Method for synthesizing a cdna in a sample in an enzymatic reaction

Abstract

The present invention relates to a method for synthesizing a cDNA in a sample in an enzymatic reaction, characterized in that the method comprises the steps: simultaneously providing of a first enzyme with polyadenylation activity, a second enzyme with reverse transcriptase activity, a buffer, at least one ribonucleotide, at least one deoxyribonucleotide, an anchor oligonucleotide, adding of a sample comprising a ribonucleic acid and incubating the agents from the preceding steps in one or more temperature steps, which are selected so that the first enzyme and the second enzyme display activity, characterized in that additionally an amplification takes place in the same reaction mixture. The invention relates further to a reaction mixture comprising a first enzyme with polyadenylation activity, a second enzyme with reverse transcriptase activity, optionally a buffer, optionally at least one ribonucleotide, optionally at least one deoxyribonucleotide, optionally an anchor oligonucleotide and an enzyme with DNA synthesis activity.

1 . A method for synthesizing a cDNA in a sample in an enzymatic reaction,
 wherein the method comprises the following steps:
 a. simultaneously providing of a first enzyme with terminal transferase activity, a second enzyme with reverse transcriptase activity, a buffer, at least one ribonucleotide, at least one deoxyribonucleotide, an anchor oligonucleotide, and an enzyme and reagents for amplification of the cDNA generated, 
 b. adding of a sample comprising a ribonucleic acid and incubating the agents from steps a) and b) in one or more temperature steps, which are selected so that the first enzyme and the second enzyme display activity, and the cDNA thus generated in the method is then amplified. 
   
     
     
         2 . The method as claimed in  claim 1 , wherein the reaction additionally comprises at least one temperature step at a higher temperature of about 65° C. to 95° C. 
     
     
         3 . The method as claimed in  claim 1 , wherein the cDNA generated in the method is then amplified with a polymerase chain reaction and the reaction comprises random primers and/or comprises specific primers and/or optionally one or more probes. 
     
     
         4 . The method as claimed in  claim 1 , wherein the terminal transferase activity is a polyadenylation activity. 
     
     
         5 . The method as claimed in  claim 1 , wherein the sample comprises a ribonucleic acid, which is selected from the group comprising prokaryotic RNA, eukaryotic RNA, viral RNA, Archaea-RNA, miRNA, snoRNA, mRNA, tRNA, non-polyadenylated RNA, and rRNA and mixtures thereof. 
     
     
         6 . The method as claimed in  claim 1 , wherein the anchor oligonucleotide is selected from the group comprising a poly(A) oligonucleotide, poly(C) oligonucleotide, poly(T) oligonucleotide, poly(G) oligonucleotide, poly(U) oligonucleotide, poly(A) oligonucleotide additionally comprising a 5′-tail, poly(C) oligonucleotide additionally comprising a 5′-tail, poly(T) oligonucleotide additionally comprising a 5′-tail, poly(G) oligonucleotide additionally comprising a 5′-tail and poly(U) oligonucleotide additionally comprising a 5′-tail. 
     
     
         7 . The method as claimed in  claim 6 , wherein the anchor oligonucleotide has a length between 6 and 150 nucleotides, and optionally has an anchor sequence at the 3′-end. 
     
     
         8 . The method as claimed in  claim 6 , wherein the anchor oligonucleotide is a deoxyribonucleic acid (DNA), a peptide-nucleic acid (PNA) or a locked-nucleic acid (LNA), a phosphorothioate-deoxyribonucleic acid, a cyclohexene-nucleic acid (CeNA), an N3′-P5′-phosphoroamidate (NP), or a tricyclo-deoxyribonucleic acid (tcDNA). 
     
     
         9 . The method as claimed in  claim 1 , wherein the ribonucleotide can be selected from the group comprising adenosine-5′-triphosphate, thymine-5′-triphosphate, cytosine-5′-triphosphate, guanine-5′-triphosphate, uracil-5′-triphosphate, a ribonucleotide with a base analog, and wherein the ribonucleotide can optionally be modified or labeled. 
     
     
         10 . The method as claimed in  claim 1 , wherein the deoxyribonucleotide can be selected from the group comprising deoxyadenosine-5′-triphosphate (dATP), deoxythymine-5′-triphosphate (dTTP), deoxycytosine-5′-triphosphate (dCTP), deoxyguanine-5′-triphosphate (dGTP), deoxyuracil-5′-triphosphate (dUTP) and where the deoxyribonucleotide can optionally be modified or labeled. 
     
