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WO2021013534A1 - Procédé et trousse d'analyse pour la modification de l'adn au bisulfite - Google Patents

Procédé et trousse d'analyse pour la modification de l'adn au bisulfite Download PDF

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Publication number
WO2021013534A1
WO2021013534A1 PCT/EP2020/069148 EP2020069148W WO2021013534A1 WO 2021013534 A1 WO2021013534 A1 WO 2021013534A1 EP 2020069148 W EP2020069148 W EP 2020069148W WO 2021013534 A1 WO2021013534 A1 WO 2021013534A1
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Prior art keywords
dna
bisulfite
solid phase
buffer
rough surface
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP2020/069148
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German (de)
English (en)
Inventor
Timo Hillebrand
Magdalena GRUNT
Vipul Patel
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AJ Innuscreen GmbH
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AJ Innuscreen GmbH
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Filing date
Publication date
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Priority to EP20750595.9A priority Critical patent/EP3994277A1/fr
Publication of WO2021013534A1 publication Critical patent/WO2021013534A1/fr
Anticipated expiration legal-status Critical
Ceased 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 application relates to a method and a test kit for carrying out a reaction for bisulfite modification of DNA to determine the
  • Methylation pattern of the DNA characterized in that after
  • the modified DNA is bound to a rough solid phase and all further necessary process steps are carried out on this rough solid phase. Finally, the modified, highly pure DNA is detached from this solid phase.
  • the method can be carried out manually or automatically.
  • the rough solid phase is a non-mineral material with a rough surface.
  • a rough surface is understood to be a non-smooth or structured surface of a material that is preferably made of metal, plastic or rubber.
  • Cavity produce a structure which functions as a non-smooth surface and can therefore also be used according to the invention.
  • DNA methylation is an epigenetic mechanism that occurs in most
  • Organisms is found. Its main functions in prokaryotes are aimed at protecting against parasitic DNA and controlling the accuracy of replication. In
  • the predominant methylation site is the 5-carbon of cytosine within the CpG dinucleotide sites. More than half of the genes contain short ( ⁇ 1 kb) CpG-rich regions called CpG islands, while the rest of the genome has no CpGs. While CpG sites in CpG islands are predominantly unmethylated, most CpG sites outside of CpG islands are normally methylated. The elucidation of the function of CpG methylation has proven difficult and appears to vary with context. The CpG methylation is on several genes.
  • hypomethylation while several specific genes are methylated (gene-specific hypermethylation). Hypomethylation typically affects repetitive elements of DNA, while hypermethylation usually occurs within CpG islets. nowadays DNA methylation analysis has become an important tool for studying cancer, gene expression, genetic diseases, and many other important aspects of biology. The study of DNA methylation is also in the
  • the prior art also includes the document WO 2004/053115 A1.
  • This describes a method and kit for isolating a target cell, a cell organelle or a virus from a sample, using, inter alia, a non-specific or little specific binding between a target cell, a cell organelle or a virus with a magnetic microsphere.
  • the object of the invention was to eliminate the disadvantages of the solutions described in the prior art.
  • the aim of the present invention was to provide a
  • the present invention achieves these objects in all of the points defined.
  • the essence of the invention is the knowledge that free nucleic acids or nucleic acids released by lysis can be bound to a solid phase with a rough surface under specific binding conditions, where they are washed and finally eluted from this rough surface.
  • the term "rough surface” is to be understood as meaning that by touching the
  • Non-mineral solid phases in the sense of this
  • Invention are materials that do not consist of glass, silicon dioxide, an ion exchanger or hydroxyapatite.
  • An arrangement of per se non-rough materials in a cavity can also lead to the creation of a “rough” or structured surface due to corners or edges being formed.
