Reverse transcriptase with enhanced properties

Patent metadata

Field	Value
Publication number	US-9920305-B2
Application number	US-201414516124-A
Country	US
Kind code	B2
Filing date	Oct 16, 2014
Priority date	Oct 16, 2013
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

Compositions and methods are provided for improved reverse transcriptases and their uses in reverse transcription where the improvement may include increased temperature, increased salt, increased activity and/or increased dUTP tolerance.

First claim

What is claimed is:

1. A non-naturally occurring reverse transcriptase comprising an amino acid sequence at least 96% identical to SEQ ID NO:18,
and wherein the amino acid sequence of the reverse transcriptase differs from SEQ ID NO:17 at all of the amino acid positions corresponding to Valine at position 35, Glutamic acid at position 122, Aspartic acid at position 177, Glutamine at position 207, Arginine at position 211, Leucine at position 214, Valine at position 245, Arginine at position 277, Threonine at position 286, Glutamic acid at position 291 Isoleucine at position 293, Glycine at position 359 Alanine at position 371, Threonine at position 376, Lysine at position 390, Lysine at position 431, Asparagine at position 460, Threonine at position 468, Aspartic acid at position 471, Tyrosine at position 483, Leucine at position 491, Glutamine at position 512, Alanine at position 534, and Alanine at position 554.

2. The reverse transcriptase of claim 1 , wherein the amino acid sequence is at least 98% identical to SEQ ID NO:18.

3. The reverse transcriptase of claim 1 , wherein the amino acid sequence is 100% identical to SEQ ID NO:18.

4. A reverse transcriptase according to claim 1 , wherein the reverse transcriptase further comprises a second, heterologous amino acid sequence.

5. An enzyme preparation comprising a reverse transcriptase according to claim 1 , wherein the enzyme preparation is substantially free of contaminating nucleic acids.

6. An enzyme preparation comprising a reverse transcriptase according to claim 1 and a detergent.

7. The enzyme preparation of claim 6 , wherein the detergent is selected from the group consisting of a non-ionic detergent, a cationic, an anionic detergent, and a zwitterionic detergent.

8. The enzyme preparation according to claim 5 , further comprising at least 0.35M KCl.

9. The enzyme preparation according to claim 5 , further comprising dUTP.

10. The enzyme preparation according to claim 5 , further comprising a target RNA.

11. The enzyme preparation according to claim 10 , wherein the target RNA is from a cell.

12. The enzyme preparation according to claim 10 , wherein the target RNA further comprises a synthetic RNA adapter.

13. The enzyme preparation according to claim 10 , wherein the RNA is the product of two RNA molecules ligated to form a single RNA molecule.

14. The enzyme preparation according to claim 10 , further comprising at least two primers.

15. The enzyme preparation of claim 14 , wherein at least one primer is attached to a solid support.

16. The enzyme preparation according to claim 14 , wherein the primer comprises a first nucleic acid sequence for hybridizing to a target RNA in a sample and a second nucleic acid sequence for encoding information about the target RNA upon amplification of the target RNA.

17. The enzyme preparation according to claim 5 , further comprising a DNA-dependent DNA polymerase.

18. The enzyme preparation of claim 17 , wherein the molecular ratio of the reverse transcriptase and the DNA-dependent DNA polymerase is less than 2:1.

19. The enzyme preparation according to claim 5 , further comprising a buffer.

20. The enzyme preparation of claim 19 , wherein the buffer has a pH between 8.3 and 9.3.

21. A method of reverse transcribing RNA, the method comprising incubating a reaction comprising RNA, the non-naturally occurring reverse transcriptase of claim 1 , a buffer, and deoxyribonucleotides, to reverse transcribe the RNA to produce a complementary strand of DNA.

22. The method of claim 21 , wherein the method shows improved activity, salt tolerance, thermostability, and/or dUTP tolerance as compared to a method comprising exposing the same RNA to AMV reverse transcriptase or MuMLV reverse transcriptase under the same conditions.

23. The method according to claim 21 , further comprising purifying the RNA prior to the incubation step.

24. The method according to claim 23 , wherein the purifying comprises hybridizing the RNA to a complementary oligonucleotide attached to a solid support.

25. The method according to claim 21 , further comprising exposing the complementary strand of DNA to a DNA-dependent DNA polymerase in the presence of primers, deoxyribonucleotides, and buffer.

26. The method of claim 25 , wherein the DNA-dependent DNA polymerase amplifies the DNA by loop-mediated isothermal amplification.

27. A kit comprising the reverse transcriptase according to claim 1 and, as one or more separate components, at least one element selected from the group consisting of: a buffer, a nucleic acid, deoxyribonucleotides, potassium chloride, magnesium sulfate, ammonium sulfate, a nucleic acid binding dye, a DNA-dependent DNA polymerase, and a uracil-DNA glycosylase.

28. A method for one-step RT-PCR, said method comprising:
(a) mixing an RNA template with a composition comprising the reverse transcriptase according to claim 1 and one or more DNA polymerases;
(b) incubating the mixture under conditions sufficient to amplify a DNA molecule complementary to all or a portion of the RNA template.

Assignees

• New England Biolabs Inc

Inventors

• Zhang Yinhua
• Evans Jr Thomas C

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12N9/1276 Primary

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

• C12Q2521/107

  RNA dependent DNA polymerase,(i.e. reverse transcriptase) · CPC title

• C12Y207/07049

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