WO2024083474A1 - Marquage et enrichissement combinés spécifiques de cellules de biomarqueurs - Google Patents

Marquage et enrichissement combinés spécifiques de cellules de biomarqueurs Download PDF

Info

Publication number
WO2024083474A1
WO2024083474A1 PCT/EP2023/077002 EP2023077002W WO2024083474A1 WO 2024083474 A1 WO2024083474 A1 WO 2024083474A1 EP 2023077002 W EP2023077002 W EP 2023077002W WO 2024083474 A1 WO2024083474 A1 WO 2024083474A1
Authority
WO
WIPO (PCT)
Prior art keywords
microcavity
cell
amplification
primer
biomarker
Prior art date
Application number
PCT/EP2023/077002
Other languages
German (de)
English (en)
Inventor
Anke Timm
Samir KADIC
Christian GRUMAZ
Sebastian PILSL
Michael Knapp
Astrid LUX
Original Assignee
Robert Bosch Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Robert Bosch Gmbh filed Critical Robert Bosch Gmbh
Publication of WO2024083474A1 publication Critical patent/WO2024083474A1/fr

Links

Classifications

    • 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
    • C12Q1/686—Polymerase chain reaction [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
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/119—Strand displacement amplification [SDA]
    • 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
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/159—Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/629—Detection means characterised by use of a special device being a microfluidic device

