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Compositions and methods for reverse transcription of nucleic acid molecules

a nucleic acid molecule and reverse transcription technology, applied in the field of molecular and cellular biology, can solve the problems of inefficient use of unmodified rt to catalyze reverse transcription, heterodimeric rt formation, etc., and achieve the effect of reducing or substantially reducing the activity of rnase h

Inactive Publication Date: 2007-11-22
LIFE TECH CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports heretofore of the simultaneous expression of cloned ASLV RT α and β genes resulting in the formation of heterodimeric αβ RT.
However, the use of unmodified RT to catalyze reverse transcription is inefficient for at least two reasons.

Method used

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  • Compositions and methods for reverse transcription of nucleic acid molecules
  • Compositions and methods for reverse transcription of nucleic acid molecules
  • Compositions and methods for reverse transcription of nucleic acid molecules

Examples

Experimental program
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Effect test

example 1

Cloning and Expression of RSV RNase H− RT

[0153] General Methods

[0154] RSV H− RT is a cloned form of retrovirus RT, in which both the α and the β subunits are mutated by a single amino acid change to eliminate RNase H activity. An RSV RT exhibiting substantially reduced RNase H activity is also produced when only the α subunit is mutated to substantially reduce RNase H activity (with the β subunit not being mutated in the RNase H domain). Mutations and plasmid constructions were conducted using standard molecular biology methods (see, e.g., Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: Laboratory Press (1989)), modified as described below.

[0155] Plasmid preparation. Plasmid preparations from 1 ml E. coli cultures were made by the alkaline lysis procedure (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Laboratory Press (1989)). From 10 ml cultures, the preparation was additionally t...

example 2

Isolation of RSV RNase H− RT

[0182] To provide purified recombinant RSV H− RT, cloned RSV H− RT was overexpressed in cultured insect cells as described in Example 1 and purified by affinity and ion-exchange chromatography. The RSV RT produced comprises the α and β subunits. Isolation of RSV RT provides a substantially pure RSV RT in which contaminating enzymes and other proteins have been substantially removed, although such contaminants need not be completely removed.

[0183] Buffers. The pH of all buffers was determined at 23° C., and buffers were stored at 4° C. until use. Crack Buffer contained 50 mM Tris-HCl (pH 7.9), 0.5 M KCl, 0.02% (v / v) Triton X-100 and 20% (v / v) glycerol. Just before use, the following protease inhibitors (Boehringer Mannheim; Indianapolis, Ind.) were added to Crack Buffer at the final concentrations indicated: leupeptin (2 mg / ml), Pefabloc (48 μg / ml), pepstatin A (2 μg / ml), benzamidine (800 μg / ml) and PMSF (50 μg / ml). Buffer A contained 20 mM Tris-HCl (pH ...

example 3

Preparation of Full-Length cDNA Molecules

[0188] Enzymes. SuperScript II RT (SS II RT), a cloned form of Moloney murine leukemia virus (M-MLV) RT lacking demonstrable RNase H activity (i.e., an “RNase H− RT”), was from Life Technologies, Inc. (Rockville, Md.). M-MLV RT, a cloned murine RT with full RNase H activity (i.e., an “RNase H+ RT”), was also from Life Technologies, Inc. AMV RT, an RNase H+ uncloned form of avian myeloblastosis virus RT, was from Seikagaku America, Inc. RSV H− RT was prepared as described in Examples 1 and 2. Recombinant Tth DNA polymerase, a cloned, thermophilic DNA polymerase from Thermus thermophilus with reverse transcriptase activity, was from Perkin Elmer.

[0189] Synthetic mRNA. A 7.5 kilobase (Kb) synthetic mRNA with a 120-nucleotide 3′ poly(A) tail (Life Technologies, Inc.; Rockville, Md.) was used as template to test the efficiency of various enzymes alone or in combination.

[0190] cDNA Synthesis Reaction Mixtures. Reaction mixtures (20 μl each) cont...

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Abstract

The present invention is generally related to compositions and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also relates to methods for producing Rous Sarcoma Virus (RSV) and Avian Myeloblastosis Virus (AMV) RTs or other Avian Sarcoma-Leukosis Virus (ASLV) RTs (α and / or β subunits thereof), to isolated nucleic acid molecules encoding such RSV RT, AMV RT or other ASLV RT subunits, to vectors and host cells comprising these isolated nucleic acid molecules and to RSV RT, AMV RT and other ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant heterodimeric RT holoenzymes, particularly heterodimeric RSV RTs, AMV RTs or other ASLV RTs (which may be αβ RTs, ββ RTs, or α RTs), vectors (particularly baculovirus vectors) and host cells (particularly insect and yeast cells) comprising these nucleic acid molecules, methods for producing these heterodimeric RTs and heterodimeric RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, or RSV RTs, AMV RTs or other ASLV RTs of the invention.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of U.S. Provisional Application Nos. 60 / 044,589, filed Apr. 22, 1997, and 60 / 049,874, filed Jun. 17, 1997, the disclosures of which are entirely incorporated by reference herein.FIELD OF THE INVENTION [0002] The present invention is in the fields of molecular and cellular biology. The invention is generally related to reverse transcriptase enzymes and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of reverse transcriptase enzymes, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these reverse transcriptase enzymes or compositions. The invention also relates to nucleic acid molecules produced by these methods and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also con...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/00C12N1/15C12N15/09C12N1/19C12N1/21C12N5/10C12N9/12C12N15/10C12Q1/68
CPCC12N9/1276C12N15/1096C12N2740/10011C12N2740/11011C12Q1/6848C12Q2527/101C12Q2521/301C12Q2521/107
Inventor GERARD, GARY F.SMITH, MICHAEL D.CHATTERJEE, DEB K.
Owner LIFE TECH CORP
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