EP2326431A1 - Verfahren zur normierung des gehalts von biomolekülen in einer probe - Google Patents

Verfahren zur normierung des gehalts von biomolekülen in einer probe

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Publication number
EP2326431A1
EP2326431A1 EP09783068A EP09783068A EP2326431A1 EP 2326431 A1 EP2326431 A1 EP 2326431A1 EP 09783068 A EP09783068 A EP 09783068A EP 09783068 A EP09783068 A EP 09783068A EP 2326431 A1 EP2326431 A1 EP 2326431A1
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EP
European Patent Office
Prior art keywords
reaction
biomolecules
pcr
vessel
reaction vessel
Prior art date
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EP09783068A
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German (de)
English (en)
French (fr)
Inventor
Christoph Erbacher
Peter GRÜNEFELD
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Qiagen GmbH
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Qiagen GmbH
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Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP2326431A1 publication Critical patent/EP2326431A1/de
Withdrawn legal-status Critical Current

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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

Definitions

  • the present invention relates to a method, a use and a device for normalizing the content of biomolecules in a sample.
  • the method, the use and the device are suitable, for example, for applications in biochemistry, molecular biology, molecular genetics, microbiology, molecular diagnostics and / or molecular forensics.
  • the standardization of the content of biomolecules in a sample plays a major role in the analysis of samples, for example in molecular diagnostics, gene expression analyzes, in drug-related transcript level analysis, molecular forensics, sequencing or genotyping.
  • biomolecules to be detected-in particular nucleic acids and / or proteins-can occur in different amounts in the sample.
  • preparatory sample treatment steps e.g. a lysis, a cell disruption, an isolation step or a reverse transcription - to provide the biomolecules concerned with different levels of efficiency.
  • PCR polymerase chain reaction
  • RNA content of a sample is usually determined before the beginning of the reverse transcription, for example by means of OD or fluorescence measurements.
  • a quantification at the cDNA level is usually not carried out, since existing nucleotides, ribosomal RNA u.a. Components after completing reverse transcription complicate a quantification of the cDNA.
  • Such a quantification approach can be used in particular in so-called two-step methods in which the sample preparation (eg by lysis, cell disruption, isolation step or reverse transcription) and the sample further treatment (eg by PCR) takes place in separate steps and / or different vessels, so that a pipetting step can take place between the two steps.
  • sample preparation eg by lysis, cell disruption, isolation step or reverse transcription
  • sample further treatment eg by PCR
  • a pipetting step can take place between the two steps.
  • a pipetting step is suitable for those methods in which a possibly already prepared sample is directly subjected to a PCR process.
  • One-step methods from the prior art use an optionally spectroscopically quantified amount of biomolecules (for example mRNA).
  • biomolecules for example mRNA
  • Both the reverse transcription and the PCR procedure itself are carried out in the same reaction vessel. Since the latter is not opened during the procedure as far as possible, so the complete batch of reverse transcription is converted into the PCR reaction.
  • the biomolecule content for example, of mRNA
  • the efficiency of the sample preparation step for example, reverse transcription
  • is also subject to fluctuations which leads in some cases to very different product quantities after completion of the sample preparation step (for example, cDNA). which are then introduced into the subsequent reaction (for example PCR), and thus leads to non-reproducible reaction results.
  • the additional reaction batch volume of usually 20 .mu.l also can not be used in a customary for a PCR volume of 25 ul.
  • single-stage RT-PCRs are performed in a 50 ⁇ l or 100 ⁇ l scale.
  • the method according to the invention or the device according to the invention e.g. normalizes an absolute amount of nucleic acid by a binding area produced according to the invention in a device, instead of quantifying it with OD measurements or fluorescence measurements.
  • the so-called "housekeeper approach” is an internal or endogenous reference that can also be used to make a statement about the state of the biological system. The quality of the assay can be assessed with the housekeeper gene, because it exists for certain housekeeper genes associated with certain cell types are indicative of their level of expression.
  • Nucleic acids can then be bound.
  • a pH above the pKa value of the surface groups on the other hand, a change in the charge from the positive to the neutral or negative, so that negatively charged biomolecules, in particular nucleic acids, can be released again.
  • suitable buffers with different pH values which otherwise have low salt concentrations (“low-salt buffer”), the binding and release process can be controlled via the pH value.
  • suitable materials are not permanently covalent, for example, on the surface of
  • Micro-reaction vessels for example made of polypropylene, fasten.
  • RNA isolation of RNA from biological samples under these conditions is problematic with charge-switch materials and generally with anion exchangers due to the ubiquitous RNAs, which remain intact under the prevailing low salt conditions, degrading RNA significantly within a few seconds and providing detection difficult or even impossible.
