COMPOSITIONS AND METHODS FOR cDNA SYNTHESIS

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.

1 . A method for reverse transcription of one or more nucleic acid molecules comprising incubating one or more nucleic acid templates in a buffer under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said one or more templates, wherein said buffer comprises:
 (a) at least one reverse transcriptase;   (b) an effective amount of a mixture of random primers, wherein said random primers are present in a concentration of at least about 5 ng/[11;   (c) an effective amount of oligo(dT), wherein said oligo(dT) is present in a concentration less than about 211M.   
     
     
         2 . The method according to  claim 1  wherein said random primers are present in a concentration of between about 5 ng/111 and about 20 ng/pl 
     
     
         3 . The method according to  claim 1  or  2 , wherein said oligo(dT) is present in a concentration of between about 25 nM and about 2 1110/1 
     
     
         4 . The method according to any of  claims 1 - 3 , wherein said random primers are between 5 and 10 nucleotides long. 
     
     
         5 . The method according to any of  claims 1 - 4 , wherein said random primers are random hexamers. 
     
     
         6 . The method according to any of  claims 1 - 5  wherein said oligo(dT) consists essentially of between about 12 and about 25 dT residues. 
     
     
         7 . The method according to any of  claims 1 - 6 , wherein said oligo(dT) is oligo(dT)12-1 8 or oligo(dT) 20 . 
     
     
         8 . The method according to any of  claims 1 - 7 , wherein said at least one reverse transcriptase is a viral reverse transcriptase. 
     
     
         9 . The method according to any of  claims 1 - 8 , wherein said reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, TTH DNA polymerase,  C. hydrogenoformans  DNA polymerase, Superscript II RT, Superscript I RT, Thermoscript RT MMLV and Rnase 1-1″ mutants thereof. 
     
     
         10 . A method for reverse transcription of one or more nucleic acid molecules comprising incubating one or more nucleic acid templates in a buffer under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said one or more templates, wherein said buffer comprises:
 (a) at least one reverse transcriptase,   (b) one or more primers suitable for priming reverse transcription of said one or more templates, and   (c) an effective amount of Li ion.   
     
     
         11 . The method according to  claim 10 , wherein said reverse transcriptase is a viral reverse transcriptase. 
     
     
         12 . The method according to  claim 11 , wherein said viral reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, Superscript 11 RT, Superscript 1 RT, Thermoscript RT MMLV and Rnase if mutants thereof. 
     
     
         13 . The method according to any of  claims 10 - 12 , wherein said Li ion is present in a concentration of between about 5 mM to about 200 mM. 
     
     
         14 . The method according to  claim 13 , wherein said buffer further comprises at least one additional monovalent cation in a concentration between about 20 mM and 200 mM, wherein said monovalent cation is selected from the group consisting of Na, K, and N114, and wherein the total concentration of said Li on and said further monovalent cation is less than or equal to about 200 mM. 
     
     
         15 . The method according to  claim 14 , wherein said additional monovalent cation is K. 
     
     
         16 . A reagent mixture suitable for use in a reverse transcription reaction of at least one template nucleic acid, comprising:
 glycerol in a concentration between about 10% and about 40%,   a buffer and   a reverse transcriptase,   wherein said reagent mixture demonstrates prolonged stability when stored at −20° C. and may be used directly for a reverse transcription reaction without adding additional reverse transcriptase.   
     
     
         17 . The mixture according to  claim 16 , wherein said buffer comprises:
 a monovalent cation selected from the group consisting of Li Na, K and NH4;   a magnesium salt;   a reducing agent,   nucleoside triphosphates,   and at least one non-ionic detergent.   
     
     
         18 . The mixture according to  claim 17 , wherein said buffer further comprises at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase. 
     
     
         19 . The mixture according to  claim 18 , wherein said buffer comprises an RNAse inhibitor protein. 
     
     
         20 . The mixture according to any of  claims 16 - 19 , wherein said buffer comprises a potassium salt, a magnesium salt, nucleoside triphosphates, DTT, at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase, at least one non-ionic detergent, and an RNAse inhibitor protein. 
     
     
         21 . (canceled)