Compositions, methods and kits for nucleic acid synthesis and amplification

Abstract

The present invention is directed to compositions, methods and kits useful for the synthesis of nucleic acid molecules. More specifically, compositions, methods and kits are provided for the amplification of nucleic acid molecules in a one-step RT-PCR procedure comprising one or more agents used to increase tolerance to PCR inhibitors.

1 . A composition comprising:
 at least one DNA polymerase,   at least one reverse transcriptase (RT), and   a plurality of PCR inhibitor blocking agents,   wherein said PCR inhibitor blocking agents increases tolerance to one or more PCR inhibitors, and   wherein at least one of the PCR inhibitor blocking agents is a gelatin and is provided at a concentration of about 0.4% to about 4% (w/v).   
     
     
         2 . (canceled) 
     
     
         3 . The composition of  claim 1 , wherein said reverse transcriptase is an M-MLV reverse transcriptase or a mutant thereof. 
     
     
         4 . The composition of  claim 3 , wherein said MMLV RT comprises one or more mutations selected from the group consisting of: Y64, R116, D124, H126, Y133, K152, Q190, T197, H204, V223, M289, T306, or F309. 
     
     
         5 . (canceled) 
     
     
         6 . The composition of  claim 1 , wherein said DNA polymerase Taq DNA polymerase or a mutant thereof. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The composition of  claim 1 , wherein said gelatin is fish gelatin. 
     
     
         10 . The composition of  claim 1 , wherein the plurality of PCR inhibitor blocking agents further comprises an albumin. 
     
     
         11 . The composition of  claim 10 , wherein said albumin is bovine serum albumin (BSA). 
     
     
         12 . (canceled) 
     
     
         13 . The composition of  claim 11 , wherein BSA is included at a concentration of about 500 ng/μL to 5000 ng/μL. 
     
     
         14 - 22 . (canceled) 
     
     
         23 . The composition of  claim 1 , further comprising glycerol. 
     
     
         24 . The composition of  claim 23 , wherein the concentration of said glycerol is between 5%-50%. 
     
     
         25 . The composition of  claim 1 , further comprising RNase inhibitor protein (RIP) at a concentration of between 0.1 U/μL to 1.0 U/μL. 
     
     
         26 . (canceled) 
     
     
         27 . The composition of  claim 1 , further comprises a detergent at a concentration of between 0.005% to 0.05% (w/v). 
     
     
         28 . The composition of  claim 27 , wherein said detergent is NP-40 or TWEEN 20. 
     
     
         29 - 63 . (canceled) 
     
     
         64 . A reaction mixture comprising:
 at least one primer;   at least one reverse transcriptase;   at least one polymerase;   a plurality of PCR inhibitor blocking agents; and   at least one nucleotide,   wherein at least one of the PCR inhibitor blocking agents is a gelatin and is provided at a concentration of about 0.4% to about 4% (w/v).   
     
     
         65 . The reaction mixture of  claim 64 , further comprising a labeled probe. 
     
     
         66 - 67 . (canceled) 
     
     
         68 . The reaction mixture of  claim 64 , further comprising a nucleic acid template. 
     
     
         69 - 70 . (canceled) 
     
     
         71 . A kit, comprising in a single container a composition comprising:
 at least one reverse transcriptase,   at least one DNA polymerase, and   a plurality of PCR inhibitor blocking agents,   wherein at least one of the PCR inhibitor blocking agents is a gelatin and is provided at a concentration of about 0.4% to about 4% (w/v).   
     
     
         72 - 78 . (canceled) 
     
     
         79 . The kit of  claim 71 , further comprising in at least one other container a composition comprising at least one probe and/or at least one primer. 
     
     
         80 - 85 . (canceled) 
     
     
         86 . The kit of  claim 71 , wherein said reverse transcriptase is M-MLV or a mutant thereof having reverse transcriptase activity. 
     
     
         87 . The kit of  claim 71 , wherein said polymerase is Taq DNA polymerase or a mutant thereof having DNA polymerase activity.