  • nucleic acid is understood to mean, in particular but not limited to, natural, preferably linear, September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • branched or circular nucleic acids such as RNA, in particular mRNA, single-stranded and double-stranded viral RNA, siRNA, miRNA, snRNA, tRNA, hnRNA or ribozymes, genomic, bacterial or viral DNA (single-stranded and double-stranded), chromosomal and episomal DNA, free-circulating nucleic acid and the like , synthetic or modified nucleic acids, for example plasmids or oligonucleotides, in particular primers, probes or standards used for the PCR, nucleic acids labeled with digoxigenin, biotin or fluorescent dyes or so-called PNAs ("peptide nucleic acids").
  • RNA in particular mRNA, single-stranded and double-stranded viral RNA, siRNA, miRNA, snRNA, tRNA, hnRNA or ribozymes, genomic, bacterial or viral DNA (single-stranded and double-stranded
  • normalizing the content of biomolecules in a sample is understood to mean a step in which it is ensured that the content of biomolecules in the sample is a predetermined (according to the invention by the size and binding properties of at least part of the vessel surface, preferably at the Inside the vessel) is a measure of quantifying the content of biomolecules to a predetermined value, which implies that biomolecules beyond this level are subsequently discarded, and further implies that if the sample contains fewer biomolecules than the predetermined measure described above, the normalization is unsuccessful.
  • immobilization in the sense of the present invention is understood to mean, in particular but not limited to, a reversible immobilization to a suitable solid phase.
  • membranes are meant, in particular but not limited to, solid phases capable of reversibly binding biomolecules. September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • high salt buffer in particular, but not limited to, a buffer having a high salt concentration (preferably chaotropic substances), preferably> 100 mM, more preferably> 500 mM and more preferably> 1 M.
  • high salt conditions is understood below to mean a medium which uses a high salt buffer, preferably a high salt buffer containing chaotropic salts
  • High salt preferably containing chaotropic salts
  • a polar surface as a hydrogen bond donor
  • the nucleic acids bind to this surface, where they undergo better stabilization than in water Water again a better hydrogen bond donor than the polar surface, and the nucleic acids can be detached again from the surface.
  • chaotropic substances or “chaotropic salts” are in particular - but not limited to - substances containing the secondary,
  • Tertiary and / or quaternary structure of proteins and / or nucleic acids change and at least leave the primary structure intact, the solubility more polar
  • Preferred chaotropic substances are guanidine hydrochloride, guanidinium (iso) thiocyanate, sodium iodide,
  • silica amorphous, crystalline
  • polysilicic acid with the September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • the material can be surface-functionalized.
  • the silanol groups may have been treated by silanization with silanes.
  • the surface can be hydrophobized or anionic and / or cationic groups and / or chelators can be applied.
  • a nitrilotriacetic acid (NTA) can be applied as a chelating group. This allows an adaptation of the adsorber surface to the biomolecules to be bound.
  • halogen-containing atom transfer radical initiators to the silanol groups by means of a silanization process so that polymer chains can be generated by a "grafting from” process on the silanol groups.
  • This process also referred to as graft copolymerization, requires polymerization processes in which the tendency to chain break, disproprionation or recombination has a low frequency.
  • initiators must be applied to the silanol groups. This can be done by treating the PECVD silicate layer with halogenated silanes. Alternatively, the initiators are introduced directly in the PECVD process. In the process, volatile, halogen-containing compounds are added to the process gas (precursor in the PECVD process). September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • polymers which are covalently bonded to the surface can be generated in situ.
  • Suitable monomers are radically polymerizable compounds, e.g. Acrylates, methacrylates, styrene and styrene derivatives.
  • the atom transfer radical polymerization is a form of living, radical polymerization.
  • the radicals are formed via a Cu-I / CuII redox equilibrium from an organohalide through an atom transfer process.
  • the redox balance ensures a strong reduction in the concentration of free radicals. Chain termination reactions by disproportionation or recombination are therefore strongly suppressed.
  • amplification reaction is understood to mean a process which makes it possible to at least double the concentration of one or more analytes, preferably nucleic acids.
  • thermocyclic amplification reactions A distinction is made here between isothermal and thermocyclic amplification reactions. In the former, the temperature remains the same throughout the process, while in the latter, thermal cycles are passed through which the reaction and amplification are controlled.
  • Preferred isothermal amplification reactions are e.g.
  • LAMP Loop mediated isothermal amplification
  • NASBA Nucleic Acid Sequence Based Amplification
  • TMA Transcription mediated amplification
  • thermocyclic amplification reactions are e.g.
