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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions

Definitions

• the invention relates to the field of molecular biology and research in this area but also human and non-human diagnostics.
• RNA molecules such as bacterial RNAs or small RNAs, such as the so-called microRNAs (miRNAs)
• miRNAs microRNAs
• One possible method has recently been described in the literature. This process involves several sequential enzymatic steps, i. first, "tailing" the RNA with poly (A) polymerase and a suitable substrate, typically ATP, followed by stopping the poly (A) reaction and purifying the reaction product, then generating the generated poly (A ) RNA into a reverse transcriptase reaction and transcribed into cDNA with appropriate primers.
• MicroRNAs vary in size from about 20 to 25 nucleotides and represent a new family of noncoding RNAs.
• RNAs Tiny regulators with great potential, Cell 107, 823 - 826) .MiRNAs are initially considered as long "primary transcripts” (they are also used as primary miRs). NAs) (Lee, Y., Jeon, K. et al., 2002, MicroRNA maturation: stepwise processing in subcellular localization, Embo J. 21, 4663-4670).
• Pre-miRNAs are exported to the cytoplasm exporting enzyme is called exportin-5, which is further processed here to give a mature miRNA molecule of about 22 nucleotides in length (Lee, Y., et al., 2003, The nuclear RNA's Drosha initiated microRNA processing, Nature 425, 415-419.)
• miRNAs play an important role in development and differentiation, and in principle, microro- mRNAs can regulate in two different ways: In plants, miRNAs complement with their corresponding rnRNAs exact complement This leads to destruction of the target mRNA by a mechanism involving RNA interference (RNAi) .In animals, miRNAs prevent gene expression a mechanism involving Lin-4 and Let-7.
• RNAi RNA interference
• the miRNAs are not exactly complementary to their corresponding rRNAs, but they prevent the synthesis and function of the proteins (Ambros, V., 2004, The functions of animal microRNAs, Nature, 431, 350-355). Because of the critical role played by the recently discovered miRNAs, their detection or analysis plays a key role.
• rRNAs small nucleolar RNAs
• snoRNPs small nucleolar ribonucleo protein particles
• snoRNAs All hitherto characterized snoRNAs, with the exception of RNase MRP, fall into two families. These are the box c / D and box li / ACA slow RNAs, which allow them to distinguish common sequence motifs (Ballakin, AD et al., 1996, The RNA world of the nucleolus: two major families of small nucleolar RNAs defined by different box elements with related functions, Cell, 86, 823-834). The genomic organization of the snoRNA genes has a great diversity in different eukaryotes.
• snoR-NAs are inserted within introns via "host genes.” Exceptions such as U3 are transcribed independently, in yeast there are snoRNAs inserted in introns, but the majority of snoR-NAs are inserted into introns. transcribed the snoRNAs as single gene with its own promoter. Cloned snoRNA genes are transcribed upstream by common promoters. Due to the small size and the lack of polyadenylation, the detection or analysis of snoRNAs is a molecular biological challenge.
• PCR is a widely used tool for studying microbial organisms and is also used to analyze 16S rRNA genes.
• discovery of new genes in microbial samples is limited by the limited possible synthesis of primers.
• primers for 16S RNA genes are derived from those already known from cultured microbes (Olson, DJ, 1986, Microbial ecology and evolution: A ribosomal RNA approach, Annu., Rev. Microbial, 40: 337 -365). Due to the systematics that one uses sequences for the extraction of 16S rRNA genes from previously unknown organisms on which one already knows, it is probable that the microbial diversity is strongly underestimated and also not isolated.
• prokaryotic mRNA molecules are difficult to isolate due to a lack of knowledge of the sequence and especially lack of poly A tail.
• RNA molecule is reacted by taking the enzyme poly-A polymerase and the substrate adenosine triphosphate so that a polyadenylated Ribonukleinkladegalyzed.
• This polyadenylated ribonucleic acid molecule is purified in a further step before reverse transcription takes place in a third step.
• Reverse transcription takes the form of the polyadenylated tail, with a homopolymeric oligonucleotide, usually a poly-T oligonucleotide, complementing the polyadenylated RNA tail.
• the 3 'end of the poly-T oligonucleotide is now used by the polymerase to create a deoxyribonucleic acid strand that is complementary to the ribonucleic acid strand present.
• the resulting strand is called "Fürst Strand cDNA.”
• This cDNA can be used in a PCR reaction, using either random primers or specific primers to generate an amplicon, and Shi et al teaches specifically miRNA detection via an oligo-dT adapter primer using an adapter of specific primers in the PCR (Shi, R. and Chiang, VL (Shi, R. et al., Facile means for quantifying microRNA expression by real-time PCR , Biotechniques, 2005, 39, 519-25).
• This recently published method has significant disadvantages with respect to the specific ribonucleic acid molecules mentioned above.
• the two-step process generally may require contamination.
• the clearance step results in losses of rare RNAs.
• the two-step process requires inactivation of the first enzyme as well as incubation time of the first and second enzymes, resulting overall in a very large amount of time.
• the two-stage process has the further disadvantage that a likelihood of confusion of samples can occur when two or more samples are processed simultaneously.
• ribonucleic acids are relatively sensitive to attack by nucleases.
• the two-step process in particular the step of purification according to the first process, involves the risk of introducing nucleases. Ultimately, two or more steps always increase the risk of pipetting errors.
• the present object is achieved by a method of synthesizing a cDNA in a sample, in an enzymatic reaction, the method comprising the steps of: (a) simultaneously providing a first enzyme having polyadenylation activity, a second enzyme having reverse transcriptase activity, a buffer, at least one ribonucleotide, at least one deoxyribonucleotide, an anchor oligonucleotide, (b) adding a sample comprising a ribonucleic acid and (c) incubating the agents of steps (a) and (b) at one or more temperature steps chosen such that the first and second enzyme show activity.
• a further object of the present invention is to provide a simple process which enables cDN A synthesis, and couples this reaction optionally with a third enzymatic reaction which allows specific detection of the generated cDNA in the same reaction vessel.
• This "3-in-1" method is very easy to handle, especially when large numbers of samples have to be analyzed for one or a few analytes, for example, coupled with real-time PCR rapid procedure to analyze large numbers of samples, minimizing the need for additional handling and contamination, minimizing the risk of sample confusion, minimizing the risk of nuclease entry, and ultimately eliminating the risk of pipetting errors. These benefits are of great importance in diagnostics.
• the object of the "3inl" reaction is solved by a method of synthesizing a cDNA in a sample, in an enzymatic reaction, followed by another enzymatic reaction, optionally an amplification, optionally coupled with the detection, either in real-time during the amplification or downstream, the method comprising the steps of: (a) simultaneously providing a first enzyme having polyadenylation activity, a second enzyme having reverse transcriptase activity, a buffer, at least one ribonucleotide, at least one deoxyribonucleotide, an anchor oligonucleotide, at least one third enzyme having nucleic acid Synthesis Activity, at least one primer, optionally a probe (b) addition of a sample comprising a ribonucleic acid and (c) incubation of the agents of steps (a) and (b) in one or more teraturation steps chosen such that the first and second show the second enzyme activity and opti onal the third enzyme is active or inactive.
• the substrate of poly (A) polymerase used in vivo is adenosine triphosphate (ATP).
• ATP adenosine triphosphate
• Some poly (A) polymerases has been shown that the attachment of short tails with other NTPs may be possible as a substrate (Martin, G. and Keller, W., tailing and 3'-end labeh 'ng of RNA with yeast poly (A) Polymerase and various nucleotides, RNA, 1998, 4, 226-30).