  • rough surface is to be understood in such a way that by touching or looking at the surface it can be seen that it is not smooth. However, it can also be a surface that has a structure (e.g. grooves). This structure removes the smoothness of the surface, even if the structure, i.e. the grooves, itself can be smooth. According to the invention, such surfaces are referred to as “structured surfaces”. If it is not possible to tell whether a surface is smooth or rough by looking at or touching the surface, a test can be carried out in which a laser beam is aimed at that surface. If the surface is smooth, the laser will only work in
  • the process can be carried out both manually and automatically.
  • automated bisulfite modification of DNA there are currently no suitable solutions.
  • the manual method according to the invention for bisulfite modification of DNA is carried out in a preferred variant as follows.
  • Materials for binding the DNA are plastic granules with a rough surface, which are preferably paramagnetic.
  • plastic granulates, the surface of which has been mechanically roughened are placed in a 2.0 ml reaction vessel for this purpose.
  • the bisulfite modification reaction (deamination and / or desulfonation step as well as necessary washing steps) are carried out with reagents from a commercially available kit for bisulfite modification of DNA (innuCONVERT Bisulfite Basic Kit; Analytik Jena AG).
  • the reaction mixture is transferred to the 2.0 ml reaction vessel with the granules.
  • the binding of the DNA to the surface of the rough plastic granulate is initiated by adding a binding buffer, which contains an alcoholic component and other additives, or by adding just one alcohol. After a short incubation, the supernatant is removed from the plastic granulate by placing the reaction vessel in a magnetic trap.
  • the bound modified DNA is washed with an alcoholic washing buffer
  • a desulfonation buffer is added to the reaction vessel with the granulate
  • the modified DNA is finalized by adding a low salt buffer from
  • Granules according to the invention detached.
  • Bisulfite reagent all process steps necessary for a bisulfite modification of DNA are of the kind that none of the previously used mineral
  • the means and method according to the invention also reveal further advantages. For the processing of larger sample volumes, it is necessary to parallelize processes and, in the most effective case, to automate them. With regard to the process of bisulfite modification of DNA, there are a large number of commercially available products for manual processing, but very few products for processing large sample sizes. Automated solutions cannot be found commercially and must be implemented individually by users on automation platforms.
  • the agent according to the invention is ideally suited to carry out an automated bisulfite modification of DNA.
  • the material with the rough surface is located within a pipette tip, so that reagents can flow past this material.
  • the same material that was used for manual bisulfite modification can also be placed in a pipette tip.
  • the method according to the invention runs in the form of a "walk-away process" as follows:
  • the pipette tip according to the invention is empty. 5.
  • the desulphonation reaction of the DNA bound to the plastic granulate then takes place inside the pipette tip. This is done by pipetting up and down a desulfonation buffer (innuCONVERT Bisulfite Basic Kit; Analytik Jena AG).
  • Rough materials can be used, modified plastic materials whose surfaces are not smooth but structured. This also includes so-called composite materials, which are mixtures of polymers and, for example, organic components or metallic components. It is only essential to provide a roughened or structured surface (not a smooth
  • the architecture of the plastic material is also not limiting (round, rectangular, etc.). It is only important that the material can be brought into a pipette tip and also remains in this position and that a liquid can be washed around it at any time without the liquid having to pass through the material introduced.
  • a pipette tip can also be used in which there is (from an injection molded part manufactured) that the binding material is already in place and no longer has to be brought into the tip.
  • a device platform that is a commercially available magnetic particle processor (e.g. KingFisher Flex for DNA / RNA extraction and processing of 96 samples) is used and misappropriated.
  • a commercially available magnetic particle processor e.g. KingFisher Flex for DNA / RNA extraction and processing of 96 samples
  • the device uses plastic combs into which magnetic bars move, which then move magnetic particles in a walk-away process and immerse them in the buffers required for a standard extraction. These plastic combs were used for the method according to the invention for purposes other than those intended by their lower end being mechanically roughened. The method according to the invention for bisulfite modification of DNA was then implemented automatically by means of the device. In contrast to the
  • the pipette tip variant binds the DNA to the rough surface of the plastic combs and goes through all the necessary steps following the initial deamination reaction in a walk-away workflow.
