Compositions and methods for cDNA synthesis
First Claim
1. A reagent mixture comprisinga ready to use reagent solution that demonstrates prolonged stability when stored at −
- 20°
C., wherein said solution comprises;
(a) glycerol in a concentration between about 10% and about 40%, and(b) a viral reverse transcriptase in a concentration sufficient for use in a reverse transcription reaction without adding additional reverse transcriptase, wherein said viral reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, THERMOSCRIPT RT, ASLV and Rnase H−
mutants thereof, SUPERSCRIPT II RT, and SUPERSCRIPT I RT,in a buffer suitable foruse in a reverse transcription reaction, wherein said buffer further comprises;
a metal ionnecessary for reverse transcriptase activity;
nucleoside triphosphates, andwherein said buffer does not contain Taq polymerase in a concentration suitable for a subsequent PCR reaction.
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Accused Products
Abstract
Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided. Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.
51 Citations
50 Claims
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1. A reagent mixture comprising
a ready to use reagent solution that demonstrates prolonged stability when stored at − - 20°
C., wherein said solution comprises;(a) glycerol in a concentration between about 10% and about 40%, and (b) a viral reverse transcriptase in a concentration sufficient for use in a reverse transcription reaction without adding additional reverse transcriptase, wherein said viral reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, THERMOSCRIPT RT, ASLV and Rnase H−
mutants thereof, SUPERSCRIPT II RT, and SUPERSCRIPT I RT,in a buffer suitable foruse in a reverse transcription reaction, wherein said buffer further comprises; a metal ionnecessary for reverse transcriptase activity; nucleoside triphosphates, and wherein said buffer does not contain Taq polymerase in a concentration suitable for a subsequent PCR reaction. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 45, 46, 47)
- 20°
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25. A reagent mixture comprising
a ready to use reagent solution that demonstrates prolonged stability when stored at − - 20°
C., wherein said solution comprises;
(a) glycerol in a concentration between about 10% and about 40%, and (b) a viral reverse transcriptase in a concentration sufficient for use in a reverse transcription reaction without adding additional reverse transcriptase, in a buffer suitable for use in a reverse transcription reaction, wherein said solution does not contain Taq DNA polymerase in a concentration suitable for a subsequent PCR reaction, and wherein addition of one volume of said solution to 4 volumes of a solution comprising an RNA template will permit cDNA synthesis from said RNA template. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 49, 50)
- 20°
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48. A reagent mixture comprising
a ready to use reagent mixture that demonstrates prolonged stability when stored at − -
20°
C. wherein said solution comprises;(a) glycerol in a concentration between about 10% and about 40%, and (b) a viral reverse transcriptase in a concentration sufficient for use in a reverse transcription reaction without adding additional reverse transcriptase, wherein said reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, THERMOSCRIPT RT, ASLV and Rnase H−
mutants thereof, SUPERSCRIPT II RT, and SUPERSCRIPT I RT,in a buffer suitable for use in a reverse transcription reaction, wherein said buffer further comprises; nucleoside triphosphates;
a potassium salt, a magnesium salt, nucleoside triphosphates, DTT, at least one random primer, oligo dT, at least one non-ionic detergent, and an RNAse inhibitor protein.
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20°
Specification