• the sample is a ribonucleic acid which is selected from the group comprising prokaryotic ribonucleic acids, eukaryotic ribonucleic acids, viral ribonucleic acids, ribonucleic acids whose origin is an archae organism, micro ribonucleic acids (miRNA), small nucleolar ribonucleic acids (snoRNA), messenger ribonucleic acid (mRNA), transfer ribonucleic acids (tRNA), non-polyadenylated ribonucleic acids in general, and ribosomal ribonucleic acids (rRNA)
• a mixture of two or more of said ribonucleic acids -A RNA may be included.
• the sample is a ribonucleic acid selected from the group comprising prokaryotic ribonucleic acids, miRNA, snoRNA and rRNA.
• the sample comprises a ribonucleic acid which is selected from the group comprising miRNA and snoRNA. Preference is also given to mixed samples of different amounts of ribonucleic acids of different types, along with other substances.
• the inventors of the present invention have found that it is possible, under certain conditions, to run the two distinctly different enzymatic reactions simultaneously in one reaction vessel and to additionally couple this with a third enzymatic reaction for specific detection of the generated cDNA, which preferred is a nucleic acid synthesis activity.
• the sample is a ribonucleic acid which is selected from the group comprising prokaryotic ribonucleic acids, eukaryotic ribonucleic acids, viral ribonucleic acids, ribonucleic acids whose origin is an archae organism, micro-ribonucleic acids (miRNA), small nucleolar ribonucleic acids (snoRNA), messenger ribonucleic acid ⁇ rnRNA), transfer ribonucleic acids (tRNA), non-polyadenylated ribonucleic acids in general, as well as ribosomal ribonucleic acids (rRNA)
• a mixture of two or more of said ribonucleic acids in the sample can of course already poly-A Be contained in RNA.
• the sample is a ribonucleic acid selected from the group comprising prokaryotic ribonucleic acids, miRNA, snoRNA and rRNA.
• the sample comprises a ribonucleic acid selected from the group comprising miRNA and snoRNA. Preference is also given to mixed samples of different amounts of ribonucleic acids of different types, along with other substances.
• the anchor oligonucleotide is a homopolymeric oligonucleotide selected from the group consisting of a poly (A) oligonucleotide, poly (C) oligonucleotide, poly (T) oligonucleotide , Poly (G) - An 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.
• Preference is given to a poly (T) oligonucleotide, which may optionally additionally have a 5 'tail as stated
• the anchor oligonucleotide according to the invention is generally between 6 and 75 nucleotides in length. However, it can be up to about 150 nucleotides long. If the anchor oligonucleotide is synthetic, the maximum length results from the technical limitations of DNA synthesis.
• the anchor oligonucleotide comprises a 5 'tail and / or an anchor sequence.
• a 5 'tail is an additional nucleotide sequence at the 5' end of the oligonucleotide which, for example, serves to introduce a cloning sequence, primer and / or probe binding sites, or any other sequence. Identification of suitable sequences for the 5 'tail is possible for those skilled in the art based on the requirements of the particular application.
• the anchor oligonucleotide may be an additional anchor sequence, typically one to five additional nucleotides in length.
• the anchor sequence may be at least one base in length, the first position in a preferred embodiment being a degenerate base containing all bases except for the base used in the homopolymer portion of the anchor oligonucleotide. This can be followed by other bases. These can also be degenerate.
• the anchor oligonucleotide is a deoxyribonucleic acid (DNA).
• the anchor oligonucleotide may also be a peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• LNA locked nucleic acids
• NP cyclohexene nucleic acids
• tcDNA tricyclo-deoxyribonucleic acid
• the anchor oligonucleotide is a poly (T) oligonucleotide which additionally comprises a 5 'tail, a Deoxyribonucleic acid is 15-150 nucleotides in length and is present as a mixture.
• an additional anchor sequence typically one to five additional nucleotides in length, may be included.
• the anchor sequence may be at least one base in length, the first position in a preferred embodiment being a degenerate base containing all the bases other than the base used in the homopolymer portion of the anchor oligonucleotide. This can be followed by other bases. These can also be degenerate.
• Example 1 (SEQ ID NO: 10): 5 'TGG AAC GAG ACG ACG ACA GAC ⁇ CAA GCT
• Example 3 (SEQ ID NO: 12): 5 'AACGAGACGACGACAGAC (T) X V 3'
• Example 4 (SEQ ID NO: 13): 5 'AACGAGACGACGACAGAC (T) X N 3'
• Example 7 (SEQ ID NO: 16): 5 'AACGAGACGACGACAGAC (T) X VNNN 3 !
• Example 8 (SEQ ID NO: 17): 5 'AACGAGACGACGACAGAC (T) X NNN 3'
• Example 9 (SEQ ID NO: 18): 5 'TGG AAC GAG ACG ACG ACA GAC CAA GCT
• Example 10 (SEQ ID NO: 19): 5 'TGG AAC GAG ACG ACG ACA GAC CAA GCT
• the optional 5 'tail contains additional 1-100 nucleotides, which can be used for subsequent analyzes.
• the 5 'tail may contain the binding sequence for an oligonucleotide such as one or more DNA probes and / or one or more PCR primers.
• the sequences used for the 5 'tail are preferably selected so that they are compatible with the method according to the invention. This includes, for example, the selection of those sequences which do not cause undesired side reactions, both in the method according to the invention and in subsequent analysis methods.
• FIG. 1 Anchor oligonucleotides according to the invention are shown in FIG. 1
• the enzymatic reaction according to the invention can take place on a carrier or in a container, i. the reaction can take place in a reaction vessel.
• a reaction vessel may be a reaction tube or, for example, a microtiter plate.
• the reaction can take place on a chip. If it takes place on a chip, one or more components can be immobilized.
• the reaction can take place on a test strip or in a microfluidic system.
• the ribonucleotide is an adenosine 5'-triphosphate, a thymidine 5'-triphosphate, a cytosine 5'-triphosphate, a guanine 5'-triphosphate and / or a uracil 5 ' triphosphate.
• the ribonucleotide may also be a base analog.
• the ribonucleotide may be modified or labeled. In principle, it is essential that the ribonucleotide can be reacted by the enzyme in polyadenylation activity as a substrate.
• the deoxyribonucleotide according to the invention may be selected from the group comprising a deoxyadenosine 5'-triphosphate (dATP), a deoxythymine 5'-triphosphate (dTTP), a deoxycytosm-5'-triphosphate (dCTP), a deoxyguanosine S ' Triphosplate (dGTP), deoxyuracil 5'-triphosphate (dUTP) and modified deoxyribonucleotides and labeled deoxyribonucleotides.
• dATP deoxyadenosine 5'-triphosphate
• dTTP deoxythymine 5'-triphosphate
• dCTP deoxycytosm-5'-triphosphate
• dGTP deoxyguanosine S ' Triphosplate
• dUTP deoxyuracil 5'-triphosphate
• Applications are also conceivable in which, in addition to or in exchange, one or more deoxyribonucleotides
• dATP dCTP
• dTTP dTTP
• dGTP dGTP
• desoxyuracil 5'-triphosphate can also be used in the mixture. This can be combined with an enzymatic reaction taking place after the actual reaction. see reaction, which uses the uracil-DNA-glycosilase and can degrade unused enzymatically produced reaction product.
• the label may be selected from the group comprising 32 P, 33 P, 35 S, 3 H, a fluorescent dye such as fluorescein isothiocyanates (FITC), 6-carboxyfluorescein (FAM), xanthene, rhodamine, , 6-carboxy-2 ', 4', 7 ', 4,7-hexachlorofluorescein (HEX), 6-carboxy-4 ⁇ 5'-dichloro ⁇ 2 ⁇ 7'-dimemodyfluorescein (JOE), N, N, N' , N'-tetramethyl-6-carboxy rhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyhodamine-6G (R6G5), 6-carboxy rhodamine-6G (RG6), Rhodamine 1 10; Coumarins such as umbelliferones, benzimides such as
• labels such as the insertion of biotin or one or more haptens such as digoxigenin, which allow a direct or indirect detection of the nucleic acid.
