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 at least one PCR inhibitor blocking agent, wherein said PCR inhibitor blocking agent increases tolerance to one or more PCR inhibitors. 
     
     
         2 . The composition of  claim 1 , wherein said reverse transcriptase is a thermostable reverse transcriptase. 
     
     
         3 . The composition of  claim 2 , wherein said thermo stable reverse transcriptase is an M-MLV reverse transcriptase or a mutant, variant, or derivative 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 . The composition of  claim 1 , wherein said DNA polymerase is a thermostable DNA polymerase. 
     
     
         6 . The composition of  claim 5 , wherein said thermo stable DNA polymerase Taq DNA polymerase or a mutant, variant, or derivative thereof. 
     
     
         7 . The composition of  claim 1 , wherein said PCR inhibitor blocking agent is a protein. 
     
     
         8 . The composition of  claim 7 , wherein said protein is a gelatin. 
     
     
         9 . The composition of  claim 8 , wherein said gelatin is fish gelatin. 
     
     
         10 . The composition of  claim 7 , wherein said protein is an albumin. 
     
     
         11 . The composition of  claim 10 , wherein said albumin is bovine serum albumin (BSA). 
     
     
         12 . The composition of  claim 1 , wherein said composition comprises at least both fish gelatin and BSA. 
     
     
         13 . The composition of  claim 12 , wherein BSA is at a concentration of about 500 ng/μL to 5000 ng/μL. 
     
     
         14 . The composition of  claim 12 , wherein said fish gelatin is at a concentration of about 0.4% to 4%. 
     
     
         15 . The composition of  claim 1 , wherein said composition is a liquid or a gel at −20° C. 
     
     
         16 . The composition of  claim 1 , wherein said composition is not a solid at −20° C. 
     
     
         17 . The composition of  claim 1 , wherein said composition is not frozen at −20° C. 
     
     
         18 . The composition of  claim 1 , wherein said composition does not require thawing prior to use. 
     
     
         19 . The composition of  claim 1 , wherein said PCR inhibitor is selected from the group consisting of hematin, humic acid, heparin or EDTA. 
     
     
         20 . The composition of  claim 1 , further comprising one or more nucleotides (dNTPs). 
     
     
         21 . The composition of  claim 20 , wherein said nucleotides are selected from the group consisting of dTTP, dATP, dCTP, dGTP or dUTP. 
     
     
         22 . The composition of  claim 20 , wherein the concentration of each of said nucleotides is about 0.5 mM to 5 mM. 
     
     
         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). 
     
     
         26 . The composition of  claim 25 , wherein said concentration of said RIP is between 0.1 U/μL to 1.0 U/μL. 
     
     
         27 . The composition of  claim 1 , further comprises a detergent. 
     
     
         28 . The composition of  claim 27 , wherein said detergent is NP-40 or TWEEN 20. 
     
     
         29 . The composition of  claim 27 , wherein said concentration of said detergent is between 0.005% to 0.05%. 
     
     
         30 . The composition of  claim 1 , further comprising a passive reference control. 
     
     
         31 . The composition of  claim 30 , wherein said passive reference control is a ROX dye. 
     
     
         32 . The composition of  claim 1 , wherein said composition is a concentrated stock solution. 
     
     
         33 . The composition of  claim 32 , where said concentrated stock solution is a 2× to 5× stock solution. 
     
     
         34 . The composition of  claim 1  or  32 , wherein said composition is for use in nucleic acid synthesis, nucleic acid amplification or RT-PCR methods. 
     
     
         35 . A method of performing RT-PCR of a nucleic acid sample comprising:
 mixing a composition comprising:
 at least one active reverse transcriptase; 
 at least one active DNA polymerase; and 
 at least one PCR inhibitor blocking agent, wherein said PCR inhibitor blocking agent increases tolerance to one or more PCR inhibitors, with: 
 a) a nucleic acid sample; 
 b) one or more labeled probes; 
 c) one or more primers; and 
   performing a RT-PCR on said nucleic acid sample.   
     
     
         36 . The method of  claim 35 , wherein the nucleic acid sample is obtained from a source containing a PCR inhibitor. 
     
     
         37 . The method of  claim 36 , wherein said nucleic acid source is selected from the group consisting of blood, sweat, tears, soil, saliva, urine, and feces. 
     
     
         38 . The method of  claim 35 , wherein said labeled probe is a TaqMan® probe. 
     
     
         39 . The method of  claim 35 , wherein said one or more PCR inhibitors is selected from the group consisting of hematin, humic acid and heparin. 
     
     
         40 . The method of  claim 35 , wherein at least one of said PCR inhibitor blocking agents is fish gelatin. 
     
     
         41 . The method of  claim 35 , wherein at least one of said PCR inhibitor blocking agents is BSA. 
     
     
         42 . The method of  claim 35 , wherein said RT-PCR is performed in a single tube or reaction. 
     
     
         43 . The method of  claim 35 , wherein said composition is not thawed prior to said mixing with a, b or c. 
     
     
         44 . The method of  claim 34 , wherein said increased tolerance is indicated by a decrease in C t . 
     
     
         45 . The method of  claim 34 , wherein said increased tolerance is by at least 10% when compared to methods using compositions without PCR inhibitor blocking agents. 
     