  • Ligase chain reaction (LCR), and / or
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence based amplification
  • an RNA matrix is added to a reaction mixture, and a first primer binds to the complementary sequence near the 3 'end of the matrix.
  • the complementary to the matrix DNA strand is polymerized with a reverse transcriptase.
  • RNase H the RNA matrix is then digested (RNase H digests only RNA in RNA-DNA hybrids, not single-stranded RNA).
  • a second primer is attached to the 5 'end of the DNA strand. This is used by the T7 RNA polymerase as a starting point for the synthesis of a DNA strand complementary to the RNA molecule, which can then be used again as the starting matrix.
  • NASBA is maintained at a constant temperature of normally 41 ° C. performs and delivers faster and better results than PCR under certain circumstances.
  • TMA Transcription Mediated Amplification
  • rolling circle chain reaction (RCCR) or “rolling circle amplification” (RCA) refers to an amplification method that mimics the general nucleic acid replication according to the rolling circle principle and is described inter alia in US5854033.
  • immuno-PCR is understood to mean, in particular, a method for detecting target molecules in which chimeric conjugates of target-specific antibodies and nucleic acid molecules are used.
  • target molecules are above all proteins and / or oligopeptides, since they are the simplest against these molecular species September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • target molecules may also be other biomolecule species, e.g. Oligo- and polysaccharides, or lipids, as long as against these biomolecule lekülspezies spaspezif ⁇ sche antibodies can be produced so that they can be detected using the immuno-PCR.
  • biomolecule species e.g. Oligo- and polysaccharides, or lipids
  • the nucleic acid molecules serve as markers or probes which are amplified for signal generation by means of polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the tremendous efficiency of the nucleic acid amplification and the high specificity of binding can result in a 100 to 10,000-fold increase in sensitivity compared to standard methods for detection of target molecules (e.g., ELISA methods).
  • the IPCR was developed in 1992 (Sano et al., (1992)).
  • reverse transcription is understood to mean a method for rewriting mRNA into DNA (the so-called “cDNA”), in which a reverse transcriptase (also RNA-dependent DNA polymerase) is generally used. This first synthesizes an RNA-DNA hybrid strand from a single-stranded RNA by means of an RNA-dependent DNA polymerase activity. For the subsequent degradation of the RNA portion, a separate section of the protein is responsible, the RNase H-share. This is followed by the completion of the single-stranded DNA strand to double-stranded by DNA-dependent DNA polymerase activity. The cDNA generated in this way can then be amplified and detected by means of PCR.
  • cDNA reverse transcriptase
  • a reverse transcriptase primer also requires the initiation of DNA synthesis.
  • a so-called oligo-d (T) primer is used here, ie several thymine bases, which are complementary to the poly (A) tail at the 3 'end of the mRNA.
  • RT-PCR reverse transcription followed by PCR
  • PCR also referred to as RT-PCR
  • different primers are used for reverse transcription and the subsequent PCR
  • the gene-specific primers are already used in the reverse transcription instead which are also used for the subsequent PCR and both reactions are carried out consecutively in the same vessel, making use of the fact that the reverse transcriptase used (usually of viral origin) denatures at a lower temperature than the DNA polymerase used (eg Taq polymerase), which is known to denature only at relatively high temperatures, and accordingly performs the reverse transcription at a lower temperature than the subsequent PCR
  • a so-called hot-start DNA polymerase is used, which is thermoreversibly inhibited When switching from the lower temperature level of the reverse Transcription to the higher temperature level of the PCR
  • thermoreversible inhibition may e.g. be realized by an antibody which binds in the active center of the DNA polymerase, as well as e.g. by reversible covalent or non-covalent chemical modification of the polymerase, for example with aldehydes (see, e.g., Applicant's U.S. Patent 6,183,998).
  • aldehydes see, e.g., Applicant's U.S. Patent 6,183,998.
  • Birch et al. (1996).
  • PCR polymerase chain reaction
  • the quantification is performed by fluorescence measurements taken during a PCR cycle (hence the name "Real Time") .
  • the fluorescence increases proportionally with the amount of PCR products at the end of a run Cycles)
  • the quantification in the exponential phase of the PCR is carried out on the basis of the fluorescence signals obtained, only in the exponential phase of the PCR (which lasts a few cycles in one run) the correct quantification is possible, since during this phase the optimal reaction conditions prevail
  • the method differs from other quantitative PCR methods, which perform a quantitative evaluation (eg competitive PCR) only after the end of the PCR, usually involving gel electrophoretic separation of the PCR fragments.