• Indirect evidence such as antibodies, which in turn, for example, enzymatic detection of an antibody coupled to the enzyme. Indirect detection is also possible by introducing nanoparticles which are eg coupled to antibodies or an affinity ligand.
• Modification of the deoxyribonucleotide may also be via the 5 'phosphate, which allows for easier cloning.
• reactive groups e.g. an amino linker (also biotin)
• the deoxyribonucleotide may e.g. be immobilized or made available to direct or indirect evidence.
• Particularly preferred modifications are selected from the group comprising fluorescence dyes, haptens, 5'-phosphate, 5'-biotin, 5'-amino linker.
• the concentration of a deoxyribonucleotide in the reaction is at least 0.01 mM and at most 10 mM. This concentration is the concentration of the single deoxyribonucleotide.
• the deoxyribonucleotides are each dATP, dCTP, dGTP and dTTP in a concentration of 0.2 mM to 2 mM. This concentration is the concentration of the individual deoxyribonucleotide in the mixture.
• the single deoxyribonucleotide, dATP, dCTP, dGTP and dTTP are present in a concentration of 0.5 mM each.
• the inventors have surprisingly found that the one-step enzyme reaction as subject of the present invention can take place in a narrow buffer pH range of 6 to 10 in the presence of magnesium ions (Mg 2+ ).
• a pH of 6 to 10 is present.
• the buffer according to the invention has a pH of 6.8 to 9.
• the buffer according to the invention additionally comprises ions which can be selected from the group comprising Mn 2+, K ⁇ NH 4+ Na + UBD.
• Buffers according to the invention include, for example, MgCl 2 , MgSO 4 , magnesium acetate, MnCl 2 , KCl, (NH 4 J 2 SO 4 , NH 4 Cl, NaCl Suitable buffer substances are Tris, Tricine, Bicine, Hepes and also other buffer substances in the pH range according to the invention or mixtures of two or more suitable buffer substances.
• enzymes having polyadenylation activity are selected from the group comprising enzymes of prokaryotic origin, of eukaryotic origin, of viral origin and of archae origin, and also of enzymes of plant origin.
• a polyadenylation activity in the sense of this invention is an enzymatic activity which uses as substrate the 3 'end of a ribonucleic acid and in a suitable buffer is capable of enzymatically adding to this 3' end ribonucleotides, preferably at least 10 to 20 ribonucleotides.
• the enzyme is an enzyme which is capable of using adenosine 5'-triphosphate as a substrate. According to the invention, this includes enzymes and reaction conditions which have a polyadenylation activity in the context of the invention when using single-stranded or double-stranded RNA, for example hairpin RNA such as, for example, pre-miRNA.
• RNAs eg., mature miRNAs
• double-stranded RNAs eg, pre-miRNAs
• Polyadenylation activity in the sense of the invention is generally a transcriptase activity.
• the enzyme having polyadenylation activity is an enzyme selected from the group consisting of poly (A) polymerase from Escherichia coli, poly (A) polymerase from yeast, poly (A ) Beef polymerase, frog poly (A) polymerase, human poly (A) polymerase, and plant poly (A) polymerase. Others are known to those of skill in the art or newly identified by analysis of homology to known poly (A) polymerases. In a particularly preferred embodiment, the enzyme having polyadenylation activity is a poly (A) polymerase derived from Escherichia coli.
• the enzyme according to the invention having reverse transcriptase activity is selected according to the invention from the group comprising enzymes from viruses, bacteria, archae bacteria and eukaryotes, in particular from thermostable organisms. These include e.g. also enzymes from introns, retrotransposons or retroviruses.
• an enzyme with reverse transcriptase activity is an enzyme which is able to incorporate deoxyribonucleotides complementary to ribonucleic acid at the 3 'end of a deoxyoligonucleotide or ribo-oligonucleotide hybridized to the ribonucleic acid hybridase under suitable buffer conditions. This includes, on the one hand, enzymes which naturally have this function but also enzymes which have such a function only by changing their gene sequence, e.g. Mutagenesis or obtained by appropriate buffer conditions.
• the enzyme with reverse transcriptase activity an enzyme which is selected from the group comprising HTV reverse transcriptase, M-MLV reverse transcriptase, EAFV reverse transcriptase, AMV reverse transcriptase, Thermus thermophilus DNA polymerase I, M-MLV RNAse H, Superscript, Superscript ⁇ , Superscript IH, monster script (Epicenter), Omniscript, Sensiscript reverse transcriptase (Qiagen), ThermoScript and Tliermo-X (both in vitro).
• enzymes which have reverse transcriptase activity as enzyme only after a modification of the gene sequence. It is also possible to use a reverse transcriptase activity which has an increased error accuracy.
• AccuScript reverse transcriptase (Stratagene) may be mentioned here. It is for the It will be apparent to those skilled in the art that the use of mixtures of two or more enzymes having reverse transcriptase activity is also possible.
• those enzymes with reverse transcriptase activity require a divalent ion.
• those enzymes which require a divalent ion have a divalent ion.
• Preferred are Mg 2+ , Mn 2+ .
• thermophilus DNA polymerase I or M-MLV RNAse H Superscript, Superscript ⁇ , SuperScript UI or monster script (Epicenter) or Omniscript reverse transcriptase (Qiagen) or Sensiscript reverse transcriptase (Qiagen), ThermoScript, Thermo-X (both Invitrogen) or a mixture of two or more enzymes with reverse transcriptase activity and poly (A) polymerase from Escherichia coli.
• a reverse transcriptase which is mammalian.
• An enzyme is preferred which has an optimum nucleic acid synthesis activity at between 45 0 C and 85 0 C, more preferably between 55 0 C and 8O 0 on Trentzugtesten preferably C between 60 0 C and 75 0 C.
• the process can be carried out in several temperature steps the first temperature step may be at a temperature which is the optimum temperature for the enzyme having polyadenylation activity and the second temperature step may be at a temperature which is the optimum temperature for the enzyme having reverse transcriptase activity.
• the AMV reverse transcriptase is used, the second temperature step takes place at 42 0 C, whereas the first temperature step, which belongs to the primary activity of the enzyme with polyadenylation, at a temperature of 37 ° C instead.
• the first temperature step which belongs to the primary activity of the enzyme with polyadenylation, at a temperature of 37 ° C instead.
• thermostable a non-thermostable enzyme can be combined with a thermostable enzyme.
• the temperature steps depend on which of the two enzymes has polyadenylation activity.
• the enzyme with reverse transcriptase activity is thermostable.
• the incubation at high temperature in the polyadenylation step may in part or totally inactivate the enzyme having reverse transcriptase activity.
• both enzymes are thermostable.
• the method of the invention preferably additionally comprises poly (C) polynucleotides.
• the method according to the invention particularly preferably additionally comprises poly (C) -polyribonucleotides.
• the reaction of the invention may further comprise reagents such as Volume Excluder, a single strand binding protein, DTT and / or competitor nucleic acids.
• reagents such as Volume Excluder, a single strand binding protein, DTT and / or competitor nucleic acids.
• volume excluder is selected from the group comprising dextran, polyethylene glycol, and EP 1411133A1 mentions the volume excluder according to the invention.
• the competitor nucleanic acid is a homopolymeric ribonucleic acid, most preferably polyadenoribonucleic acid. Examples are disclosed in US 6,300,069.
• the method according to the invention preferably additionally comprises poly (C) polynucleotides.