     
         11 . The method as claimed in  claim 10 , wherein the marker of the deoxyribonucleotide can be selected from the group comprising a radioactive marker, for example  32 P,  33 P,  35 S,  3 H, a fluorescent dye such as, for example, fluorescein-isothiocyanate (FITC), 6-carboxyfluorescein (FAM), xanthene, rhodamines, 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethodyfluorescein (JOE), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine 110; coumarins, such as umbelliferones, benzimides, such as Hoechst 33258; phenanthridines, such as Texas Red, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, cyanin dyes, such as Cy3, Cy5, Cy7, BODIPY dyes, and quinoline dyes and Alexa dyes. 
     
     
         12 . The method as claimed in  claim 11 , wherein the modification is selected from the group comprising biotinylation, digoxigenin labeling, and haptens. 
     
     
         13 . The method as claimed in  claim 10 , wherein the concentration of a deoxyribonucleotide is at least 0.01 mM in the reaction and is at most 10 mM in the reaction. 
     
     
         14 . The method as claimed in  claim 13 , wherein the deoxyribonucleotides dATP, dCTP, dGTP and dTTP are present at a concentration from 0.2 mM to 2 mM. 
     
     
         15 . The method as claimed in  claim 1 , wherein the buffer has a pH from 6 to 10 and comprises Mg 2+  ions. 
     
     
         16 . The method as claimed in  claim 1 , wherein the enzyme with terminal transferase activity or polyadenylation activity is selected from the group comprising enzymes of prokaryotic origin, eukaryotic origin, viral origin, Archaea origin and vegetable origin. 
     
     
         17 . The method as claimed in  claim 16 , wherein the enzyme with polyadenylation activity is selected from the group comprising poly(A) polymerase from  Escherichia coli , poly(A) polymerase from yeast, poly(A) polymerase from cattle, poly(A) polymerase from frog and human poly(A) polymerase. 
     
     
         18 . The method as claimed in  claim 1 , wherein the enzyme with reverse transcriptase activity is selected from the group comprising enzymes from viruses, bacteria, archaebacteria, eukaryotes and enzymes, in particular from thermostable organisms and enzymes that only acquire such a function through alteration of their gene sequence mutagenesis or as a result of corresponding buffer conditions. 
     
     
         19 . The method as claimed in  claim 18 , wherein the enzyme with reverse transcriptase activity is selected from the group comprising HIV reverse transcriptase, M-MLV reverse transcriptase, EAIV reverse transcriptase, AMV reverse transcriptase,  Thermus thermophilus  DNA Polymerase I, M-MLV RNase H −  (Superscript, Superscript II, Superscript III), Monsterscript (Epicentre), Omniscript, Sensiscript Reverse Transcriptase (Qiagen), ThermoScript and Thermo-X (both Invitrogen), AccuScript Reverse Transcriptase (Stratagene). 
     
     
         20 . The method as claimed in  claim 1 , wherein incubation of the agents in step c) takes place in one or more temperature steps, which are selected so that the first enzyme and the second enzyme display activity. 
     
     
         21 . A reaction mixture comprising a first enzyme with polyadenylation activity, a second enzyme with reverse transcriptase activity, optionally a buffer, optionally at least one ribonucleotide, optionally at least one deoxyribonucleotide and optionally an anchor oligonucleotide, optionally random primers, optionally homopolymeric nucleic acid, and an enzyme with DNA synthesis activity. 
     
     
         22 . The reaction mixture as claimed in  claim 21 , comprising a first enzyme with polyadenylation activity, a second enzyme with reverse transcriptase activity, a buffer, at least one ribonucleotide, at least one deoxyribonucleotide, an anchor oligonucleotide, optionally random primers, optionally a homopolymeric nucleic acid, and an enzyme with DNA synthesis activity, optionally at least one specific primer, characterized in that an essential component for the amplification step such as primer or enzyme with DNA synthesis activity is optionally in the same container, but either is temporarily inactivated or at first is spatially separate from the other reagents. 
     
     
         23 . A kit comprising a reaction mixture as claimed in  claim 21 .