Definitions

  • the present invention relates to a microfluidic method for combined cell-specific labeling and enrichment of biomarkers, as well as to a control device configured to carry out and/or control the steps of the method, according to the class of the independent claims.
  • the present invention also relates to a computer program.
  • a biomarker is a measurable biological characteristic with prognostic or diagnostic significance. In molecular diagnostics, diseases can be detected on the basis of nucleic acid biomarkers and appropriate therapies can be initiated. These biomarkers can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) biomarkers.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the query of targeted, multiple biomarkers is of great value and is used, for example, in infection diagnostics, in which different species are queried simultaneously, or in tumor diagnostics, in which different mutation sites in the genome are queried simultaneously.
  • biomarker targets vary from a few dozen to several tens of thousands depending on the assay.
  • the aim is always to specifically enrich these biomarker targets compared to the non-informative, but usually highly concentrated background in order to make the reading of the biomarker targets easier and more robust, for example using quantitative real-time polymerase chain reaction (qPCR), digital polymerase chain reaction (ddPCR) or parallel sequencing (next-generation sequencing, NGS).
  • qPCR quantitative real-time polymerase chain reaction
  • ddPCR digital polymerase chain reaction
  • parallel sequencing non-generation sequencing
  • nucleic acids are required as starting material, which, for example, requires additional pre-amplification in the case of low-input sample material (whole genome amplification, WGA and/or whole transcriptome amplification, WTA), which in turn requires additional process steps and time.
  • WGA whole genome amplification
  • WTA whole transcriptome amplification
  • Droplet-based, microtub-based or valve-based microstructured substrates are known with which cells can be spatially isolated.
  • none of these systems meets the requirements of carrying out a highly parallelized enrichment of DNA and/or RNA-encoded biomarkers with a clear molecular labeling of the target amplificates in just a few process steps in order to be able to carry out point-of-care analyses, for example.
  • microfluidic method for the combined cell-specific labeling and enrichment of biomarkers in a reaction compartment with the features of the independent patent claim is provided.
  • the microfluidic method for combined cell-specific labeling and enrichment of biomarkers in a reaction compartment comprises at least steps a) - g).
  • a microfluidic device as described in the as yet unpublished application with the file number 102022203848.7 is provided.
  • This has at least one carrier substrate for receiving a sample liquid.
  • the carrier substrate has at least one microcavity and furthermore at least one electrode which is arranged on or in the microcavity in order to generate an electric field which is designed to capture a cell, in particular a nucleated cell, and/or a primer particle in the microcavity.
  • microcavities For example, around 30,000 individually actuatable microcavities or reaction compartments per square centimeter can be integrated into this microfluidic device. These microcavities have, for example, a square base area and a cavity width of around 50 pm.
  • the microcavities are arranged in a regular matrix, for example.
  • the microcavities can capture or repel individual cells and/or particles. Individual cells can sediment in a microcavity in a controlled manner.
  • irrelevant cells can be removed from the microcavities, for example, while relevant cells can be retained by using an electric field to create a dielectrophoresis cage (D EP cage) in the microcavity, which can be opened, closed or switched off depending on the current strength, for example.
  • D EP cage dielectrophoresis cage
  • the DEP cages can be implemented using active components, such as transistors or memory elements. These are integrated within individual electrodes using Complementary Metal Oxide Semiconductor technology (CMOS technology).
  • CMOS technology Complementary Metal Oxide Semiconductor technology
  • step b) the at least one microcavity of the microfluidic device is loaded with a cell, in particular a nucleated cell, and the cell is trapped in the microcavity.
  • the DEP cage is closed by a voltage change. Other cells located outside the DEP cage can no longer enter it. while the cell is stably trapped within the DEP cage. This ensures that only a single cell is present within the microcavity.
  • the at least one microcavity of the microfluidic device is loaded with a primer particle (primer bead) and the primer particle is trapped in the microcavity.
  • the primer particle comprises, for example, at least one primer population coupled to it.
  • the DEP cage is closed by a voltage change. Additional primer particles located outside the DEP cage can no longer enter it, while the cell and the primer particle are stably trapped inside the DEP cage. This ensures that there is only a single primer particle inside the microcavity.
  • Steps b), c) and d) can also be carried out in any reversed order; in particular, steps b) and c) can be carried out in the reverse order.
  • step d) an amplification mixture is introduced into the at least one microcavity.
  • the components in the amplification mixture diffuse into the at least one microcavity.
  • the amplification mixture comprises a buffer compatible with its components, a polymerase, in particular an SD polymerase, such as Bst, Bst2.0, Bst3.0, EquiPhi, Phi29, Vent exo and/or others.
  • the amplification mixture optionally comprises a reverse transcriptase (RT), e.g. RTx, SSIV and/or others.
  • RT reverse transcriptase
  • the amplification mixture comprises deoxyribonucleoside triphosphates (dNTPs) and/or analogues thereof.
  • the dNTPs can also be modified.
  • the amplification mixture can also contain components that have a positive effect on the amplification reaction and/or cell lysis and/or primer release, for example.
  • Such components are, for example, additives such as polyethylene glycol (PEG), dithiothreitol (DTT), detergents or enzymes such as restriction enzymes or single-strand break-inducing enzymes (nicking enzymes).
  • step e) the amplification mixture above the at least one microcavity is displaced by a non-aqueous phase, in particular by an oil phase or air, so that the microcavity is closed in the region of its opening by the non-aqueous phase and thus represents a closed reaction compartment.
  • the microfluidic device comprises several microcavities, all microcavities represent represent isolated and closed reaction compartments, in each of which one cell and one primer particle with the amplification mixture are enclosed.
  • step f conditions are created which lead to lysis of the cell and to release of the primers from the primer particle.
  • Such conditions are created, for example, by increasing the temperature, for example to above 70°C for more than one minute, and/or by electroporation and/or by ultrasound and/or by other conditions or reagents which promote this reaction, which have been introduced, for example, by means of the amplification mixture.