• the method according to the invention particularly preferably additionally comprises poly (C) -polyribonucleotides.
• 1 ng to 300 ng of poly (C) -polyribonucleotides are used per 20 ⁇ l, preferably 10 ng to 150 ng of poly (C) -p olyribonucleotides per 20 ⁇ l of reaction, more preferably 25 ng to 100 ng of poly ( C) -PoI yribonucleotide used per reaction and most preferably 50 ng to 75 ng of poly (C) -Polyribonucleotide per 20 ul reaction used.
• the invention further relates to a reaction mixture comprising a first enzyme having polyadenylation activity, a second enzyme having reverse transcriptase activity, optionally a buffer, optionally at least one ribonucleotide, optionally at least one deoxyribonucleotide and optionally an anchor oligonucleotide.
• the anchor oligonucleotide preferably comprises a homopolymer portion, an anchor sequence and / or a tail.
• the reaction mixture additionally comprises random primers. The additional use of random primers has the advantage that even 5 'ends of long transcripts are efficiently rewritten, which is important in quantitative analyzes.
• the reaction mixture may contain the same agents as used for the method according to the invention.
• the anchor oligonucleotide is a homopolymeric oligonucleotide selected from the group consisting of 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.
• Preference is given to a poly (T) oligonucleotide, which may optionally additionally have a 5 'tail, as already explained above.
• the anchor oligonucleotide according to the invention is generally between 6 and 75 nucleotides in length. However, it can be up to about 150 nucleotides long. If the anchor oligonucleotide is synthetic, the maximum length results from the technical limitations of DNA synthesis.
• the anchor oligonucleotide comprises a 5 'tail and / or an anchor sequence.
• a 5 'tail is an additional nucleotide sequence at the 5' end of the oligonucleotide which, for example, serves to introduce a cloning sequence, primer and / or probe binding sites, or any other sequence. The identification of suitable sequences for the 5 'tail is possible for the skilled person based on the requirements of the respective application.
• an additional anchor sequence typically one to five additional nucleotides in length, may be included.
• the anchor sequence may be at least one base in length, the first position in a preferred embodiment being a degenerate base containing all bases except the base used in the homopolymer portion of the anchor oligonucleotide. This can be followed by other bases. These can also be degenerate.
• the optional 5'-tail contains additional 1-100 nucleotides, which can be used for subsequent analyzes.
• the 5 'tau may be the binding sequence for an oligonucleotide, such as an oligonucleotide.
• the sequences used for the 5'Tail are preferably selected so that they are compatible with the method according to the invention. This includes e.g. the selection of those sequences which do not cause any unwanted side reactions, both in the method according to the invention and in subsequent analysis methods.
• the reaction mixture according to the invention comprises the anchor oligonucleotide according to the invention which has a length of between 10 and 150 nucleotides and optionally carries at the 3 'end a one to five nucleotide long anchor sequence according to the invention.
• the reaction mixture according to the invention comprises the anchor oligonucleotide which, as described above, is for example a deoxyribonucleic acid (DNA), is a peptide nucleic acid (PNA) or is a locked nucleic acid (LNA).
• the reaction mixture according to the invention comprises an anchor according to the invention, OHgonukleotid, which is a poly (T) - oligonucleotide and additionally carries a 5 ' ⁇ Tail, wherein the oligonucleotide is a deoxyribonucleic acid, which is 10 to 75 nucleotides long and is present as a mixture, wherein at the 3 'end an anchor sequence is present, each consisting of one nucleotide, which is selected from the group comprising A, G and C, optionally followed by one to five further nucleotides comprising all four bases A, C. , G, and T or analogous
• the reaction mixture according to the invention further comprises at least one ribonucleotide as described above for the process according to the invention.
• this includes reaction mixture according to the invention at least one ribomicule selected from ATP, TTP, CTP, GTP, UTP or corresponding base analogs.
• the ribonucleotides may optionally be modified or labeled as described above.
• the reaction mixture according to the invention comprises deoxyribonucleotides as described for the process according to the invention.
• the reaction mixture according to the invention comprises one or more deoxyribonucleotides such as dATP, dCTP, dGTP, dUTP and / or dTTP.
• a mixture of deoxyribonucleotides is used which allow cDNA synthesis. These deoxyribonucleotides may optionally be modified or labeled.
• the label can be selected from the group consisting of 32 P, 33 P, 35 S, 3 H, a fluorescent dye such as fluorescein isothiocyanates (FITC), 6-carboxyfluorescein (FAM), xanthene, rhodamia , 6-Carboxy-2 ', 4', 7 ', 4,7-hexachlorofluorescein (HEX), 6-carboxy-4 ⁇ 5'-diceworo-2 ⁇ 7'-dimemodyfluorescein (JOE), N, N, N ', N'-tetramethyl-6-carboxy rhodamine (TAMRA), ⁇ -carboxy-X-rhodium (ROX), 5-carboxyhodamine-6G (R6G5), 6-carboxy rhodamine-6G (RG6), Rhodamine 110; Cy3, Cy5, Cy7, coumarin
• labels such as the inclusion of biotin or one or more haptens such as digoxigenin, which allow direct or indirect detection of the nucleic acid.
• Indirect evidence such as antibodies, which in turn an example enzymatic detection of an antibody coupled to the enzyme.
• Indirect detection is also possible via the introduction of nanoparticles which are eg coupled to antibodies or an affinity ligand.
• the reaction mixture according to the invention in each case comprises a deoxyribonucleotide at a concentration of 0.01 mM to 10 mM.
• the single deoxyribonucleotide A, C, G and T are preferably present in a concentration of 0.2 mM to 2 mM in each case. It is particularly preferred if the deoxyribonucleotides A 1 C, G and T are present together. Each is present in a concentration of 0.5 mM in this preferred embodiment.
• the Itemssgen ⁇ isch invention further comprises a buffer. This buffer has a pH of 6 to 10. In the reaction mixture according to the invention are still Mg 2+ - ions.
• the reaction mixture according to the invention has a buffer having a pH of 6.8 to 9.
• the reaction mixture may further comprise additionally ions which may be selected from the group comprising Mn 2+ , K + , NH 4+ and Na + , Essential for the reaction mixture according to the invention is the presence of two different enzyme activities.
• the reaction mixture according to the invention comprises at least one first enzyme activity with polyadenylation activity and, secondly, a second enzyme activity with reverse transcriptase activity. The preferred embodiments of these activities have already been described for the method above.
• the reaction mixture like the method above, may further comprise additional substances such as a volume excluder, a single strand binding protein, DTT or one or more competitor nucleic acids.
• volume excluder it is preferred that this is selected from the group comprising dextran, polyethylene glycol. Further volume excluders according to the present invention can be found in EP1411133Al.
• reaction mixture optionally comprises a competitor nucleic acid, this is selected from the group comprising homopolymeric ribonucleic acids and polyadenoribonucleic acid.
• a competitor nucleic acid this is selected from the group comprising homopolymeric ribonucleic acids and polyadenoribonucleic acid.
• Further competitor nucleic acids according to the invention are disclosed in US Pat. No. 6,300,069.
• the reaction mixture according to the invention preferably additionally comprises poly (C) polynucleotides.
• the reaction mixture according to the invention particularly preferably additionally comprises poly (C) -polyribonucleotides.
• the invention further relates to a kit comprising a reaction mixture as described above.
• the reaction mixture is present in a preferred embodiment in a single reaction vessel.
• the kit comprises a reaction vessel comprising the enzyme having polyadenylation activity, the enzyme having reverse transcriptional activity. taseactivity, optionally the deoxyribonucleotides, optionally at least one ribonucleotide, optionally a buffer containing Mg 2+ and optionally one or more oligodeoxyribonucleotides according to the invention.
