-50281906-A
Country	US
Kind code	B2
Filing date	Aug 10, 2006
Priority date	Aug 10, 2005
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

The invention relates to the generation and characterization of stable MMLV reverse transcriptase mutants. The invention also discloses methods of using stable MMLV reverse transcriptase mutants.

First claim

What is claimed is:

1. An isolated mutant Moloney Murine Leukemia Virus (MMLV) reverse transcriptase having increased reverse transcriptase activity comprising the sequence of SEQ ID NO: 19 with the exception of a mutation at at least one of the following amino acid positions: E69, E302, W313, L435, N454, and M651,
wherein the mutant MMLV reverse transcriptase comprises at least one of the following mutations:
a glutamic acid to lysine mutation at position E69,
a glutamic acid to lysine mutation at position E302,
a glutamic acid to arginine mutation at position E302,
a tryptophan to phenylalanine mutation at position W313,
a leucine to glycine mutation at position L435,
a leucine to methionine mutation at position L435,
an asparagine to lysine mutation at position N454,
an asparagine to arginine mutation at position N454, and
a methionine to leucine mutation at position M651.

2. The mutant MMLV reverse transcriptase of claim 1 , comprising at least two of said mutations.

3. An isolated mutant MMLV reverse transcriptase having reverse transcriptase activity comprising the sequence of SEQ ID NO:19 with the exception that the sequence has a combination of mutations selected from the following groups of combinations of mutations: E302R or K/E69K/W313F/L435G or M/N454K or R; E302R or K/W313F/L435G or M/N454K or R; E302R or K/W313F/L435G or M; E302R or K/E69K/N454K or R; E302R or K/W313F; and E69K/E302R or K/W313F/L435G or M/N454K or R/D524N.

4. The mutant MMLV reverse transcriptase of claim 1 , further comprising a C-terminal extension.

5. The mutant MMLV reverse transcriptase of claim 4 , wherein said C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1).

6. The mutant MMLV reverse transcriptase of claim 1 , wherein said reverse transcriptase lacks RNase H activity.

7. The mutant MMLV reverse transcriptase of claim 1 , wherein the reverse transcriptase has at least one of the following characteristics: increased stability, increased accuracy, increased processivity, and increased specificity.

8. An isolated polynucleotide comprising a nucleotide sequence encoding a mutant MMLV reverse transcriptase of claim 1 .

9. The isolated polynucleotide of claim 8 , wherein the mutant MMLV reverse transcriptase comprises at least two of said mutations.

10. An isolated polynucleotide comprising a nucleotide sequence encoding a mutant MMLV reverse transcriptase of claim 3 .

11. The isolated polynucleotide of claim 8 , further encoding a C-terminal extension.

12. The isolated polynucleotide of claim 11 , wherein said C-terminal extension is RDRNKNNDRRKAKENE.

13. A composition comprising an isolated mutant MMLV reverse transcriptase having increased reverse transcriptase activity comprising the sequence of SEQ ID NO: 19, with the exception that at least one of the following amino acid positions comprises a mutation: E69, E302, W313, L435, N454, and M651,
wherein the mutant MMLV reverse transcriptase comprises at least one of the following mutations:
a glutamic acid to lysine mutation at position E69,
a glutamic acid to lysine mutation at position E302,
a glutamic acid to arginine mutation at position E302,
a tryptophan to phenylalanine mutation at position W313,
a leucine to glycine mutation at position L435,
a leucine to methionine mutation at position L435,
an asparagine to lysine mutation at position N454,
an asparagine to arginine mutation at position N454, and
a methionine to leucine mutation at position M651.

14. The composition of claim 13 , wherein the mutant MMLV reverse transcriptase comprises at least two of said mutations.

15. A composition comprising an isolated mutant MMLV reverse transcriptase having reverse transcriptase activity and comprising the sequence of SEQ ID NO:19 with the exception that the sequence has a combination of mutations selected from the following groups of combinations of mutations: E302R or K/E69K/W313F/L435G or M/N454K or R; E302R or K/W313F/L435G or M/N454K or R; E302R or K/W313F/L435G or M; E302R or K/E69K/N454K or R; E302R or K/W313F; and E69K/E302R or K/W313F/L435G or M/N454K or R/D524N.

16. The composition of claim 13 , wherein the reverse transcriptase further comprises a C-terminal extension.

17. The composition of claim 16 , wherein said C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1).

18. The composition of claim of claim 13 , wherein the reverse transcriptase has at least one of the following characteristics: increased stability, increased accuracy, increased processivity, and increased specificity.

19. The composition of claim 13 , wherein said reverse transcriptase lacks RNase H activity.

20. The composition of claim 13 , further comprising an epsilon subunit from an eubacteria.

21. The composition of claim 20 , wherein said epsilon subunit is from Eschericia coli.

22. The composition of claim 20 , wherein said epsilon subunit is epsilon 186 from Eschericia coli.

23. The composition of claim 13 , further comprising formamide, betaine, or dimethyl sulfoxide (DMSO).

24. A kit comprising
an isolated mutant MMLV reverse transcriptase having increased reverse transcriptase activity and comprising the sequence of SEQ ID NO: 19 with the exception of a mutation at at least one of the following amino acid positions: E69, E302, W313, L435, N454, and M651,
wherein the mutant MMLV reverse transcriptase comprises at least one of the following mutations:
a glutamic acid to lysine mutation at position E69,
a glutamic acid to lysine mutation at position E302,
a glutamic acid to arginine mutation at position E302,
a tryptophan to phenylalanine mutation at position W313,
a leucine to glycine mutation at position L435,
a leucine to methionine mutation at position L435,
an asparagine to lysine mutation at position N454,
an asparagine to arginine mutation at position N454, and
a methionine to leucine mutation at position M651.

25. The kit of claim 24 , wherein the mutant MMLV reverse transcriptase comprises at least two of said mutations.

26. A kit comprising
an isolated mutant MMLV reverse transcriptase having reverse transcriptase activity and comprising the sequence of SEQ ID NO:19 with the exception that the sequence has a combination of mutations selected from the following groups of combinations of mutations: E302R or K/E69K/W313F/L435G or M/N454K or R; E302R or K/W313F/L435G or M/N454K or R; E302R or K/W313F/L435G or M; E302R or K/E69K/N454K or R; E302R or K/W313F; and E69K/E302R or K/W313F/L435G or M/N454K or R/D524N, and
packaging materials therefor.

27. The kit of claim 24 , wherein said reverse transcriptase lacks RNase H activity.

28. The kit of claim 24 , wherein said mutant MMLV reverse transcriptase further comprises a C-terminal extension.

29. The kit of claim 28 , wherein said C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1).

30. The kit of claim 24 , wherein said reverse transcriptase has at least one of the following characteristics: increased stability, increased accuracy, increased processivity, and increased specificity.

31. The kit of claim 24 , further comprising an epsilon subunit from an eubacteria.

32. The kit of claim 31 , wherein said epsilon subunit is from Eschericia coli.

33. The kit of claim 31 , wherein said epsilon subunit is epsilon 186 from

Assignees

• Hogrefe Holly
• Arezi Bahram
• Xing Weimei
• Agilent Technologies Inc

Inventors

• Hogrefe Holly
• Arezi Bahram
• Xing Weimei

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12Y207/07049

  RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase · CPC title

• C12N9/1276 Primary

  RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title