  • step g) the at least one primer population binds to biomarker regions of the DNA and/or RNA which were released from the cell during cell lysis, so that an amplification of the biomarker target region occurs and the biomarker target amplificate is cell-specifically labeled.
  • DNA double strands and/or secondary and/or tertiary structures of the RNA are denatured so that the DNA and/or RNA are present as single strands, for example by using suitable temperatures.
  • the primers bind to the DNA and/or RNA, thereby initiating amplification of the corresponding DNA and/or RNA.
  • the method according to the invention is an automated, lab-on-chip-compatible method with which DNA and/or RNA-encoded biomarkers can be enriched in a highly parallelized manner and the biomarker target amplificate can be clearly assigned to its original cell via molecular cell barcode.
  • the biomarker target amplificates are thus available for downstream detection methods, such as NGS analyses, within a few minutes to hours, making the method suitable for point-of-care analyses.
  • Another big advantage is that only a few process steps are required. For example, there is no need for prior amplification/WGA/WTA or prior pooling of several pre-sorted individual cells. This leads to a significant reduction in the required process time, reagents, costs and work steps.
  • the method ensures a unique marking of each individual cell, and thus an almost 100% mating efficiency, with an also almost 100% Loading efficiency. This means that each microcavity can be loaded with exactly one cell and exactly one primer particle. In addition, several process steps can be carried out one after the other, since the microcavities can be loaded and unloaded multiple times.
  • the simultaneous enrichment of the relevant DNA and/or RNA biomarker target regions can be achieved with a degree of parallelization of, for example, 30,000 microcavities per cm 2 .
  • sample input volume is theoretically unlimited and different samples can be entered, such as blood or saliva.
  • the method only allows those cells that are relevant to be analyzed downstream.
  • Another advantage is that the primer particle that has entered the microcavity is trapped in the microcavity together with the cell and no further cells or primer particles can enter the microcavity.
  • different populations of primers are reversibly coupled to the primer particle.
  • the primer particle comprises, for example, a polystyrene, in particular in the form of a polystyrene bead.
  • the different primer populations are in particular biomarker-specific forward primers (Specific Forward Primer, SFP), biomarker-specific reverse primers (Specific Reverse Primer, SRP) and/or randomized primers (Random Primer, RP).
  • the primer populations are coupled to the primer particle, for example, via base pairings that can be dissolved by certain temperatures or via specific sequences or other chemical modifications in the base composition that can be dissolved by enzymes or light of a certain wavelength.
  • This can be, for example, a cleavage site for a restriction enzyme or the base analogue dUTP for the uracil-DNA glycosylase (UDG).
  • the SFP and SRP each comprise a selection marker coupled to a universal sequence, a unique cell barcode sequence and specific target sequences flanking the biomarker target regions in forward and reverse orientation.
  • Each primer particle contains a clonal, unique cell barcode sequence (BCi- n ) with a length of > 8 bases.
  • the selection marker is, for example, biotin at the 5' end of the SFP or SRP.
  • the selection marker is coupled to an internal, for example modified base of the universal sequence, wherein the universal sequence is extended towards the 5' end by a single-strand break sequence (nicking site) which induces a single-strand break via a corresponding nicking enzyme.
  • the selection marker enables subsequent positive selection of the biomarker target amplification products.
  • the universal sequence is required, for example, for subsequent amplification of a sequencing library or a known sequence with a function of choice, such as a restriction site, in particular for releasing the biomarker target amplification product from a capture molecule, for example.
  • Another advantage is that, since the individual biomarker target amplification products are marked with a cell barcode, they can be clearly assigned to the individual cells. Further analyses of a large number of cells, for example of a molecular genetic nature using NGS, in which relevant biomarkers can be assigned to the individual cells, for example from isolated cells from tissue biopsies, can be of great benefit, for example in the diagnosis and therapy monitoring of tumor patients.
  • each primer particle is equipped with a unique, molecular cell barcode.
  • biomarker target region several, in particular 2-4, SFP and SRP with different specific forward and reverse target sequences flanking the biomarker target region are used for each biomarker target region. These can also be used in different concentrations, for example.
  • the advantage here is that, for example, with an increasing number of SFPs or SRPs per biomarker target region, not only the amplification rate but also the chances of binding of the SFPs or SRPs to the target region increase or, in another example, by adjusting the concentration of the SFPs or SRPs of individual biomarker target regions that are, for example, more difficult to amplify, a more uniform amplification is achieved compared to other biomarker target regions.
  • SFP and SRP further comprise a unique molecular identifier (UMI) in the form of a randomized sequence.
  • UMI unique molecular identifier
  • the UMI can be, for example, between the universal sequence and the cell barcode sequence or between the cell barcode sequence and the Forward or reverse target sequence.
  • the UMI has a length of > 6 bases.
  • the primer particles are designed in such a way that the SFP and SRP are already reversibly bound to the primer particles via the selection marker.
  • the SFP and SRP are, for example, via antibodies, terminal chemical modifications, such as a biotin coupling.
  • the SFP and SRP can also be reversibly bound to the primer particles via aptamers, temperature-stable groups or pH-sensitive groups or light-cleavable groups such as o-nitrobenzyl.
  • the random primers are supplied with the amplification mixture for amplification, in particular for an ssMDA (described below).
  • ssMDA ssMDA
  • the advantage here is that the target amplificates are produced directly on the primer particle and can be held (captured) by means of the DEP forces after removal of the non-aqueous phase and purified directly in the microcavity.
  • no capture molecules are necessary, since the amplification of the biomarker target region takes place via the SFP and SRP bound to the primer particle.
  • a selection marker can be omitted here.
  • a further advantage is that the biomarker target amplificates, immobilized on the primer particles held in the DEP cage, can be further processed in the microcavity if required, for example by adding additional primer particles or liquid reagents.
  • the universal sequence of the SFP and SRP is selected such that a direct linkage point in the form of a specific sequence is introduced for further amplification, in particular for the completion of a sequencing library.