• the reaction vessel in the kit according to the invention contain further constituents, as indicated for the reaction mixture according to the invention.
• the kit may further comprise a probe for the 5 'tail of the anchor oligonucleotide of the invention.
• the kit may contain one or more other deoxyribonucleotides, such as a generic primer for detecting the tail sequence introduced by reverse transcription.
• the reaction mixture can be in "pellet form", ie lyophilized, for example, and the person skilled in the art will be familiar with further methods of preparation which include, for example, non-liquid forms.
• kit can optionally be combined with reagents as they are necessary for the PCR reaction or real-time PCR reaction.
• reagents are preferably reagents for at least one PCR reaction which allows the detection of at least one of the cDNAs generated according to the invention.
• the kit may further optionally include random primers and optionally one or more primers or primers / probes for detection of additional target genes in singleplex or multiplex PCR reactions and / or real-time singleplex or multiplex PCR reactions.
• the reaction mixture, the method according to the invention or the kit may further contain target-specific primers.
• the length of the target-specific primers should be selected so that a specific detection in a PCR reaction is possible, the sequence of the target primer should be so specific that binding to only one point in the generated cDNA sequence is possible , In general, such a primer has a length of 15 to 30 nucleotides, preferably from 17 to 25 nucleotides.
• the reaction mixture comprises the enzyme with polyadenylation activity and the enzyme with reverse transcriptase activity as Zweienzyme Pre-mix.
• the kit comprises in this particularly preferred embodiment the two-enzyme pre-mix reaction mixture in a reaction vessel and in a separate vessel a buffer, Mg 2+ , rNTP (s), dNTP (s), optionally an anchor oligonucleotide according to the invention and optionally random primer and optionally also a volume exclusion reagent and / or a competitor nucleic acid.
• the erfmdungswele method can, as already stated above, take place in one or more temperature steps.
• the process according to the invention takes place at a single temperature step for the incubation and at a further temperature step for the inactivation for the enzymes.
• the incubation period is about 1 to 120 minutes, preferably 5 to 90 minutes, more preferably 10 to 75 minutes, more preferably 15 to 60 minutes, more more preferably 20 to 60 minutes, most preferably 50 to 70 minutes.
• An additional incubation period is usually not harmful.
• a temperature of at least 65 ° C, but at most 100 0 C is used.
• a temperature of about 80-95 0 C is used.
• the denaturation occurs for a period of at least 1 minute, but at most for 30 minutes. Li of a preferred embodiment is denatured for a period of 5 minutes.
• the method according to the invention for the generation of cDNA can subsequently comprise a polymerase chain reaction. If this is the case, it is preferable to add a tail-specific primer and / or a specific primer to the reaction mixture according to the invention for the tail introduced during cDNA synthesis.
• the reaction mixture will then continue to contain a thermostable DNA polymerase.
• reaction components are already present in the tube before the first reaction is started (see Example 9).
• no template controls e.g.
• the third reaction is a PCR, it is preferably a real-time PCR.
• a number of methods are known in the art, which allow reversibly inactivate the thermostable DNA polymerase used. This process, called hot-start PCR, offers a number of technical solutions.
• Known hot-start procedures for PCR enzymes are: Chemical (see also DE69928169T), antibodies, aptamers, DNA oligos, affinbodies, microencapsulation (sa DE69930765T).
• the OHgonukleotide required only in the PCR i. one or more target specific primers, the Tai! Specific primers, and optionally one or more fluorescence-labeled probes, are available only at the beginning of the third reaction.
• target specific primers i. one or more target specific primers, the Tai! Specific primers, and optionally one or more fluorescence-labeled probes
• fluorescence-labeled probes are available only at the beginning of the third reaction.
• a preferred option is the temporary compartmentalization of individual reagents. These are then released when needed. It is particularly preferable to separate the amplification enzyme or the amplification primer.
• side groups or complexes with the aim of providing the relevant oligonucleotides for the third reaction, preferably a PCR or real-time PCR, a number of methods are conceivable or known to the person skilled in the art.
• This activation can be carried out with the aid of a series of physical parameters such as temperature, pH, ionic strength, mechanical influences, chemical or enzymatic, or a combination of two or more of the parameters; examples of possible embodiments of the hot-start primers are taken from the examples.
• the prior art describes various embodiments for a primer Hot Start (see DE69930765T, US6274353, EP0866071).
• the enzyme used for activation may also be provided in an incipient form.
• Suitable hot-start processes are known to the person skilled in the art and have already been described above (inter alia EP0962526). It is obvious to the person skilled in the art that adaptation of the enzymatic activation to the requirements of the third reaction is possible by using a thermostable enzyme which has been isolated eg from a thermopile or hyperthermophilic organism in combination with a suitable hot-start method such as EP0962526 is.
• the activation can by means of a series of physical parameters such as temperature, pH, ionic strength, mechanical influences, chemical or enzymatic, or a combination of two or more of the parameters.
• the discussed particle size distribution of the reactions is conceivable.
• conditions are provided under which the 2inl PAP-RT reaction proceeds.
• reactants are provided for a third reaction without handling steps of the experimenter, preferably without opening the vessel, e.g. by removing a physical blockage (e.g., thermal, mechanical).
• a thermolabile barrier is used.
• a clear polymer such as e.g. Wax with defined melting point for use.
• This is mixed with the oligonucleotides relevant for the third reaction under suitable conditions, so that a solid phase is formed during the reaction conditions of the first and second reaction.
• the solid phase can be achieved in a variety of ways. It can e.g.
• a solid drop at the bottom of the vessel one or more particles, a deposit on the surfaces of the reaction compartment or another form.
• the relevant oligonucleotides are released and are available for the third reaction.
• Another form of compartmentalization of the reactions is a physical barrier that prevents or at least minimizes the contact of the reactants uncritically.
• This can be a septum, e.g. from wax, polymer, which can then be influenced by a number of physical parameters such as temperature, pH, ionic strength, mechanical influences, chemical or enzymatic in its permeability.
• one or more components of the third reaction are separated by a wax barrier from the components of the 2-in-1 PAP-RT reaction.
• one or more components of the third reaction are present at the bottom of the reaction vessel, overlaid with at least one septum for the purposes of this invention, then the components of the 2-in-1 PAP-RT Reaction.
• the components of the third reaction may be both in liquid form and dried or otherwise stabilized, or as multiple concentrates, or combinations of one or more formats.
• the primers were used in a modified form, as so-called “hot-start” primers. These are then so-called “CleanAmp” primers, available from Trilinkbiotech.com. "CleanAmp” primers have the property of becoming functional only through the initial heat step at the beginning of the PCR and are therefore available in functional form only in the PCR.
• the primers are inactivated as described in WO2007 / 139723 (Applicant: Trilink), in particular in claim 1 and the subsequent claims.
• the subsequent PCR reaction may also be a quantitative PCR reaction. It can take place on an array, take place in a microfluidic system, take place in a capillary, or be a real-time PCR. Other variants of the PCR are known to the person skilled in the art and are likewise encompassed by the method according to the invention.
• the DNA polymerase is preferably thermostable. It may be selected from the group comprising enzymes originating from thermophilic bacteria or from thermophilic archae. It is preferably selected from the group comprising polymerases from Thermus aquaticus, Thermus paeificus, Thermus thermophilus, Pyroccocais fimosus, Thermus brockianus, Aquifex aeolicus and other DNA polymerases which are thermostable.
• amplification methods may also be used; these may be selected from the group comprising the rolling circle amplification (as described in Liu, et al., Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerase). Soc., 118: 1587-1594 (1996)), the “isothermal amplification” (as described in Walker, et al., “Strand displacement amplification-an isothermal, in vitro DNA amplification technique,"”Nucleic Acids Res.