  • the procedure is extremely simple and allows up to 96 samples to be processed in parallel.
  • the present invention thus ideally enables the implementation of the objective already mentioned.
  • the method of bisulfite modification of DNA including all the necessary process steps does not require any mineral materials known from the prior art, but only roughened surfaces.
  • the automated bisulfite modification was carried out with the InnuPure C16 automatic extraction device (Analytik Jena AG). This automatic extraction system is based on a magnetic particle nucleic acid extraction, but was used for the process “for a different purpose” without magnetic particles.
  • Cavity 4 1 ml Washing Solution LS (Analytik Jena AG)
  • the reaction mixture of the cavity 1 was carried out by means of the
  • the DNA bound to the rough plastic granules was brought into contact with desulfonation buffer (4 times AuF and pipetting off). An incubation time of 2 minutes with Desulfonation Buffer was observed for each pipetting step.
  • the DNA bound to the plastic granules was then washed successively in three additional cavities containing alcoholic washing buffers (Washing Solution LS, 80% ethanol, 80% ethanol) by pipetting in and out.
  • alcoholic washing buffers Wash Solution LS, 80% ethanol, 80% ethanol
  • the tip according to the invention and the rough plastic granules contained in it were dried by pipetting air in and out 60 times and the remaining ethanol was thus removed.
  • the nucleic acids were eluted by pipetting the AuF 150 times and pipetting off with 100 ml elution buffer which had previously been tempered to 50 ° C. by the device.
  • the total volume of elution BuFfer was 100 m3.
  • Automatic extraction machines can be used for carrying out the method according to the invention with the agent according to the invention corresponding thereto.
  • the time required compared to a spin column-based extraction is less.
  • a real-time PCR assay specific for the actin beta gene locus was used to quantify the total amount of bisulfite-modified DNA.
  • the Actin Beta gene locus does not contain any CpG sites, therefore, after the bisulfite modification reaction, all cytosines from this gene locus are converted into uracils and these are detected as thymines in a real-time PCR assay. This assay was used to verify that the genomic DNA is bisulfite modified.
  • Taq-man probe 5 '-F AM- ACC ACC ACCC AACACAC AAT AACAAAC ACA-BHQ- 1 -3'
  • Reverse primer 50 pmol / m ⁇ : 0.1 m ⁇
  • Taq-man probe (25 pmol / m ⁇ ): 0.1 m ⁇
  • the real-time PCR was carried out in the CFX96 real-time system by the company Biorad.
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 amplification 40 cycles (95 ° C / 4 "; 57 ° C / 40")
  • FIG. 1 shows a real-time PCR from the ⁇ -actin assay.
  • gray curves genomic DNA
  • magenta curves no template control
  • blue and green curve samples DNA after the respective bisulfite modification reaction.
  • the cytosine free flagment (CFF-1) assay amplifies genomic and bisulfite-modified DNA with the same efficiency and is therefore suitable for quantifying the yield of bisulfite-modified DNA. It enables the direct comparison of input DNA and output DNA of the bisulite modification reaction.
  • genomic DNA the sense and the antisence strand serve as templates.
  • the antisense strand serves as a template in bisulfite-modified DNA, because the antisense strand (contains cytosines) is not available for the amplification reaction after the bisulfite modification.
  • Primem and Taq-man probe are as follows. Forward primer: 5 '-T AAG AGT AAT AATGGATGG ATG ATG-3'
  • Taq-man probe 5'- F AM- ATGGATG AAGAAAGAAAGGATG AGT-BHQ- 1 -3 'Real-Time PCR approach:
  • the real-time PCR was carried out in the CFX96 real-time system by Biorad: Amplification conditions:
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 amplification 40 cycles (95 ° C / 4 "; 52 ° C / 40")
  • FIG. 2 shows a real-time PCR from the CFF-1 assay. (magenta curves: No.