  • the advantage here is that additional amplification or preparation of the purified biomarker target amplificates is possible using other sequencing technologies, for example from another manufacturer.
  • the necessary asymmetric design can be implemented, for example, in such a way that all SFPs receive a first universal sequence and all SRPs receive a second universal sequence that differs from the first universal sequence.
  • all SFPs receive a first universal sequence
  • all SRPs receive a second universal sequence that differs from the first universal sequence.
  • a highly standardized PCR for example an index PCR
  • a flexible By using universal sequences, it can be ensured that the method is compatible with downstream, preferred analysis methods, such as various NGS technologies, for single-cell analysis.
  • the amplification mixture supports cell lysis. This can be achieved, for example, by a reagent that lyses the cells and releases nucleic acids, but does not inhibit the subsequent reaction or even promotes it, such as with a detergent or by changing the osmolarity.
  • the advantage here is that no individual process steps are required for cell lysis and mixing with the amplification mixture, and the respective reagents can be stored pre-mixed. This leads to time, reagent and cost savings. Furthermore, the reduced steps minimize dilution effects and losses that would otherwise result from repeated rinsing. This results in an increase in efficiency.
  • cell lysis is carried out after introduction of the amplification mixture by brief heating, for example to >50 °C for >1 min. In a further alternative or additional embodiment, cell lysis is carried out by increasing the pressure within the microcavities, for example by applying an overpressure.
  • the amplification is an isothermal amplification, in particular a semi-specific multiple displacement amplification (ssMDA).
  • ssMDA semi-specific multiple displacement amplification
  • the ssMDA offers the option of simultaneously enriching DNA and RNA from minimal amounts both across the entire genome and the entire transcriptome, as well as preferentially amplifying and enriching individual biomarker-relevant regions, i.e. the biomarker target regions.
  • the SFP and SRP in the ssMDA have a single-strand break cleavage site.
  • This single-strand break cleavage site provides an additional amplification starting point for a polymerase after the single-strand break has been generated by a suitably added single-strand break-inducing endonuclease (nicking enzyme). This allows biomarker target amplification to continue at these sites without further primer hybridization, which increases processivity.
  • a single-strand break-inducing endonuclease is added to the polymerase.
  • the endonuclease is preferably selected from the group consisting of Nt.Alwl, Nb.BbvCI, Nt.BbvCI, Nb.Bsml, Nt.BsmAI, Nt.BspQI, Nb.BsrDI, Nb.BssSI, Nt.BstNBI, Nb.BtsI and Nt.CviPII.
  • the RPs in ssMDA are preferably primers that have at least one modification selected from the group consisting of LNA (Locked Nucleic Acid), MGB (Minor Groove Binder), C-5 Propynyl-Deoxycytidine, C-5 Propynyl-Deoxyuridine, Aminoethyl-Phenoxazine-Deoxycytidine, 5-Methyl-Deoxycytidine, 2-Amino-Deoxyadenosine, Trimethoxystilbene, Pyrene and Spermine.
  • ZNA primers Zip Nucleic Acids
  • These are spermine-modified primers.
  • the ssMDA occurs isothermally, for example at temperatures between 40-72°C for more than 10 minutes.
  • an SD polymerase is preferably used.
  • the SD polymerase can, for example, be an enzyme mutant of the DNA polymerase of Bacillus subtilis phage Phi29, in particular Equi Phi29, or of Thermococcus litoralis, in particular Vent (exo-).
  • the SD polymerase Vent (exo-) has the advantage that it is stable and processive up to temperatures of 100°C. It can therefore be added before denaturing the DNA and/or RNA without being damaged by the high denaturation temperature. However, these SD polymerases do not exhibit RNA processivity.
  • the SD polymerase is preferably an enzyme mutant of the DNA polymerase of Bacillus stearothermophilus. In addition to their processivity towards DNA, these SD polymerases also have processivity towards RNA. Particularly preferred enzyme mutants of the DNA polymerase of Bacillus stearothermophilus are selected from the group consisting of Bst, Bst 2.0 and Bst 3.0. Among these enzyme mutants, Bst 3.0 has the highest RNA processivity, which is why Bst 3.0 is particularly preferred.
  • SD polymerase is a DNA polymerase that is suitable for isothermal amplification reactions and is already known for its high strand displacement activity in amplification reactions.
  • SD polymerase has several advantages. It enables isothermal amplification. In addition, some SD polymerases tolerate high temperatures during isothermal amplification, which increases the reaction speed to such an extent that the reaction time can be significantly shortened. Finally, some SD polymerases can use RNA as a template for amplification in addition to DNA.
  • RNA as well as DNA it is preferable that at least one reverse transcriptase is also used in ssMDA. In particular, this is RTx or SSIV. In principle, however, all reverse transcriptases that process under similar reaction conditions to the SD polymerase used are conceivable. This specifically supports the amplification of RNA, so that the required amplification period can be shortened even further. If an SD polymerase is used that does not have RNA processivity, then the use of a reverse transcriptase is necessary in order to process RNA as well as DNA.
  • the DNA and/or RNA to be amplified is denatured, in particular by heating to a suitable temperature.
  • a suitable temperature for example Vent (exo-) polymerase
  • the Denaturation preferably at a temperature in the range of 75°C to 98°C and most preferably at a temperature in the range of 80°C to 95°C.
  • the heating is carried out for a period of more than 10 seconds, most preferably for a period in the range of 30 seconds to 120 seconds.
  • temperatures of 65-85°C are preferably used, and particularly preferably temperatures of 72-80°C.
  • the primers for hybridizing the single-stranded DNA and/or RNA can preferably be brought together with the DNA and/or RNA before denaturation, particularly preferably by already dissolving them in the amplification mixture.
  • the hybridization of the primers to the DNA and/or RNA single strands then preferably takes place at a temperature in the range of 4°C to 65°C for a period of time that is preferably more than 10 seconds.
  • the ssMDA is preferably carried out at a constant temperature with a value in the range of 40°C to 72°C.
  • a preferred period of amplification is in the range of 10 minutes to 120 minutes. The higher the temperature during amplification, the faster it proceeds and the shorter the amplification period can usually be advantageously chosen.
  • ssMDA DNA and RNA biomarkers can be simultaneously amplified and enriched from single cells in the same reaction compartment, which represents an enormous improvement and simplification of biomarker enrichment, also with regard to the effort required, the duration of the experimental procedure, the number of work steps and the associated costs.