• rolling circle amplification as described in Liu, et al., Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerase. Soc., 118: 1587-1594 (1996)
• the “isothermal amplification” as described in Walker, et al., “Strand displacement amplification-an isothermal, in vitro DNA amplification technique,"”Nucleic Acids Res.
• the invention also relates to a method for the reverse transcription of RNA into DNA, the method comprising the steps of providing a sample comprising RNA, adding a first enzyme having reverse transcriptase activity, a buffer, at least one deoxyribonucleotide, an oligonucleotide, incubating the agents at one or more temperature steps selected so that the enzyme exhibits activity, wherein the reaction additionally comprises poly (C) polynucleotides.
• the enzyme having reverse transcriptase activity is HIV reverse transcriptase, M-MLV reverse transcriptase, EATV reverse transcriptase, AMV reverse transcriptase, Thermus thermophilus DNA polymerase I, M-MLV RNAse H ⁇ ( Superscript, Superscript ⁇ , Superscript
• Transcriptase (Qiagen), ThermoScript, Thermo-X (both Invitrogen) or a mixture of two or more enzymes with reverse transcriptase activity and poly (A) polymerase from Escherichia coli. Particularly preferred is HTV reverse transcriptase.
• the reaction comprises poly (C) -polyribonucleotides.
• poly (C) -polyribonucleotides Preferably, from 1 ng to 300 ng of poly (C) -polyribonucleotides are used per 20 ⁇ l, preferably from 10 ng to 150 ng of poly (C) -polyribonucleotides per 20 ⁇ l of reaction, more preferably from 25 ng to 100 ng of poly (C ) Polyribonucleotides per reaction, and most preferably from 50 ng to 75 ng of poly (C) -polyribonucleotides per 20 ⁇ l of reaction.
• a hompolymeric nucleic acid in the 3inl reaction, a hompolymeric nucleic acid can be used.
• the sample is a ribonucleic acid which is selected from the group comprising prokaryotic ribonucleic acids, eukaryotic ribonucleic acids, viral ribonucleic acids, ribonucleic acids whose origin is an archae organism, micro-ribonucleic acids (miRNA), small nucleolar ribonucleic acids (snoRNA), messenger ribonucleic acid (mRNA), transfer ribonucleic acids (tRNA), non-polyadenylated ribonucleic acids in general, and ribosomal ribonucleic acids (rRNA)
• miRNA micro-ribonucleic acids
• snoRNA small nucleolar ribonucleic acids
• mRNA messenger ribonucleic acid
• tRNA transfer ribonucleic acids
• rRNA ribosomal ribonucleic acids
• the template used may be an RNA which is selected from the group comprising eukaryotic ribonucleic acids, mRNA, prokaryotic ribonucleic acids, miRNA, snoRNA and rRNA.
• the sample comprises a ribonucleic acid selected from the group comprising miRNA and snoRNA. Preference is also given to mixed samples of different amounts of ribonucleic acids of different types, along with other substances.
• RNA in general can be reversibly transcribed by the method.
• the invention further relates to a kit for reverse transcription comprising an enzyme with reverse transcriptase activity and poly (C) polynucleotides preferred, poly (C) - polyribonucleotides.
• the RNA is first polyadenylated before the sample is reverse transcribed.
• PAP poly A polymerase RT: reverse transcription rATP: adenosine 5'-triphosphates
• rATP adenosine 5'-triphosphates
• the PCR protocol consisted of an initial reactivation of the HotStarTaq polymerase contained in the QuantiTect SYBR Green PCR Master Mix for 15 min at 95 ° C followed by 40 cycles of 15 sec 94 0 C, 30 sec at 52 ° C and 30 sec at 72 0 C (see Table 5).
• the fluorescence data were recorded during the 72 ° C extension step.
• the PCR analyzes were carried out with an ABI PRISM 7700 (Applied Biosystems) in a reaction volume of 20 ⁇ l.
• PCR products were then subjected to melting curve analysis. This was performed on an ABI PRISM 7000 Real-time PCR instrument.
• the coupled one-step process was carried out under different conditions. These were one of the poly (A) -polymerase-supplied buffer and the reverse transcriptase-supplied buffer (Table 7, 8).
• Table 8 Composition of the combined poly (A) polymerase / reverse transcription reaction and the standard reverse transcription reaction
• the detected 22-mer RNA corresponds in its sequence of human Ieu7a miRNA (EMBL Acc #: AJ4217241 and may be expressed in human blood cells such as leukocytes However, such small RNAs are be ⁇ dingt by the purification technology of the RNeasy used for the RNA isolation procedure.
• the RNeasy method only ensures efficient binding of RNAs larger than 200 bases to the silica core of the RNeasy column (QIAGEN RNeasy Midi / Maxi Handbook, 06/2001, p as miRNAs depleted heavily.
• the fluorescence data were recorded during the 72 ° C extension step.
• the PCR analyzes were carried out with an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems) in a reaction volume of 20 ⁇ l, followed by a melting curve analysis.
• Unwanted artifacts such as poly (A) tailing of the primer used for cDNA synthesis are also undetectable.
• reaction a add 293 RNA and in reaction b) water as negative control (neg control).
• negative control a standard reverse transcription reaction with the reagents listed in Table 14 was set up using the scheme in Table 17.
• reaction in reaction c add 293 RNA and in reaction d) water as negative control (neg control). The samples were then incubated for one hour at 37 ° C. In order to stop the reaction, the reactions were incubated for 5 min at 93 ° C, by this temperature step, the enzymes are inactivated.
• the batches were diluted 1: 2 with water and 2 ⁇ l each of the batches a) to d) were used as template in a real-time SYBR Green PCR.
• the materials for the PCR are given in Table 15.
• Reactions 1-5 (Table 18) each employed the miRNA-specific or ⁇ -actin 3 'primer and the tail primer (Hum Uni). Table 18: SYBR GREEN PCR reaction 1-5
• the PCR protocol consisted of an initial reactivation of the HotStarTaq polymerase contained in the QuantiTect SYBR Green PCR Master Mix for 15 min at 95 ° C, followed by 40 cycles of 15 sec 94 0 C, 30 sec at 52 ° C and 30 sec at 72 0 C (see Table 21). The acquisition of the fluorescence data was done during the C 72 0 extension step.
• the PCR analyzes were carried out with an Applied Biosystems 7000 Fast Real-Time PCR system (Applied Biosystems) in a reaction volume of 20 ⁇ l, followed by a melting curve analysis. Table 21: 3 step PCR protocol
• the real-time PCR when using the cDNA prepared under standard conditions, yields very high Ct values of more than 38, which in the case of a real-time PCR performed with SybrGreen means a very good specificity of the detection (FIG. 6).
• PCR products of the expected size were detected (FIG. 8), or no product was detected using the cDNA prepared under standard conditions.
• a real-time PCR was performed using a Taqman probe that has a specific binding site on Taü primer (Uni Gap dT).
• Evidence of a probe represents a conceivable alternative for detection by SYBR Green real-time PCR.
• the use of the probe offers the additional possibility of multiplex PCR, i. a co-amplification of one or more additional target nucleic acids, such as an internal control, e.g. a housekeeping gene can be.
• reaction mixture was incubated at 37 ° C, followed by inactivation of the enzymes for 5 min at 93 ° C.
• the PCR protocol consisted of an initial reactivation of the HotStarTaq polymerase contained in the QuantiTect Probe PCR Master Mix for 15 min at 95 0 C, followed by 45 cycles of 15 sec at 94 ° C and 30 sec at 52 ° C (see Table 26).
• the fluorescence data were recorded during the 52 ° C annealing step.
