Compositions and methods for cDNA synthesis
First Claim
Patent Images
1. A method for reverse transcription of one or more nucleic acid molecules comprising contacting a solution comprising one or more nucleic acid templates with a reagent mixture, wherein said reagent mixture comprises a ready to use reagent solution that demonstrates prolonged stability when stored at −
- 20° and
that was stored at −
20°
C. for at least two weeks prior to contacting said template solution comprising one or more nucleic acid templates with said reagent mixture, wherein said ready to use reagent mixture comprises;
(a) glycerol, 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(c) a buffer suitable for use in a reverse transcription reaction, wherein said buffer further comprisesa non-ionic detergent,a metal ion necessary for reverse transcriptase activity in a concentration sufficient for reverse transcriptase activity andnucleoside triphosphates;
and incubating the resulting solution at a temperature suitable for cDNA synthesis.
2 Assignments
0 Petitions
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.
35 Citations
9 Claims
-
1. A method for reverse transcription of one or more nucleic acid molecules comprising contacting a solution comprising one or more nucleic acid templates with a reagent mixture, wherein said reagent mixture comprises a ready to use reagent solution that demonstrates prolonged stability when stored at −
- 20° and
that was stored at −
20°
C. for at least two weeks prior to contacting said template solution comprising one or more nucleic acid templates with said reagent mixture, wherein said ready to use reagent mixture comprises;(a) glycerol, 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(c) a buffer suitable for use in a reverse transcription reaction, wherein said buffer further comprises a non-ionic detergent, a metal ion necessary for reverse transcriptase activity in a concentration sufficient for reverse transcriptase activity and nucleoside triphosphates; and incubating the resulting solution at a temperature suitable for cDNA synthesis. - View Dependent Claims (2, 3, 4, 5, 6)
- 20° and
-
7. A method for reverse transcription of one or more RNA molecules comprising contacting a solution comprising one or more RNA templates with a reagent mixture previously stored at −
- 20°
C., and incubating the resulting solution at a temperature suitable for cDNA synthesis,wherein said reagent mixture comprises a ready to use reagent solution in a buffer suitable for use in a reverse transcription reaction, wherein said reagent solution is stable for 2.5 months when stored at −
20°
C. and comprises glycerol, a non-ionic detergent, a magnesium salt in a concentration sufficient for reverse transcriptase activity, nucleoside triphosphates, a reducing agent, an RNAse inhibitor protein, random primers, a monovalent cation selected from the group consisting of Li, Na, K, and NH4,and 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. - View Dependent Claims (8)
- 20°
-
9. A method for reverse transcription of one or more RNA molecules comprising contacting a solution comprising one or more RNA templates with a reagent mixture previously stored at −
- 20°
C., and incubating the resulting solution at a temperature suitable for cDNA synthesis,wherein said reagent mixture comprises a ready to use reagent solution in a buffer suitable for use in a reverse transcription reaction, wherein said reagent solution is stable for 2.5 months when stored at −
20°
C. and comprises glycerol, a non-ionic detergent, a magnesium salt in a concentration sufficient for reverse transcriptase activity, nucleoside triphosphates, a reducing agent, an RNAse inhibitor protein, oligo dT, random primers, a monovalent cation selected from the group consisting of Li, Na, K, and NH4,and 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.
- 20°
Specification
-
- Resources
Litigation Campaign Assessment
Thank you for your request. You will receive a custom alert email when the Litigation Campaign Assessment is available.
×
-
Current AssigneeQiagen Beverly, Inc. d/b/a Enzymatics Inc. (Qiagen NV)
-
Original AssigneeQuanta Biosciences Incorporated
-
InventorsRashtchian, Ayoub, Schuster, David
-
Primary Examiner(s)Chunduru, Suryaprabha
-
Application NumberUS12/632,802Publication NumberTime in Patent Office1,225 DaysField of SearchNoneUS Class Current435/6.12CPC Class CodesC12N 15/1096 cDNA Synthesis; Subtracted ...C12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6846 Common amplification featuresC12Q 2521/107 RNA dependent DNA polymeras...C12Q 2525/179 incorporating arbitrary or ...C12Q 2527/143 Concentration of primer or ...