  • the amplification is carried out using a reverse transcriptase PCR.
  • a reverse transcriptase PCR for example, only one SFP and SRP are used per biomarker target region.
  • the amplification mixture comprises, for example, a Taq polymerase and a reverse transcriptase.
  • an amplification reaction is understood to mean the reaction solution in the reaction compartment after the amplification.
  • the at least one microcavity has a further opening in the wall, in particular one directed downwards.
  • a further opening is closed, for example, by an actuatable membrane during the reaction.
  • the further opening is opened or the actuatable membrane is removed in order to transfer the amplification reaction, in particular downwards, into a microfluidically connected system and to combine the individual amplification reactions if necessary.
  • the microfluidically connected system is, for example, a microfluidically connected reaction chamber with a volume of, for example, 20 pl.
  • the biomarker target amplificates can be further enriched and purified, for example.
  • the biomarker target amplificates are amplified again with the universal sequence if necessary and read out, for example, using NGS.
  • the biomarkers can be clearly assigned to the individual cells again using the cell barcode sequence queried during the readout.
  • the biomarker target amplificates are purified using capture molecules, in particular using capture molecules immobilized on magnetic beads.
  • the capture molecules in particular streptavidin, bind, for example, to the selection marker of the SFP and SRP, in particular biotin.
  • appropriate binding conditions are created for the capture molecules and the biomarker target amplificates comprising the selection marker, and a "bind-wash-elute" process that can be carried out according to known protocols is used.
  • a restriction enzyme can also release the biomarker target amplificates, for example from the capture molecule.
  • a prerequisite for this is that corresponding interface sequences are incorporated into the SFP and SRP in the universal sequence.
  • the protelomerase TelN or an enzyme mutant thereof is used when eluting the biomarker target amplificates from the capture molecule, which covalently links the two single-stranded molecules of the DNA double helix (sense and antisense strands) at the interface, leaving behind a hairpin structure.
  • a hairpin structure is required, for example, for the production of NGS libraries, for example for the sequencing library of the sequencing technology from Pacific Biosciences. The advantage here is that no further steps for adapter ligation need to be carried out.
  • biomarker target amplifications can be achieved using isothermal rolling circle amplification (RCA) without the need for additional sample preparation steps for the amplification.
  • RCA isothermal rolling circle amplification
  • the cells are circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • the molecular genetic single cell analysis of these CTCs is, for example, a highly sensitive option for monitoring the therapy of cancer patients in order to collect information early on about the course of therapy (prognostic) and about the adjustment of therapy (diagnostic).
  • the rare CTCs (for example 10 1 to 10 2 per mL of blood; individually or a few as a pool) must be isolated from a high background of "healthy" blood cells (>10 6 leukocytes, >10 9 erythrocytes per mL of blood).
  • the genetic material of these individual, isolated cells must be pre-amplified in order to generate sufficient input for subsequent analyses.
  • the method according to the invention eliminates process steps for pre-amplification of the DNA and/or RNA of the CTCs, as well as previous steps for merging several pre-sorted cells, which significantly shortens the process time, requires fewer work steps, requires fewer reagents and thus also results in lower costs.
  • Cells used in the method according to the invention can be previously stained cells, such as EpCAM-positive CTCs. These carry the surface antigen EpCAM (epithelial cell adhesion molecule). However, all cells without prior staining can also be examined, such as all individual cells released from the cell network of a tissue.
  • EpCAM-positive CTCs carry the surface antigen EpCAM (epithelial cell adhesion molecule).
  • EpCAM epidermal cell adhesion molecule
  • the cells can be microbial cells or single-cell cells.
  • the invention further relates to a control device configured to carry out and/or control the steps of the method according to the invention in corresponding units, in particular in a microfluidic cartridge.
  • the invention further relates to a computer program configured to carry out and/or control the steps of the microfluidic method, as well as a machine-readable storage medium on which the computer program is stored.
  • Fig. 1 the schematic representation of an embodiment of a method according to the invention for the combined cell-specific labeling and enrichment of biomarkers in a reaction compartment
  • Fig. 2 Schematic representation of a biomarker-specific forward or
  • Figure 1 shows the method according to the invention for the combined cell-specific labeling and enrichment of biomarkers in a reaction compartment in an embodiment, which is described using an isothermal ssMDA.
  • a device with which the method according to the invention can be carried out is described in the as yet unpublished German application with the file number 102022203848.7.
  • This device has a carrier substrate for receiving a sample liquid.
  • the carrier substrate has at least one microcavity 110 and furthermore at least one electrode which is arranged on or in the microcavity 110 in order to generate an electric field which is designed to capture a cell 510, in particular a nucleated cell 510, and/or a primer particle 512 and/or another particle in the microcavity 110.
  • Figure 1 of the present invention shows only one microcavity 110 of this device.
  • a first sample liquid with cells 510 is introduced into the microfluidic device or placed on the carrier substrate.
  • the cells 510 sediment in the direction of the microcavities.
  • the DEP cage is switched to an open state or switched off so that this cell 510 can enter the microcavity.
  • the DEP cage is closed again so that no further cells can enter it and a maximum of one cell is contained per microcavity.
  • the sample liquid is flushed away.
  • the DEP cage remains closed so that the cell 510 is held until further notice.
  • a cell 510 with a cell nucleus 511 here for example a CTC
  • a second sample liquid with primer particles 512 is introduced into the microfluidic device or placed on the carrier substrate.
  • the first sample liquid located above the carrier substrate, which comprises the cells 510 is displaced.
  • the primer particles 512 comprise, for example, a particle 513 made of polystyrene, with different primer populations reversibly coupled to them, here, for example, biomarker-specific forward primer (SFP) 517 and biomarker-specific reverse primer (SRP) 517 with a selection marker 514 and random primer (RP) 516.
  • SFP biomarker-specific forward primer
  • SRP biomarker-specific reverse primer
  • RP random primer
  • step S2 the DEP cage is transferred to an open or switched off state so that a primer particle 512 can sediment in the microcavity 110.