• the PCR analyzes were carried out with a 7700 Sequence Detection System (Applied Biosystems) in a reaction volume of 20 ⁇ l. The PCR results are shown in Table 27.
• Detection using a tail-specific probe is possible and provides the expected result.
• reaction a 2 U poly-A polymerase
• reaction b 0.5 U poly-A polymerase
• the PCR protocol consisted of an initial reactivation of the HotStarTaq polymerase contained in the QuantiTect SYBR Green PCR Master Mix for 15 min at 95 0 C, followed by 40 cycles of 15 sec 94 0 C, 30 sec at 52 ° C and 30 sec at 72 ° C (see Table 32 above). The fluorescence data were recorded during the 72 ° C extension step.
• the PCR analyzes were carried out with an Applied Biosystems 7000 Real-Time PCR system (Applied Biosystems) in a reaction volume of 20 ⁇ l, followed by a melting curve analysis.
• the method according to the invention was carried out with a total of five different reverse transcripts (see Table 35) in the buffer RT (Qiagen) (reaction 1-5), or in each case in addition to the comparison in the buffer supplied with the reverse transcriptase (reaction 6-9). ,
• reaction buffer buffer RT (QIA gene) as in Table 36, and for reaction 6).
• 9 Frther reverse transcriptases, each in the supplied buffer
• Table 36 PAP + RT reaction in buffer RT (Qiagen)
• Table 37 PAP + RT reaction in the buffer supplied with the reverse transcriptase
• the samples were incubated for 1 h at 37 ° C. Thereafter, the reactions were heated for 5 min at 93 0 C, thereby inactivating the enzymes.
• the PCR protocol consisted of an initial reactivation of HotStarTaq included in the QuantiTect SYBR green PCR Master Mix polymerase for 15 min at 95 0 C, followed by 40 cycles of 15 sec 94 ° C, 30 sec at 52 ° C and 30 sec at 72 ° C (see Table 39). The fluorescence data were recorded during the 72 ° C extension step.
• the PCR analyzes were carried out with an Applied Biosystems 7000 Real-Tirne PCR system (Applied Biosystems) in a reaction volume of 20 ⁇ l, followed by a melting curve analysis.
• reaction was carried out in a two-step process based on FIG. 1B.
• the reaction protocol consisted first of conditions for the combined reaction of poly-A polymerase and reverse transcription with the QuantiTect Multiplex Reverse Transcriptase Mix (45 min 37 0 C and 15 min 50 0 C), followed by incubation for 15 min at 95 ° C, with the aim of inactivating the poly-A polymerase and reverse transcriptase, and to activate the HotStarTaq DNA polymerase contained in the QuantiTect Multiplex RT-PCR Master Mix. This was followed by 45 PCR cycles with 15 sec at 94 0 C and 30 sec at 52 0 C (see Table 43) to the generated let7a ⁇ cDNA to amplify in a real-time PCR.
• a fluorescence-labeled probe specific for the 5 'tail of the Uni Gap dT primer was used for detection.
• the fluorescence data were recorded during the 52 ° C annealing / extension step.
• the "3inl" reaction was performed on an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems) in a reaction volume of 20 ⁇ l.
• the "3inl” reaction allows for the specific proximity of the synthetic 22mer RNA oligonucleotide in the background of maize RNA
• the sample containing the synthetic 22mer RNA oligonucleotide in the background of maize RNA provides a Ct value of 20.61
• the control reaction with maize RNA gave a Ct value of 30.65
• This experiment shows that the "3inl" reaction is technically feasible under the given conditions.
• PCR primers from Table 45 are used to prepare a primer mix and pipette the required amount for each reaction into a respective lid of an optical cap (lid for real-time PCR tubes, Applied Biosystems, material number 4323032). Subsequently, the lids were incubated on a heating block at 37 0 C for about 20 min until the liquid evaporated and thus the primer had dried.
• reagents are pipetted together as in Table 44 and placed in Optical Tubes (Real-time PCR tubes, Applied Biosystems, Material Number 4316567), capped with the pretreated optical caps and PCR as in Table 46 specified performed. The reactions were tested in triplicate.
• Table 45 Composition of dried oligo mix in the PCR lid
• the reaction protocol consisted first of conditions for the combined reaction of poly-A polymerase and reverse transcription with the QuantiTect Reverse Transcriptase Multiplex Mix (45 min 37 ° C and 15 min 50 0 C). Subsequently, the reactions were heated for 3 min at 95 0 C, with the aim of inactivating the poly-A-polymerase and reverse transcriptase enzymes. Thereafter, the PCR Tubes were briefly removed from the device and inverted, with the aim to redissolve the dried primers present in the lids and make them available for the following PCR reaction. This was followed by incubation at 95 ° C for 12 min to activate the HotStar Taq DNA polymerase contained in the QuantiTect Multiplex RT-PCR Master Mix.
• a reactivation time shorter by 3 minutes was chosen since the reaction mixture was previously heated to 95 ° C. for 3 minutes to inactivate the poly-A-polymerase and reverse transcriptase enzymes. This was followed by 45 PCR cycles of 15 sec at 94 ° C and 30 sec at 52 ° C (see Table 46) to amplify the generated let7a cDNA in a real-time PCR. For detection, a fluorescence-labeled probe specific for the 5 'tail of the Uni Gap dT primer was used. The fluorescence data were recorded during the 52 ° C annealing / extension step.
• the "3inl" reaction was performed with an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) in a reaction volume of 20 ⁇ l.
• the model sample consisted of an RNA oligonucleotide with the sequence of mleu7a miRNA. This was injected into a background of maize RNA. The reactions were tested in triplicate. For this purpose, the reagents from Table 47 were pipetted together as indicated in Table 48 and the reaction was carried out as indicated in Table 49.
• the reaction protocol consisted first of conditions for the combined reaction of poly-A polymerase and reverse transcription with the QuantiTect Multiplex Reverse Transcriptase Mix (45 min 37 ⁇ C and 15 min 5O 0 C). This was followed by incubation at 95 ° C for 15 min to activate the HotStar Taq DNA polymerase contained in the QuantiTect Multiplex RT-PCR Master Mix. This was followed by 45 PCR cycles with 15 sec at 94 ° C and 30 sec at 52 0 C (see Table 49) to amplify the generated let7a cDNA in a real-time PCR. For detection, a fluorescence-labeled probe specific for the 5 'tail of the Uni Gap dT primer was used. The fluorescence data were recorded during the 52 ° C annealing / extension step.
• the "3inl" reaction was performed with an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) in a reaction volume of 20 ⁇ l.
• CleanAmp primers All of the primers listed under PCR Hot Start Primer are called “CleanAmp” primers, available from Trilinkbiotech.com. “CleanAmp” primers have the property of becoming functional only through the initial heat step at the beginning of the PCR and are therefore functional in the first PCR available.
• the model sample consisted of an RNA oligonucleotide with the sequence of mleu7a miRNA. This was spiked into a background of maize RNA. The reactions were tested in triplicate. For this purpose, the reagents from Table 50 were pipetted together as indicated in Table 51, and the reaction was carried out as indicated in Table 52.
• the reaction protocol consisted first of conditions for the combined reaction of poly-A polymerase and reverse transcription with the QuantiTect Multiplex Reverse Transcriptase Mix (45 min 37 0 C). This was followed by incubation for 15 min at 95 ° C. to activate the HotStarTaq DNA polymerase contained in the QuantiTect Multiplex RT-PCR Master Mix. This was followed by 40 PCR cycles with 15 sec at 94 ° C and 30 sec at 52 0 C (see Table 52) to amplify the generated let7a-cDNA in a real-time PCR. For the detection, a fluorescence-labeled probe specific for the 5 'tail of the Uni Gap dT primer was used. The fluorescence data were recorded during the 52 ° C. annealing / extension step.