  • dyes such as e.g. Ethidium bromide, SYBR Green I and FRET probes or so-called double-dye oligos (also referred to as TaqMan probes) in question.
  • C ⁇ value (Threshold Cycle) describes the PCR cycle at which an amplificate is detectable for the first time; In this case, the fluorescence is usually measured and the cycle is indicated, at which the latter increases significantly above the background fluorescence for the first time.
  • a low C ⁇ value indicates that even a small number of PCR cycles is sufficient for a first significant increase in fluorescence over the background noise (ie, a relatively large amount of template was present), while a high C ⁇ value accordingly indicates that there are many PCRs for this purpose - Cycles are needed (so relatively little template was present).
  • ELISA Enzyme Linked Immunosorbent Assay
  • proteins, viruses as well as low-molecular compounds such as hormones, toxins and pesticides can be detected in a sample (blood serum, milk, urine, etc.).
  • a sample blood serum, milk, urine, etc.
  • Antibody or antigen are previously labeled with an enzyme.
  • the enzyme catalyzed reaction serves as evidence of the presence of the antigen.
  • the so-called Substrate is reacted by the enzyme, the reaction product can usually be detected by color change, fluorescence or Chemo luminescence. Signal strength is generally a function of antigen concentration, so ELISA can also be used for quantitative detection.
  • hybrid capture assay is understood below to mean a process in which RNA: DNA hybrids are formed by incubation of the desired target DNA with an RNA sample. The hybrids are bound to a surface and then incubated with an enzyme-labeled antibody. "Hybrid Capture Assay” are used in particular in the HPV assays of Digene.
  • nested PCR is understood below to mean a method in which an already duplicated DNA fragment is amplified a second time; this process is done with a second primer pair located within the primer pair used in the first reaction.
  • Another object is to provide, for a wide range of applications, a method, a use, and a device for normalizing the level of a nucleic acid in a sample. September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • Another object is to provide, for a wide range of applications, a method, a use and a device for normalizing the content of a cDNA produced by means of a reverse transcription from RNA, preferably mRNA.
  • Another object is to provide, for a wide range of applications, a method, a use and a device for normalizing the content of a nucleic acid, in particular RNA in a sample.
  • Another object is to provide a method, a use and a device for normalizing the content of a nucleic acid in a sample, which allows a repeated use of the reaction vessels.
  • a method for normalizing the content of biomolecules in a sample comprising the following steps:
  • reaction vessel having a vessel surface which is at least partially functionalized - preferably on the inside of the vessel - in such a way that it can bind biomolecules reversibly under high salt conditions, b) performing at least one sample preparation step, September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • the surface of the reaction vessel functions for the first time as an adsorption surface for the reversible binding of a defined amount of biomolecules under high salt conditions, and thus as a means of standardizing the biomolecule content in the sample. It is therefore functionalized in such a way that it can reversibly bind biomolecules under high salt conditions.
  • the type of functionalization also depends in particular on the type of biomolecules to be bound. This will be discussed below. The amount of biomolecules that can be bound to the surface is adjusted by the size of the surface, the area contacted by the sample, the nature of the chemical functionalization of that surface, the incubation time of the biomolecules with the surface, and the stringency of the binding buffer used ,
  • RNA contained in a sample can be isolated as completely and intact as possible and sent to detection.
  • a follow-up reaction is performed. This step is also taken as an opportunity to perform a normalization at the cDNA level. It compensates for variations in cellular input, RNA quality and quantity, RT efficiency, allowing the results to be compared and interpreted.
  • the normalization also has the advantage of correcting the measured expression data for differences in cellular input, RNA quality / quantity and efficiency of reverse transcription of different samples (the latter principle only effective in two-step RT PCR).
  • biomolecules are nucleic acids.
  • Nucleic acids are particularly useful with conventional amplification techniques, e.g. PCR, detectable.
  • biomolecules may also be members of any biomolecule species that are detectable with antibodies.
  • proteins intended to be detected by oligonucleotide-labeled antibodies immuno-PCR
  • ELISA ELISA
  • said at least one sample preparation step is selected from the group comprising
  • Said enzymatic reactions and / or sample treatments may preferably be digestion of a sample with RNAses, DNAses and / or proteases.
  • said at least one subsequent reaction is selected from the group comprising
  • Enzyme-Linked Imunoassay ELISA
  • ELISA Enzyme-Linked Imunoassay
  • the said amplification reaction be a reaction selected from the group
  • any other possible detection reaction for at least one of the biomolecule species mentioned above can also be provided here.