  • the DEP cage is transferred to a closed state by a voltage change and the primer particle 512 is trapped in the cell 510. In this state, no further primer particles 512 can enter the microcavity 110, so that it is ensured that in addition to the single cell 510, only a single primer particle 512 is located in the microcavity 110.
  • the dashed line with the reference number 520 is intended to illustrate that the DEP cage is closed after loading with the primer particle 512.
  • the microcavity 110 can also be first loaded with a primer particle 512 and then with a cell 510.
  • the primer particles can also carry only the SFP and SRP and the RP are introduced via the amplification mixture in step S3.
  • an amplification mixture is then introduced into the microfluidic device or placed on the carrier substrate.
  • the second liquid with the unused primer particles 512 is removed from the carrier substrate beforehand and the amplification mixture is subsequently placed on the carrier substrate, or the liquid with the primer particles 512 is mixed directly with the amplification mixture on the carrier substrate.
  • the amplification mixture comprises a buffer with an SD polymerase 525 and deoxyribonucleoside triphosphates (dNTPs), and optionally a reverse transcriptase and/or nicking enzyme.
  • dNTPs deoxyribonucleoside triphosphates
  • step S3 the diffusion-based distribution of the components or the polymerase 525 of the amplification mixture into the microcavity 110 is shown.
  • the DEP cage is closed, which is illustrated by the dashed line with the reference number 520, since the components of the amplification mixture do not represent entities with dielectric properties and can therefore pass through the DEP cage in a diffusion-based manner even when it is closed.
  • the microcavity 110 now contains the cell 510, the primer particle 512 and the SD polymerase 525 as well as other components of the amplification mixture.
  • a step S4 the amplification mixture above the at least one microcavity 110 is displaced by a non-aqueous phase 526, for example an oil phase or air, so that the microcavity 110 is closed in the region of its opening by the non-aqueous phase 526 and thus represents a closed reaction compartment in which the cell 510 and the primer particle 512 are enclosed with the amplification mixture.
  • a non-aqueous phase 526 for example an oil phase or air
  • a lysed cell 510a is shown from which the cell components, for example RNAs 539, have been released, as well as a lysed cell nucleus 511a, which has released the DNA 539 located therein.
  • Such conditions are, for example, achieved by a temperature increase to, for example, over 70°C for, for example, more than one minute and/or by electroporation and/or by ultrasound.
  • amplification reaction is carried out using ssMDA.
  • the random primers 516 bind undirected to regions of the DNA 539 and/or RNA 539, so that an isothermal, strand-displacement-based amplification of the total DNA 539 and/or RNA 539 can be carried out using the polymerases 525. This produces total DNA amplificates 539 and/or total RNA amplificates 539.
  • the specific forward primers 517 and the specific reverse primers 517 bind flanking upstream and downstream of biomarker target regions 530 of the DNA 539 and/or RNA 539, so that an amplification of the biomarker target region 530 takes place, which is cell-specifically marked by the selection marker 514, for example biotin.
  • the polymerases 525 bind to the SFP and SRP, which provide the SD polymerases 525 with the starting point for amplification. This produces biomarker target amps 542.
  • the SFP and SRP carry additional nicking sequences at the 5' end, which extend the universal sequence. If a corresponding nicking enzyme is added with the amplification mixture in step S3, an additional, disproportionate increase in the biomarker target amplification can be induced, in which the biomarker target amplificates 542 continue to carry the selection markers 514.
  • RNA amplification of RNA can, for example, be further enhanced by reverse transcriptase.
  • a step S6 it is indicated that in order to combine several amplification reactions from different microcavities 110, the non-aqueous phase 526 above the microcavities 110, which had closed them in the area of their opening, is displaced.
  • the displacement can be carried out, for example, with an elution buffer or a detergent-containing buffer.
  • the amplification reactions can be transferred via the opening into a microfluidically connected system, for example a microfluidically connected reaction chamber, and the individual amplification reactions can be combined.
  • the biomarker target amplificates 542 can, for example, be further enriched and purified.
  • the amplified biomarker target amplificates 542 are purified using capture molecules 543, which are immobilized on magnetic particles (beads), for example.
  • the capture molecules 543 for example streptavidin, bind to the selection marker 514 of the SFP 517 and SRP 517, in particular biotin.
  • the biomarker target amplificates 542 can be separated from the other reaction components, such as the total DNA amplificates 539 and/or total RNA amplificates 539.
  • the biomarker target amplificates 542 are then eluted from the capture molecules 543, for example by classic elution methods or by a restriction enzyme.
  • FIG. 2 shows an example of a biomarker-specific forward primer (SFP) 517 or a biomarker-specific reverse primer (SRP) 517.
  • SFP biomarker-specific forward primer
  • SRP biomarker-specific reverse primer
  • the SFP or SRP comprises, starting at the 5' end, a selection marker 514 coupled to a universal sequence 501, followed by a unique cell barcode sequence 503 and a specific biomarker target sequence 506.
  • the SFP 517 binds to the DNA 539 and/or RNA 539 in a forward-facing manner up to 3 kb upstream of the biomarker target region 530, and the SRP 517 binds to the DNA 539 and/or RNA 539 in a backward-facing manner up to 3 kb downstream of the biomarker target region 530.
  • the unique cell barcode sequence 503 is clonally contained in each SFP 517 and SRP 517 of a primer particle 512, for example with a length of 8 or more bases.
  • the selection marker 514 is, for example, biotin at the 5' end of the SFP 517 or SRP 517.
  • the selection marker 514 enables a subsequent positive selection of the amplified biomarker target amplification products 542.
  • the universal sequence 501 is required, for example, for a subsequent amplification of a sequencing library or a known sequence with a function of choice, such as a restriction site, in particular for releasing the biomarker target amplification product 542, for example from a capture molecule 543.
  • the SFP 517 or SRP 517 can have a unique molecular identifier (UMI) in the form of a randomized sequence.
  • the UMI can be located, for example, between the universal sequence 501 and the cell barcode sequence 503 or between the cell barcode sequence 503 and the specific biomarker target sequence 506.
  • the UMI has in particular a length of 6 or more bases.
  • the SFP 517 or SRP 517 can be extended by a nicking sequence at the 5' end of the universal sequence 501. The selection marker is thus no longer located at the end of the SFP or SRP, but internally at the 5' end of the universal sequence.