• the "3inl" reaction was performed on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) in a reaction volume of 20 ⁇ l
• Fig. 1 shows a comparison between the one-step methods of the present invention (B) and the two-step process as known in the art (A).
• Pol A polymerase enzyme having polyadenyling activity in the sense of the invention
• Reverse transcriptase enzyme with reverse transcriptase activity in the sense of the invention
• rATP ribonucleotide, exemplified by adenosine 5'-triphosphate
• dNTPs deoxyribonucleotides
• Oligo dT Tail Primer Anchor oligonucleotide with various possible embodiments according to the invention
• Uni GAP dT primer special embodiment of the anchor oligonucleotide
• Tail 5 'tail as an optional part of the anchor oligonucleotide
• w defines the inventive length of homopolymer tails (greater than 10-20 bases) attached by the polyadenylation activity
• x, y defines the type and length of the 3 'anchor sequence of the anchor
• FIG. 2 shows a graphic representation of the Ct values from Table 6: Condition a) contained templates in each case; in Condition b), instead of template, only H 2 O was added (H 2 O in PAP reaction). At b), no signal was received until PCR cycle 40 (maximum number of cycles performed), hence "No Ct".
• FIG. 3 shows an agarose gel analysis of the real-time PCR products of Example 1.
• M 100 bp ladder (Invitrogen, catalog # 15628-050).
• FIG. 4 is a tabular representation of Ct values obtained by real-time PCR analysis of the reaction products of the approaches described in Table 8. At 3 and 6, no signal was received until PCR cycle 40 (maximum number of cycles performed), hence "no Ct".
• Figure S shows a plot of Ct values obtained by real-time PCR analysis of the reaction products of the approaches described in Table 8.
• Fig. 6 shows a tabular representation of Ct means obtained by real-time PCR analysis of the reaction products of b) and e) described in Table 18.
• Fig. 7 shows a tabular representation of the real-time PCR results of the b) and d) approaches from Table 18, as well as the controls with only one primer from Table 19 approaches a) -d). No signal was received until PCR cycle 40 (maximum number of cycles performed), hence no Ct.
• Figure 8 shows an agarose gel analysis of the real-time PCR products of Example 3.
• M 100 bp Ladder (Invitrogen, Catalog No. 15628-050).
• Fig. 9 shows a tabular representation of Ct averages obtained by real-time PCR analysis of the reaction products of the batches Ia) b) and 2 a) b) described in Table 30.
• Figure 10 is a tabular representation of Ct averages obtained by real-time PCR analysis of the reaction products of the formulations described in Table 36 1-5 and in Table 37 6-9.
• Lower panel Graph of Ct averages obtained by real-time PCR analysis of the reaction products of the approaches described in Table 36 1-5 and in Table 37 6-9.
• Fig. 11 shows a list of the nucleic acid sequences used.
• Fig. 12 shows anchor oligonucleotides according to the invention.
• Figure 13 shows a comparison between the one-step “3inl” methods of the present invention (B) and the three-step process as known in the art
• A Pol A polymerase: enzyme having polyadenyling activity in the sense of the invention; reverse transcriptase: enzyme rATP: ribonucleotide, exemplified here by adenosine 5'-triphosphate; dNTPs: deoxyribonucleosides; oligo dT tail primer: anchor oligonucleotide with various possible embodiments in the context of the invention: Uni GAP dT primer: with reverse transcriptase activity according to the invention; tail: 5 'tail as an optional part of the anchor oligonucleotide; w: defines the length of the homopolymer tails (greater than 10-20 bases) attached by the polyadenylation activity; x, y: defines the species and length of the invention 3 'Anchor sequence of the anchor oligonucleotide according to
• Fig. 14 shows a tabular representation of Ct averages of a 3 'reaction, ie the combined poly (A) polymerase reaction, reverse transcription and real-time PCR analysis coupled in a reaction vessel, according to the reaction mixture of Example 7 according to Table 41 and Reaction batch from Table 42.
• Lower part Graph of Ct averages of a 3 'reaction, ie the combined poly (A) -polymerase reaction, reverse transcription and real-time PCR analysis coupled in a reaction vessel, according to the reaction mixture from Example 7 according to Table 41 and the reaction mixture from Table 42.
• Fig. 14 shows a tabular representation of Ct averages of a 3 'reaction, ie the combined poly (A) polymerase reaction, reverse transcription and real-time PCR analysis coupled in a reaction vessel, according to the reaction mixture of Example 7 according to Table 41 and Reaction batch from Table 42.
• Lower part Graph of Ct averages of a 3 'reaction, ie the combined poly (A) -
• Ct means of a "3inl reaction ie the combined poly (A) polymerase reaction, reverse transcription and real-time PCR analysis coupled in a reaction vessel, according to reaction mixture reaction mixture of Example 8 according to Table 44 and The reaction mixture from Table 46.
• Bottom part Graph of Ct means of a "3inl reaction, ie the combined poly (A) polymerase reaction, reverse transcription and real-time PCR analysis coupled in a reaction vessel.
• Figure 16 shows various amounts (10 pg to 1 ⁇ g) of miRNAeasy RNA transcribed using miScript in the presence or absence of 100 ng of poly (A) or poly (C) reverse.
• the cDNA generated thereby was used in a real-time PCR; in this case miR-16 and let-7a were tested.
• Figure 17 shows various amounts (10 pg to 1 ⁇ g) of miRNAeasy RNA reverse transcribed using miScript in the presence or absence of various amounts of poly (A).
• the cDNA generated thereby was used in a real-time PCR; miR-16 was tested in this case.
• Figure 18 shows various amounts (10 pg to 1 ⁇ g) of miRNAeasy RNA transcribed using miScript in the presence or absence of various amounts of poly (C) reverse.
• the cDNA generated thereby was used in a real-time PCR; miR-16 was tested in this case.
• Figure 19 shows the use of 10 ⁇ g miRNeasy RNA reverse transcribed in the presence or absence of 50 ng poly (C) using the miScript RT kit.
• the resulting cDNA was used in a real-time PCR to detect GAPDH.
• Figure 20 shows various amounts (1-100 ng) miRNeasy RNA transcribed using miScript in the presence or absence of various amounts of poly (C) reverse.
• the cDNA generated thereby was used in a real-time PCR; in this case to test GADPH.
• Figure 21 shows the use of 10 and 100 pg miRNeasy RNA reverse transcribed in the presence or absence of 50 ng poly (C) and using the miScript RT kit. The resulting cDNA was tested in a real-time PCR to detect GAPDH.
• Figure 22 shows various amounts (1-100 ng) of miRNeasy RNA transcribed using miScript in the presence or absence of various amounts of poly (C) reverse.
• the cDNA generated thereby was used in a real-time PCR; in this case to test CDC2.
• Figure 23 shows the use of 1 ng miRNeasy RNA reverse transcribed in the presence or absence of 50 ng poly (C) and using the miScript RT kit. The resulting cDNA was tested in a real-time PCR to detect CDC2.
• FIG. 24 shows a tabular representation of Ct averages of a 3 'reaction, ie the combined poly (A) -polymerase reaction, reverse transcription and real-time PCR analysis with primer Hot Start coupled in a reaction vessel, according to the reaction mixture reaction mixture from Example 9 according to Table 48 and the reaction mixture from Table 49.
• FIG. 25 shows a tabular representation of Ct averages of a 3 ⁇ reaction, ie the combined poly (A) -polymerase reaction, reverse transcription and real-time PCR analysis with primer Hot Start coupled in a reaction vessel, according to the reaction mixture reaction mixture from Example 10 according to Table 51 and the reaction mixture from Table 52.

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