  • a particularly suitable example of the method according to the invention consists of a method comprising reverse transcription of mRNA into cDNA (sample preparation step), normalization of the cDNA formed by binding to the vascular surface (binding or standardization step), and subsequent detection amplification of the cDNA Real-time PCR (follow-up reaction). This procedure is referred to in Table 1 as "Workflow 1".
  • the reverse transcription is carried out directly in a reaction vessel according to the invention and the generated cDNA according to September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • An advantage of this method according to the invention over prior art methods is that despite the omission of quantification of the resulting cDNA amount, a normalized amount of cDNA is used for the PCR, regardless of the amount of RNA initially used. Likewise, it is possible to work in a volume of 25 ⁇ l which is customary for a PCR, which leads to a considerable cost reduction.
  • the reverse transcription as well as the subsequent PCR are preferably carried out in the same vessel as described. However, it may also be provided that, after the binding or standardization step, one or more aliquots are removed from the reaction vessel and transferred to one or more new reaction vessels in order to carry out the PCR in the other reaction vessel (s). September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • Another suitable example of the method according to the invention consists of a method comprising the sample digestion and the subsequent recovery of mRNA, for example with the QIAGEN product RNeasy, or alternatively with the isolation of mRNA, for example with the QIAGEN products Oligotex and / or TurboCapture, (Sample preparation step), the normalization of the released or isolated mRNA by binding to the vessel surface (binding or standardization step), and the subsequent reverse transcription of the mRNA in cDNA (subsequent reaction).
  • the reverse transcription may be followed by another binding or standardization step and another subsequent reaction, for example a real-time PCR of the cDNA generated (see Table 1, Workflow 6).
  • a simple hybridization reaction may be provided as a follow-up reaction;
  • the further binding or standardization step is dispensable (see Table 1, Workflow 2).
  • workflows according to the method of the invention are shown in Table 1.
  • Workflows 1-5 are one-step, because in the course of the procedure, the reaction vessel does not have to be opened to add new reagents.
  • Workflow 6 shows a two-step procedure, as it is normalized to mRNA level and cDNA level, ie between the two reactions, the vessel is opened to initiate the second binding or standardization step.
  • Both the reverse transcription and the possibly subsequent second consecutive reaction can be carried out in the same vessel as the sample preparation step. However, it can also be provided that after the first or, if appropriate, the second binding or standardization step, one or more aliquots are removed from the reaction vessel and transferred to one or more new reaction vessels in order to carry out the at least one subsequent reaction in the one or more Carry out reaction vessels.
  • biomolecules preferably RNA and / or DNA
  • a binding buffer is used in the binding or standardization step and / or b) a washing buffer is used during the washing step.
  • the at least partially functionalized vessel surface is preferably provided.
  • silanol groups a) silanol groups, b) unsaturated organic acids, c) carboxyl groups, sulfonate groups and other polar groups, and / or d) metal and semimetal oxides with hydroxyl groups
  • the said groups unlike the "batch switch materials" mentioned above, can be permanently (i.e., usually covalently) permanently attached to the surface of microreaction tubes and PCR tubes by suitable methods (see below).
  • a chaotropic substance such as guanidinium thiocyanate.
  • the cells are lysed, denaturing the proteins contained and the nucleic acids - if not yet freely available - released, and it is due to the presence of the chaotropic substance to a dissolution of the hydration shells to the nucleic acids.
  • Binding of the nucleic acids to the silica surface occurs via hydrogen bonding between the silanol groups (SiOx or SiOH groups) of the silica matrix and the negative ion charges of the phosphate backbone of the nucleic acids.
  • the remaining components of the sample can then be removed by washing.
  • the DNA or RNA is finally released again under the conditions of the PCR.
  • the binding of the nucleic acids to an anion exchange surface is based on the electrostatic interaction between the negative ion charge of the phosphate backbone of the nucleic acids and the positive surface charge of the anion exchange surface of the invention.
  • Quaternary ammonium groups belong to the category of strongly basic anion exchangers because their charge is independent of the pH of the binding buffer.
  • Primary, secondary and tertiary amines are referred to as weakly basic anion exchangers. At higher pH values, these are present in deprotonated form, resulting in the loss of the September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • silanol groups in particular SiO 2
  • metal and semimetal oxides which have hydroxyl groups on the surface, in particular titanium, aluminum and zirconium oxides, such as TiO 2 , Al 2 O 3 and ZrO 2 .
  • Nucleic acids also bind to these groups in the presence of chaotropic salts, and appropriately coated or functionalized surfaces can also be used under the given conditions to bind nucleic acids.
  • the unsaturated organic acids bound in this way provide carboxyl groups (-COO " or -COOH groups) which are capable of reversibly binding nucleic acids under high salt conditions, with the unsaturated organic acids being only the means to an end, carboxyl groups into a PECVD-coated polymer to create a polar surface with hydrogen-bond donating functionality.