  • Reverse primer 50 pmol / m ⁇ : 0.1 m ⁇
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 Amplification 40 cycles (95 ° C / 4 "; 58 ° C / 40")
  • FIG. 3 shows a real-time PCR of the CFP 415 bp assay. (magenta curves: No Template Control, green and blue curves: samples DNA after
  • the automated purification was done with the magnetic particle processor
  • Plastic combs into which magnetic rods penetrate and which then move magnetic particles in a walk-away process which are used to isolate nucleic acids. These plastic combs were used outside of their normal intended use to carry out the fiction, contemporary method.
  • the plastic material of the combs is polypropylene. According to the invention, the plastic combs were roughened using a grinding machine.
  • the reagents used for bisulfite modification of the nucleic acids were partly taken from the commercial kit “innuCONVERT Bisulfite Basic Kit”; Analytik Jena AG, taken. For the bisulfite modification 5 gg of genomic DNA with 70 m ⁇ conversion
  • Deep well plate 2 1 ml Washing Solution HS (Analytik Jena AG)
  • Deep well plate 3 1 ml desulfonation buffer (innuCONVERT Bisulfite Basic Kit; Analytik Jena AG)
  • Deep well plate 4 1 ml Washing Solution LS (Analytik Jena AG)
  • Deep well plate 7 100 ml Elution Buffer (innuCONVERT Bisulfite Basic Kit; Analytik Jena AG)
  • the reaction mixture described above was after the incubation in the cavity 1 given, which was filled with 250 m ⁇ 99.8% ethanol. A further 5 ml Binding Optimizer (Analytik Jena AG) were added to this reaction mixture. The automated process was then carried out on the KingFisher Flex machine.
  • the reaction mixture of cavity 1 was mixed by means of the rough plastic combs according to the invention by repeating vertical movements (moving in and out within the deep well plate cavity).
  • the bisulfite-modified DNA binds to the rough plastic surface of the plastic combs.
  • After binding of the DNA it was washed by vertical movement of the comb in the cavity of the deep well plate 2 with the Washing Solution HS (Analytik Jena AG) contained therein
  • the rough plastic combs were slowly moved vertically up and down in desulfonation buffer for 8 minutes.
  • the bisulfite-modified DNA bound to the rough plastic combs was then successively transferred to three further deep-well plates containing alcoholic washing buffers (Washing Solution FS, 80% ethanol, 80% ethanol), each for 30 seconds washed slowly moving up and down vertically.
  • alcoholic washing buffers Wash Solution FS, 80% ethanol, 80% ethanol
  • the plastic combs were dried on the foot for 10 minutes and the remaining ethanol was removed.
  • the bisulfite-modified DNA was eluted from the rough plastic combs by moving the plastic combs vertically for 10 minutes in elution buffer, which had previously been heated to 80 ° C. by the device.
  • the total volume of elution buffer was 100 ml.
  • the method is extremely simple and fast and shows that with the commercially available automatic extraction KingFisher Flex an automated bisulfite modification of up to 96 sample DNAs can be carried out in parallel by modifying the plastic combs alone.
  • the modified DNA was again analyzed using various real-time PCR assays.
  • a real-time PCR assay specific for the actin beta gene locus was used to quantify the total amount of bisulfite-modified DNA.
  • the Actin Beta gene locus does not contain any CpG sites, so all cytosines from this gene locus are converted into uracils after the bisulfite modification reaction and these are detected as thymine in a real-time PCR assay. This assay was used to check whether the genomic DNA was bisulfite-modified.
  • Taq-man probe 5 '-F AM- ACC ACC ACCC AACACAC AAT AACAAAC ACA-BHQ- 1 -3'
  • Reverse primer 50 pmol / m ⁇ : 0.1 m ⁇
  • Taq-man probe (25 pmol / m ⁇ ): 0.1 m ⁇
  • the real-time PCR was carried out in the CFX96 real-time system from Biorad.