Abstract

L'invention concerne un procédé microfluidique pour le marquage et l'enrichissement combinés spécifiques de cellules de biomarqueurs dans un compartiment de réaction, comprenant les étapes de procédé suivantes consistant à : a) fournir un dispositif microfluidique comprenant au moins un substrat de support pour recevoir un fluide échantillon, le substrat de support comprenant au moins une microcavité (110), et comprenant en outre au moins une électrode disposée au niveau de ou dans la microcavité (110) pour créer un champ électrique conçu pour piéger une cellule (510) et/ou une particule d'amorce (512) dans la microcavité (110) ; b) charger la ou des microcavités (110) du dispositif microfluidique avec une cellule (510) et la piéger ; c) charger la ou les microcavités (110) du dispositif microfluidique avec une particule d'amorce (512) comprenant au moins une population d'amorces couplée à celle-ci et piéger la particule d'amorce (512), les étapes b) et c) pouvant également être effectuées dans l'ordre inverse ; d) introduire un mélange d'amplification dans la ou les microcavités (110) ; e) déplacer le mélange d'amplification au-dessus de la ou des microcavités par une phase non aqueuse, de telle sorte que la microcavité (110) représente un compartiment de réaction fermé ; f) induire des conditions de lyse cellulaire et de libération d'amorce (516, 517) ; et, g) lier la ou les populations d'amorces (517) à des régions de biomarqueur de l'ADN (539) et/ou de l'ARN (539) de la cellule lysée (510a) pour un marquage et une amplification spécifiques de cellules d'une région cible de biomarqueur (530).
PCT/EP2023/077002 2022-10-19 2023-09-29 Marquage et enrichissement combinés spécifiques de cellules de biomarqueurs WO2024083474A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102022211089.7A DE102022211089A1 (de) 2022-10-19 2022-10-19 Kombinierte Zellspezifische Markierung und Anreicherung von Biomarkern
DE102022211089.7 2022-10-19

Publications (1)

Publication Number Publication Date
WO2024083474A1 true WO2024083474A1 (fr) 2024-04-25

Family

ID=88295626

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/077002 WO2024083474A1 (fr) 2022-10-19 2023-09-29 Marquage et enrichissement combinés spécifiques de cellules de biomarqueurs

Country Status (2)

Country Link
DE (1) DE102022211089A1 (fr)
WO (1) WO2024083474A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016132129A1 (fr) * 2015-02-17 2016-08-25 Touchlight Genetics Limited Procédé de synthèse d'adn
WO2016154600A1 (fr) * 2015-03-25 2016-09-29 The General Hospital Corporation Analyse numérique de cellules tumorales circulantes dans des échantillons de sang
WO2020036926A1 (fr) * 2018-08-17 2020-02-20 Cellecta, Inc. Préparation multiplex de fragments d'adn spécifiques à un gène à code à barres
WO2021194699A1 (fr) * 2020-03-24 2021-09-30 Cellecta, Inc. Analyse génétique de cellule unique
WO2021236916A1 (fr) * 2020-05-20 2021-11-25 Yale University Approche intégrée de transfert de nanopuits et de piégeage par diélectrophorèse pour permettre un séquençage d'arn à cellule unique à double sous-poisson
US20220106631A1 (en) * 2020-10-05 2022-04-07 Scribe Biosciences Barcoding in droplets for cell-cell interaction and secreted protein detection and analysis
CN115007231A (zh) * 2022-06-02 2022-09-06 北京化工大学 一种用于细胞-微珠捕获配对的微流控芯片
WO2022223566A1 (fr) * 2021-04-20 2022-10-27 Robert Bosch Gmbh Procédé et dispositif de capture d'au moins une cellule contenant un noyau en utilisant au moins une électrode pour un dispositif microfluidique

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210138453A1 (en) 2018-07-26 2021-05-13 Hewlett-Packard Development Company, L.P. Non-uniform electrical field to position objects

