Reverse transcriptase mixtures with improved storage stability

Patent metadata

Field	Value
Publication number	US-9458484-B2
Application number	US-201113277763-A
Country	US
Kind code	B2
Filing date	Oct 20, 2011
Priority date	Oct 22, 2010
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

Reverse transcriptase mixtures with improved storage stability are provided.

First claim

What is claimed is:

1. A frozen sterile aqueous reaction mixture comprising sufficient ingredients, except template RNA, for reverse transcription of RNA, the mixture comprising:
a reverse transcriptase having an optimal pH above 8;
deoxytriphosphate nucleotides; and
a buffer, wherein the buffer is selected from the group consisting of Tris, HEPES, ACES, PIPES, MOPSO, BES, MOPS, TES, TAPSO, POPSO, BICINE, TAPS, and AMPSO,
wherein said frozen sterile aqueous reaction mixture has a pH of between 6-8 and lacks template RNA.

2. The frozen sterile aqueous reaction mixture of claim 1 , wherein said buffer, when measured at a concentration of 0.1 M, has a change of no more than 0.027 pH units per degree Celsius when between 20° C. and 37° C.

3. The frozen sterile aqueous reaction mixture of claim 1 , further comprising one or more polyols having a total polyol concentration of at least 15%, wherein said polyol is a four or more carbon alcohol.

4. The frozen sterile aqueous reaction mixture of claim 3 , wherein said polyol is selected from the group consisting of sorbitol and xylitol.

5. The frozen sterile aqueous reaction mixture of claim 1 , wherein said reverse transcriptase is selected from the group consisting of murine leukemia virus (MLV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV) reverse transcriptase, Equine Infectious Anemia Virus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase, SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT I reverse transcriptase, THERMOSCRIPT reverse transcriptase and MMLV RNase H-reverse transcriptase.

6. The frozen sterile aqueous reaction mixture of claim 1 , wherein said frozen sterile aqueous reaction mixture further comprises potassium chloride, magnesium, one or more detergents, an oligo dT, an oligonucleotide other than an oligo dT, an RNase inhibitor, or a combination thereof.

7. The frozen sterile aqueous reaction mixture of claim 1 , consisting of said reverse transcriptase, said buffer, potassium chloride, MgSO 4 , one or more polyol(s), EDTA, and oligonucleotides.

8. The frozen sterile aqueous reaction mixture of claim 1 , wherein said reverse transcriptase is selected from the group consisting of SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT I reverse transcriptase, THERMOSCRIPT reverse transcriptase and MMLV RNase H-reverse transcriptase.

9. The frozen sterile aqueous reaction mixture of claim 1 , further comprising one or more detergents.

10. The frozen sterile aqueous reaction mixture of claim 1 , further comprising an oligo dT oligonucleotide.

11. The frozen sterile aqueous reaction mixture of claim 1 , wherein said buffer is at a concentration of between 100-500 mM.

Assignees

• Ma Jason
• Gong Xiao-Song
• Bio Rad Laboratories Inc

Inventors

• Ma Jason
• Gong Xiao-Song

Classifications

• C12N9/96 Primary

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12N9/1276

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

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Y207/07049

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