  • nucleic acids in water If a polar surface is offered as a water-bridged donor, the nucleic acids bind to this surface because they experience better stabilization there than in water. When the salt concentration is reduced, water again becomes a better water-bridged donor than the polar surface, and the nucleic acids can be released from the surface again.
  • a reaction vessel for carrying out a method as described above, which has a vessel surface which is at least partially functionalized - preferably on the inside of the vessel - in such a way that it can reversibly bind biomolecules under the mentioned process conditions.
  • the functionalized area of the reaction vessels according to the invention preferably has an area of 0.01 mm 2 inclusive - including 10 cm 2 per
  • Reaction vessel more preferably an area of 0.01 mm 2 inclusive
  • the binding capacity of the relevant reaction vessel for the relevant biomolecules can be precisely adjusted.
  • the amount of biomolecules that are bound can also be influenced by the contact time and stringency of the binding buffer. The goal, however, is the September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • the binding capacity per reaction vessel in the range between 1 ng and including 40 ug, more preferably between 1 ng and including 4 ug, more preferably between 1 ng and including 2 ug, most preferably between 1 ng and including 500 ng.
  • the at least partially functionalized vessel surface is preferably provided that the at least partially functionalized vessel surface
  • silanol groups a) silanol groups, b) unsaturated organic acids, c) carboxyl groups, sulfonate groups and other polar groups, and / or d) metal and semimetal oxides with hydroxyl groups
  • a) are applied to the material of the reaction vessel by plasma coating, b) by wet chemical processes on the material of the reaction vessel
  • Reaction vessel are applied, and / or c) are conditioned by the properties of the material of the reaction vessel itself.
  • the application of the silanol groups according to the invention by plasma coating onto the material of the inventive reaction vessel is preferably carried out in atmospheric pressure plasma, as described, for example, in DE102006036536B3 and DE000010322696B3 of the Fraunhofer Institute for Surface Engineering and Thin Films, the contents of which are hereby incorporated by reference. September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • PECVD Plasma Enhanced Chemical Vapor Deposition
  • CVD chemical vapor deposition
  • a strong alternating electric field is applied, by which a plasma is ignited.
  • the plasma breaks up the bonds of a gaseous deposition medium, also called reaction gas, and decomposes it into single radicals, which in the The gas phase reaction products deposit on the substrate in the form of thin layers (layer thicknesses between 50 and 300 nm.) Due to the plasma can in the PECVD process, which is often called corona discharge, a higher deposition rate at a lower deposition temperature achieved by the CVD method
  • the deposition media to be used are already in the gas phase and can thus be easily introduced from the outside of the reaction chamber gas supply system in the reaction chamber and fed to the plasma.
  • the carbonaceous gases acetylene (C 2 H 2 ) or methane are used as precursors for the production of a carbon-containing coating, such as DLC ("diamond like carbon") September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • Tetramethylsilane TMS
  • TEOS tetraethoxysilane
  • TMOS tetramethoxysilane
  • suitable precursors exist, for example, for the deposition of TiO 2 , Al 2 O 3 and ZrO 2 .
  • the precursor is fed into the discharge zone of the plasma, where the gas is split into ions and accelerated selbige. Often, oxygen is simultaneously fed to burn any organic portion in the precursor, for example, in the preparation of a silica coating with tetramethylsilane (but not, for example, in the preparation of a carbonaceous coating with acetylene).
  • gas ions then impinge on the surface of the workpiece to be coated at high speed, where they are reduced and build up the relevant coating. Frequently, covalent bonds are formed between the material of the surface and the coating materials, which ensure permanent bonding of the coating to the material.
  • the covalent attachment to PP occurs via Si-O-C and Si-C bonds.
  • the covalent attachment to PP takes place, for example, via CC and COC bonds.
  • the PECVD-V also makes it possible to generate organic polymers in the gas phase in the plasma and to deposit them on the carrier.
  • monomers e.g. Maleic anhydride, acrylates, vinyl silanes and the like.
  • polymerizable precursors are used.
  • the oxygen in the carrier gas is likewise dispensed with in order not to oxidize the organic constituents.
  • the precursor aminopropyltrimethoxysilane allows e.g. a direct preparation of silanol groups and anion exchange groups in a PECVD layer.
  • Metal and semimetal oxides can also be prepared by the PECVD process from the corresponding precursors (metal or semimetal alkoxides) in the gas phase with the addition of oxygen and, for example, deposited on polypropylene.