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016132129A1 (fr) * 2015-02-17 2016-08-25 Touchlight Genetics Limited Procédé de synthèse d'adn
WO2016154600A1 (fr) * 2015-03-25 2016-09-29 The General Hospital Corporation Analyse numérique de cellules tumorales circulantes dans des échantillons de sang
WO2020036926A1 (fr) * 2018-08-17 2020-02-20 Cellecta, Inc. Préparation multiplex de fragments d'adn spécifiques à un gène à code à barres
WO2021194699A1 (fr) * 2020-03-24 2021-09-30 Cellecta, Inc. Analyse génétique de cellule unique
WO2021236916A1 (fr) * 2020-05-20 2021-11-25 Yale University Approche intégrée de transfert de nanopuits et de piégeage par diélectrophorèse pour permettre un séquençage d'arn à cellule unique à double sous-poisson
US20220106631A1 (en) * 2020-10-05 2022-04-07 Scribe Biosciences Barcoding in droplets for cell-cell interaction and secreted protein detection and analysis
WO2022223566A1 (fr) * 2021-04-20 2022-10-27 Robert Bosch Gmbh Procédé et dispositif de capture d'au moins une cellule contenant un noyau en utilisant au moins une électrode pour un dispositif microfluidique
CN115007231A (zh) * 2022-06-02 2022-09-06 北京化工大学 一种用于细胞-微珠捕获配对的微流控芯片

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAI ZHILIANG ET AL: "An Integrated Dielectrophoresis-Trapping and Nanowell Transfer Approach to Enable Double-Sub-Poisson Single-Cell RNA Sequencing", ACS NANO, vol. 14, no. 6, 21 May 2020 (2020-05-21), US, pages 7412 - 7424, XP093112456, ISSN: 1936-0851, DOI: 10.1021/acsnano.0c02953 *
LUKAS VALIHRACH ET AL: "Platforms for Single-Cell Collection and Analysis", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 19, no. 3, 11 March 2018 (2018-03-11), pages 807, XP055532883, DOI: 10.3390/ijms19030807 *
YULING QIN ET AL: "A Self-Digitization Dielectrophoretic (SD-DEP) Chip for High-Efficiency Single-Cell Capture, On-Demand Compartmentalization, and Downstream Nucleic Acid Analysis", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, VERLAG CHEMIE, HOBOKEN, USA, vol. 57, no. 35, 27 July 2018 (2018-07-27), pages 11378 - 11383, XP072099898, ISSN: 1433-7851, DOI: 10.1002/ANIE.201807314 *

Also Published As

Publication number Publication date
DE102022211089A1 (de) 2024-04-25

Similar Documents

Publication Publication Date Title
DE69433136T2 (de) Nukleinsäure-isolierung
DE202016009134U1 (de) Systeme und Zusammensetzungen zur Nukleinsäureanalyse
US11905553B2 (en) Method for nucleic acid amplification
DE112013004650T5 (de) Multiplexpyrosequenzierung unter Verwendung nichtinterferierender, rauschbeendender Polynukleotididentifikationstags
DE10150121B4 (de) Echtzeitdetektion von DNA-Amplifikationsprodukten
EP0957177A2 (fr) Procédé amélioré de préamplification-PCR d'extension d'amorce
DE102004036285A1 (de) Verfahren zum Bestimmen der Häufigkeit von Sequenzen einer Probe
DE19754482A1 (de) Verfahren zur Herstellung komplexer DNA-Methylierungs-Fingerabdrücke
DE4441626A1 (de) Verfahren zum besonders sensitiven Nachweis von Nukleinsäuren
EP2035143B1 (fr) Procédé d'analyse d'acides nucléiques amplifiés
DE60318723T2 (de) Methode zur quantitativen bestimmung von polynukleotiden in einem gemisch
WO2024083474A1 (fr) Marquage et enrichissement combinés spécifiques de cellules de biomarqueurs
DE102008013715B4 (de) Verfahren zur DNA-Analyse
EP3830294A1 (fr) Mélange réactionnel, procédé et kit pour réaliser une pcr quantitative en temps réel
DE102021005845B4 (de) Verfahren und System zur Durchführung einer kumulativen Nukleinsäure-Amplifikationsreaktion
WO2007068305A1 (fr) Procede de determination du genotype a partir d'un echantillon biologique qui contient des acides nucleiques provenant de differents individus
DE102010043015B4 (de) Verfahren zur Konzentration von Probenbestandteilen und Amplifikation von Nukleinsäuren
DE69533670T2 (de) VERFAHREN ZUR VERRINGERUNG VON HINTERGRUNDSIGNALEn in DNA REPLIKATIONs/DETEKTIONS TESTS
DE102020103957B4 (de) Verfahren zum Nachweis von Mikroorganismen und ein fluidisches Kanalsystem
DE102006014000B4 (de) Verfahren zur Charakterisierung einer Mischprobe
DE112020001964B4 (de) Nachweis von nukleinsäuremodifikationen durch chemische desaminierung
EP1606420B1 (fr) Procede et kit pour detecter m. tuberculosis de maniere specifique
DE102008061774A1 (de) Indexierung von Nukleinsäure-Populationen
EP2877595B1 (fr) Procédé de différenciation entre cellules vivantes et mortes
EP1896615A1 (fr) Procédé de détection de séquences de nucléotides, utilisation du procédé et coffret de test