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 amplification 40 cycles (95 ° C / 4 "; 57 ° C / 40")
  • FIG. 4 shows a real-time PCR from the ⁇ -actin assay.
  • gray curves genomic DNA
  • magenta curves no template control
  • blue and green curve samples DNA after the respective bisulfite modification reaction.
  • the cytosine free fragment (CFF-1) assay amplifies genomic and bisulfite-modified DNA with the same efficiency and is therefore suitable for quantifying the yield of bisulfite-modified DNA. It enables the direct comparison of input DNA and output DNA of the bisulite modification reaction.
  • genomic DNA the sense and the antisence strand serve as templates.
  • only the sense strand is used for bisulfite modified DNA as a template because the antisense strand (contains cytosines) is not available for the amplification reaction after the bisulfite modification. That is why there is a theoretical loss of one Ct value for bisulfite-modified DNA, since the template is halved after the bisulfite modification.
  • the sequence of the primers and the Taq-man probe are as follows.
  • Taq-man probe 5'- F AM- ATGGATG AAGAAAGAAAGGATG AGT-BHQ- 1 -3 '
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 amplification 40 cycles (95 ° C / 4 "; 52 ° C / 40")
  • FIG. 5 shows a real-time PCR from the CFF-1 assay. (magenta curves: No.
  • a CFP 415 bp assay (cytosine-ffee primer binding site) with bisulfite-modified DNA was carried out as a further analysis. Since this assay is used to amplify long DNA sequences, this assay provides information about the degraded DNA during the bisulfite modification and during the refinement of bisulfite-modified DNA.
  • the sequences from the primers are as follows: Forward primers: 5 ATGGGT AAGGAT ATG AAGTT AAT-3 '
  • Reverse primer 50 pmol / m ⁇ : 0.1 m ⁇
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 Amplification 40 cycles (95 ° C / 4 "; 58 ° C / 40")
  • FIG. 6 shows a real-time PCR of the CFP 415 bp assay. (magenta curves: No Template Control, green and yellow curves: samples DNA after
  • Exemplary embodiment 3 Manual bisulfite modification using paramagnetic
  • the chemicals used for the bisulfite modification of the nucleic acids were partly taken from the commercial extraction kit innuCONVERT Bisulfite Basic Kit; Analytik Jena AG taken.
  • the reaction mixture was transferred into the 2.0 ml reaction vessel with the plastic granules.
  • 250 ml of 99.8% ethanol and 5 ml of Binding Optimizer (Analytik Jena AG) were added and the vessel was shaken for 10 minutes at room temperature and at 1400 rpm.
  • the 2.0 ml reaction vessel was placed in a magnetic trap, the paramagnetic granules were separated and the supernatant was removed
  • the 2.0 ml reaction vessel with the plastic granules was incubated at 65 ° C. for 10 minutes and 500 rpm with the lid open in order to remove the remaining alcohol.
  • the bisulfite-modified DNA was detached from the plastic granulate according to the invention by adding elution buffer (innuCONVERT Bisulfite Basic Kit; Analytik Jena AG). For this, 100 ml of elution buffer were added to the plastic granules and the reaction vessel was incubated for 10 minutes at 65 ° C. and at 500 rpm.
  • elution buffer innuCONVERT Bisulfite Basic Kit
  • the 2.0 ml reaction vessel with the plastic granules was placed in a magnetic trap, the granules were separated and the eluate was transferred to a new 2.0 reaction vessel.
  • the Spin Filter column-based extraction kit from Stiatec (InviGene Bisulfite Conversion Kit) was used for comparison. The instructions as described in the kit manual were followed exactly. After bisulfite modification reactions had been carried out, successful bisulfite modification was detected using various real-time PCR assays.
  • a real-time PCR assay specific for the actin beta gene locus was used to quantify the total amount of bisulfite-modified DNA.