  • gas phase polymers of, for example, acrylates and other unsaturated compounds can be prepared and coated in situ by means of PECVD.
  • monomers eg, HEMA, acrylic acid, maleic anhydride, etc.
  • vinylsilane mixed co-polymers of an organic monomer such as maleic anhydride and a silane (vinylsilane) can also be deposited on polypropylene.
  • hydroxyl groups (geminal, vicinal), diol groups, carboxyl groups, amino groups and silanol groups are suitable chemical functions to produce surface materials having hydrogen bond donating properties on polypropylene.
  • co-polymers of maleic anhydride and vinylsilane were also prepared in situ. It is advantageous that this precursor mixture has a sufficient vapor pressure, can be polymerized in the gas phase by means of PECVD and thus forms well-adhering layers on polypropylene to the material of the reaction vessel.
  • sulfonate groups by means of PECVD can be used as precursor e.g. Styrenesulfonic acid can be used.
  • the inventors of the present invention also show that with the aid of a suitable coating apparatus, the inner surfaces of microreaction vessels, in particular PCR reaction vessels ("8-th Strips", 96-well plates, multi-well plates), disposable reaction vessels and pipette tips, with tetraethoxysilane and carboxyl-containing Copolymers of vinylsilane and maleic anhydride can be coated as precursors
  • PCR reaction vessels 8-th Strips
  • disposable reaction vessels and pipette tips with tetraethoxysilane and carboxyl-containing Copolymers of vinylsilane and maleic anhydride
  • the device shown can be used in a parallel arrangement, so that a plurality of reaction vessels can be coated simultaneously.
  • Preferred wet chemical methods for applying silanol groups to the material are e.g. Sol-gel processes.
  • the precursors are dissolved together with a defined amount of water and any catalysts in a solvent, for example water.
  • a solvent for example water.
  • TEOS tetraethoxysilane
  • Silciumdioxid precursor is used as Silciumdioxid precursor.
  • the silanol groups according to the invention are conditioned by the properties of the material of the reaction vessel according to the invention itself; for example, if the material is glass.
  • the reaction vessel is a vessel from the group comprising a) PCR vessels, PCR 8ter strips or PCR 96-well plates, b) a capillary or a microfluidic channel, c) a disposable reaction vessel, d) a pipette tip and / or e) a multi-well plate.
  • microreaction vessels mentioned may be, for example, colloquially also termed PCR reaction vessels (ABI, Thermo, etc.), possibly closable vessels having a volume of 0.1-2 ml. These are usually made of polypropylene, polyethylene, COC, PET or polycarbonate. September 16, 2009
  • a microtiter plate is a unit comprising a multiplicity of "wells" in the sense of the invention, such microtiter plates generally having between 6 and up to 1536 wells in each case Typical microtiter plate formats are shown in Table 2.
  • kit for carrying out a method as described above wherein said kit has at least
  • reagents for carrying out a sample preparation step preferably a reverse transcription (RT)
  • reagents for carrying out a subsequent reaction preferably a polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • the washing buffer contains water, TRIS, a complexing agent, a polyol, a detergent, a polymer, copolymer and / or terpolymer.
  • the binding buffer has chaotropic substances. These are particularly preferably at least one substance selected from the group containing
  • the binding buffer for binding the cDNA to an anion exchange surface is preferably a low salt buffer.
  • a pH value below the pKa value of the surface or of the surface groups is preferably set, so that in the binding step the anion exchangers have positive surface charges and thus can bind the negatively charged nucleic acids.
  • reaction vessel according to the invention and / or a kit according to the invention for standardizing the content of biomolecules in a sample.
  • RNA preferably mRNA
  • PCR polymerase chain reaction
  • FIG. 1 shows a diagram for checking the C ⁇ values of PAXGene RNA from human whole blood using a method according to the invention
  • FIG. 3 shows a diagram for checking the C ⁇ values of QIAamp RNA from human whole blood using a reaction vessel according to the invention or comparative examples
  • FIG. 4 shows a diagram for checking the C ⁇ values of QIAamp RNA from Jurkat cells when using a reaction vessel according to the invention or comparative examples
  • FIG. 5 shows a diagram for checking the C ⁇ values of QIAamp RNA from Jurkat cells using a reaction vessel according to the invention and different incubation times
  • FIG. 7 shows a device 70 for applying a functionalized surface according to the invention to the inside of a reaction vessel.
  • the inventive surface-modified reaction vessels used (PCR vessel, 0.2 ml, thin-walled, PCR soft strips, Biozym) were coated with tetraethoxysilane (TEOS) in the atmospheric pressure plasma.