     
         46 . The method of  claim 44 , wherein the C r  is decreased by at least one C t . 
     
     
         47 . The method of  claim 41 , wherein said concentration of said BSA is at least 500 ng/μL. 
     
     
         48 . The method of  claim 46 , wherein a decrease in C t  of at least 8 is observed when at least 40 μM hematin, or by at least 7 when at least 10 ng/μL humic acid is present in said composition. 
     
     
         49 . The method of  claim 40 , wherein said concentration of fish gelatin is at least 1%. 
     
     
         50 . The method of  claim 46 , wherein a decrease in C t  of at least 3 is observed when at least 10 ng/μL humic acid, or by at least 6 when at least at least 0.06 U heparin is present in said composition. 
     
     
         51 . A method for amplifying a nucleic acid molecule, said method comprising:
 mixing a nucleic acid template with a composition comprising one or more reverse transcriptases, one or more DNA polymerases, and one or more PCR inhibitor blocking agents, to form a reaction mixture; and   incubating said reaction mixture under conditions sufficient to amplify a nucleic acid molecule complementary to all or a portion of said nucleic acid template.   
     
     
         52 . The method of  claim 51 , wherein said nucleic acid template is RNA. 
     
     
         53 . The method of  claim 51 , wherein said nucleic acid template is DNA. 
     
     
         54 . The method of  claim 51 , wherein at least one of said PCR inhibitor blocking agents is fish gelatin. 
     
     
         55 . The method of  claim 51 , wherein at least one of said PCR inhibitor blocking agents is BSA. 
     
     
         56 . The method of  claim 51 , wherein said PCR inhibitor blocking agent increases tolerance to one or more PCR inhibitors. 
     
     
         57 . The method of  claim 51 , wherein at least one of said PCR inhibitors is selected from the group consisting of hematin, humic acid or heparin. 
     
     
         58 . The method of  claim 56 , wherein said increase in tolerance is indicated by a decrease in C t . 
     
     
         59 . The method of  claim 58 , wherein said decrease in C t  is by at least 1 C t . 
     
     
         60 . A method for nucleic acid synthesis, said method comprising:
 mixing one or more first nucleic acid molecules with one or more reverse transcriptases, one or more polymerases, and one or more PCR inhibitor blocking agents; and   incubating said mixture under conditions sufficient to make one or more second nucleic acid molecules complementary to all or a portion of said one or more first nucleic acid molecules.   
     
     
         61 . The method of  claim 60 , wherein said first nucleic acid molecules is RNA. 
     
     
         62 . The method of  claim 60 , wherein said first or second nucleic acid molecules is DNA. 
     
     
         63 . The method of  claim 60 , wherein said PCR inhibitor blocking agent is fish gelatin or BSA. 
     
     
         64 . A reaction mixture comprising:
 at least one reverse transcriptase;   at least one polymerase;   at least one PCR inhibitor blocking agent; and   at least one nucleotide.   
     
     
         65 . The reaction mixture of  claim 64 , further comprising a labeled probe. 
     
     
         66 . The reaction mixture of  claim 65 , wherein said probe is a TaqMan® probe. 
     
     
         67 . The reaction mixture of  claim 64 , further comprising at least one primer. 
     
     
         68 . The reaction mixture of  claim 64 , further comprising a nucleic acid template. 
     
     
         69 . The reaction mixture of  claim 68 , wherein said nucleic acid template is RNA. 
     
     
         70 . The reaction mixture of  claim 68 , wherein said nucleic acid template is DNA. 
     
     
         71 . A kit, comprising in a single container a composition comprising at least one reverse transcriptase, at least one DNA polymerase, and at least one PCR inhibitor blocking agent. 
     
     
         72 . The kit of  claim 71 , wherein said composition is a gel at −20° C. 
     
     
         73 . The kit of  claim 71 , wherein said composition is not solid at −20° C. 
     
     
         74 . The kit of  claim 71 , wherein said composition is not frozen at −20° C. 
     
     
         75 . The kit of  claim 71 , wherein said composition is a 4× concentrated stock solution. 
     
     
         76 . The kit of  claim 71 , wherein said composition is used for RT-PCR methods. 
     
     
         77 . The kit of  claim 71 , wherein said composition is used for nucleic acid synthesis methods. 
     
     
         78 . The kit of  claim 71 , wherein said composition is used for nucleic amplification methods. 
     
     
         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 . The kit of  claim 79 , wherein said probe is a TaqMan® probe. 
     
     
         81 . The kit of  claim 71 , wherein said composition further comprises at least one nucleotide. 
     
     
         82 . The kit of  claim 71 , wherein said composition further comprises a buffer agent or a salt solution. 
     
     
         83 . The kit of any of  claims 76 - 78 , wherein said methods comprise thermal cycling. 
     
     
         84 . The kit of  claim 83 , wherein said thermal cycling is fast thermal cycling. 
     
     
         85 . The kit of any of  claims 76 - 78 , wherein said methods comprise multiplexing. 
     
     
         86 . The kit of  claim 71 , wherein said reverse transcriptase is M-MLV or any mutant, variant or derivative thereof having reverse transcriptase activity. 
     
     
         87 . The kit of  claim 71 , wherein said polymerase is Taq DNA polymerase or any mutant, variant or derivative thereof having DNA polymerase activity.