  • the Actin Beta gene locus does not contain any CpG sites, therefore, after the bisulfite modification reaction, all cytosines from this gene locus are converted into uracils and these are detected as thymines in a real-time PCR assay. This assay was used to check whether the genomic DNA was bisulfite-modified.
  • Taq-man probe 5'-F AM-ACCACCACCCAACACACAATAACAAACACA-BHQ- 1 -3 '
  • Reverse primer 50 pmol / m ⁇ : 0.1 m ⁇
  • Taq-man probe (25 pmol / m ⁇ ): 0.1 m ⁇
  • the real-time PCR was carried out in the CFX96 real-time system from Biorad.
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 amplification 40 cycles (95 ° C / 4 "; 57 ° C / 40")
  • FIG. 7 shows a real-time PCR from the ⁇ -actin assay.
  • genomic DNA magenta curves: No Template Control; red and green curve samples DNA after the respective bisulfite modification reaction). The results show that the genomic DNA could be successfully modified with the aid of the manual method of the invention.
  • the cytosine free fragment (CFF-1) assay amplifies genomic and bisulfite-modified DNA with the same efficiency and is therefore suitable for quantifying the yield of bisulfite-modified DNA. It enables the direct comparison of input DNA and output DNA of the bisulite modification reaction.
  • genomic DNA the sense and the antisence strand serve as templates.
  • the antisense strand serves as a template in bisulfite-modified DNA, because the antisense strand (contains cytosines) is not available for the amplification reaction after the bisulfite modification.
  • primers and the Taq-man probes are as follows: Forward primer: 5 '-T AAG AGT AAT AATGGATGG ATG ATG-3'
  • Taq-man probe 5'-F AM-ATGGATGAAGAAAGAAAGGATGAGT-BHQ- 1 -3 'Real-time PCR approach:
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 amplification 40 cycles (95 ° C / 4 "; 52 ° C / 40")
  • FIG. 8 shows a real-time PCR from the CFF-1 assay.
  • magenta curves No Template Control
  • gray curves genomic DNA
  • red and green curves sample DNA after bisulfite modification reaction.
  • a CFP 415 bp assay cytosine-ffee primer binding site
  • this assay provides information about the degraded DNA during the bisulfite modification and during the purification of bisulfite-modified DNA.
  • the sequences from the primers are as follows:
  • Reverse primer 50 pmol / m ⁇ : 0.1 m ⁇
  • Step 1 Denaturation 95 ° C / 120 "
  • Step 2 Amplification 40 cycles (95 ° C / 4 "; 58 ° C / 40")
  • FIG. 9 shows a real-time PCR of the CFP 415 bp assay. (magenta curves: No Template Control, red and green curves: samples DNA after

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Abstract

La présente invention concerne un procédé et une trousse d'analyse pour effectuer une réaction de modification de l'ADN au bisulfite afin de déterminer les schémas de méthylation de l'ADN, caractérisée en ce qu'après avoir effectué la réaction de désamination en utilisant du bisulfite (par exemple du bisulfite de sodium ou du bisulfite d'ammonium), l'ADN modifié est lié à une phase solide rugueuse et toutes les autres étapes du processus nécessaires sont réalisées sur cette phase solide rugueuse. Pour finir, l'ADN haute pureté modifié est séparé de cette phase solide. Le procédé peut être réalisé manuellement mais aussi automatiquement. Concernant la phase solide rugueuse, il s'agit d'un matériau non minéral présentant une surface rugueuse.
PCT/EP2020/069148 2019-07-07 2020-07-07 Procédé et trousse d'analyse pour la modification de l'adn au bisulfite Ceased WO2021013534A1 (fr)

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DE102019118332.4 2019-07-07
DE102019118332.4A DE102019118332B4 (de) 2019-07-07 2019-07-07 Verfahren und testkit zur bisulfitmodifizierung von dna

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