  • PCR vessel 0.2 ml, thin-walled, PCR soft strips, Biozym
  • TEOS tetraethoxysilane
  • the binding buffer used was 6M GuHCl, 0.1M potassium hydrogen phthalate pH 2.5, and as washing buffer 2% Nonidet® P40 and 0.1 mg / ml poly (methyl vinyl ether-altemalic acid) in TE buffer.
  • the PCR was performed with an ABI TaqMan® ⁇ Actin Probe Kit in an ABI 7700.
  • RNA used in the reverse transcription was quantified in advance with a nanodrop spectrophotometer ND-1000.
  • RNA used was varied in a small range (113-293 ng).
  • FIG. 2 likewise shows the results of a TaqMan® run on the cDNA from a QuantiTect cDNA synthesis of PAXGene RNA from human whole blood.
  • the amount of RNA was varied between 150 and 450 ng (columns 1-5), in each case one aliquot of the reverse transcription was pipetted into untreated vessels with PCR master mix (columns 6-10). For these aliquots, the starting amount of RNA contained therein is indicated. Compared to the first example, the amount of RNA used was varied over a wider range.
  • FIG. 3 shows the results of a TaqMan® run on the cDNA from an omniscript cDNA synthesis of QIAamp RNA from human whole blood.
  • the amount of RNA between 100 and 1 100 ng RNA was varied, it also control experiments were carried out in uncoated vessels.
  • RNA 4 shows the results of a TaqMan® run on the cDNA from an Omniscript cDNA synthesis of Jurkat RNA. In this case, the amount of RNA between 100 and 1100 ng RNA was varied, it was again carried out control experiments in uncoated vessels.
  • Fig. 5 shows the results of a TaqMan® run on the cDNA from an Omniscript cDNA synthesis of Jurkat RNA.
  • the amount of RNA was varied between 100 and 800 ng of RNA.
  • different incubation times were chosen, namely for 100-400 ng RNA 240 min, for 500-800 ng RNA 20 min.
  • FIG. 6a shows the general scheme of a workflow according to the invention, with the sample preparation step 2, the binding or standardization step 3, the washing step 4 and the follow-up reaction 6.
  • a second binding or standardization step and a second follow-up reaction may possibly follow said subsequent reaction connect (see Table 1).
  • Fig. 6b it is shown that said workflow can also be performed in a PCR strip.
  • FIG. 7 shows a device 70 for applying a functionalized surface according to the invention to the inside of a reaction vessel. September 16, 2009 QIAGEN GmbH V ⁇ -V 349
  • Said device has a chamber 71 in which a flat electrode de 72 is arranged. Furthermore, the chamber has a gas introduction device for a precursor gas 73 and a coating electrode 74.
  • the precursor gas is, for example, tetraethoxysilane (TEOS).
  • TEOS tetraethoxysilane
  • the gas inlet device 73 and the coating electrode 74 are combined in the apparatus of FIG. 7 to a combined device in shape to the interior of a reaction vessel to be coated 75 (here a colloquially referred to as "Eppendorf tube” PCR microreaction vessel made of polypropylene with a volume of 0.2 ml) is adjusted.
  • a high-frequency alternating voltage is now applied (for example, 13.56 MHz) with the aid of a frequency generator 76, and a plasma ignites in the interior of the reaction vessel.
  • the plasma breaks up the bonds of the percursor gas and decomposes it into individual radicals which precipitate on the substrate where they cause the chemical precipitation reaction of silica molecules.
  • the functionalized surface produced in this way brings about the binding of the biomolecules to the inside of the reaction vessel (for example the binding of nucleic acids to silanol groups of the functionalized surface in the presence of chaotropic salts).
  • the apparatus shown is also suitable for the simultaneous coating of a plurality of reaction vessels, for example a plurality of "Eppendorf vessels" or else, for example, a multi-liter plate having a plurality of wells.
  • a plurality of reaction vessels for example a plurality of "Eppendorf vessels” or else, for example, a multi-liter plate having a plurality of wells.
  • the size of the coated area of the reaction vessels is essentially dependent on the size of the electrode and may be between 10 mm 2 and 300 mm 2 in a 0.2 ml PCR vessel.
  • Polypropylene 0.2 ml polypropylene tubes were coated with tetraethoxysilane under atmospheric pressure in the PECVD method. Binding capacities for nucleic acids of up to 500ng were generated.
  • the area created during the coating is defined, inter alia, by the size and positioning of the electrodes (cathode, anode) relative to one another in the PECVD method. It was about 150 mm 2 here .

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CN104217612B (zh) * 2014-07-22 2016-05-25 刘全羽 一种停车场实时智能车辆引导系统
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