RU126704U1 - DEVICE FOR PREPARING DOUBLE EMULSION AND AMPLIFICATION IN THE INTERNAL WATER PHASE USING DIGITAL PCR SPECIFIC Fragments of Nucleic Acids - Google Patents

Abstract

1. A device for carrying out a digital polymerase chain reaction, characterized in that with the help of two flowing microfluidic chips and three syringe pumps, a double water-oil-water emulsion is created in the chip for PCR, where the polymerized external aqueous phase acts as a gel matrix , it is glued to the glass surfaces of the chip, and contains in a fixed position individual mono-drops of the internal aqueous phase, into which, when forming the emulsion, all the necessary ingredients for PCR are added, including intercalating dye SYBR Green I or the like, as well as an analyzed nucleic acid sample, each monodroplet enclosed in oil shells each, used for their spatial separation and to prevent evaporation during PCR, after which amplification is recorded using a suitable fluorescence microscope , where the luminescence of the corresponding fluorescent dye indicates the presence of the desired nucleic acid sequence in specific mono-drops .2. The device according to claim 1, characterized in that the registration of the nucleic acid amplification process is carried out using fluorescently-labeled hybridization probes.

Description

The utility model relates to physicochemical biology, biotechnology and medicine and is associated with the analysis of nucleic acid molecules by amplification of their specific fragments. It can be used in quantitative DNA diagnostics in medicine, veterinary medicine, sanitary and epidemiological studies to detect and count causative agents of dangerous infections, including possible bioterrorist attacks, in forensics to identify criminals, in the food industry in identifying and quantifying ingredients in food products from genetically modified organisms, determining the quality of raw materials for contamination with undesirable impurities, etc. The device is based on the use of microfluidic technology and is designed to prepare a double water-oil-water emulsion ("B-M-B") with a hardening (polymerizing) external phase and amplification in the internal liquid aqueous phase using a digital polymerase chain reaction ( qPCR) of specific nucleic acid fragments.

Modern DNA diagnostics is based primarily on the amplification using specific DNA thermocyclers of specific DNA or RNA fragments using PCR and its modifications, including PCR. Currently, most DNA thermal cyclers have a reaction block heated and cooled by Peltier elements made of a metal with good thermal conductivity, in which reaction polypropylene tubes with a capacity of 0.1 to 1.5 ml containing reaction volumes of 10 to 30 μl are usually located. Another direction in the amplification of nucleic acids using reactions controlled by temperature changes is the creation of various microfluidic devices in which the volumes of the reaction mixtures are much smaller and the types of reaction vessels are much more diverse. Microfluidic PCR can be divided into PCR with stationary reaction chambers in the nanoliter and picolitre ranges, PCR in a liquid stream, where the volumes of reaction mixtures are also measured in pico- and nanoliters, into solid-phase PCR on chips and in emulsion PCR, in which volumes are for separate independent reactions amplifications range from femto to nanoliters. If in ordinary PCR, as a rule, the reaction mixture occupies only a small part of the total volume of the vessel, then in microfluidic PCR the volume of vessels of one type or another is almost equal to the volume of the reaction mixture. Moreover, a vessel with solid walls can act as a vessel, as well as emulsified drops of the aqueous phase, which are in the water-oil-immiscible phase, or parts of the gel space, or parts of the aqueous solution, which are in direct contact with places where amplification occurs.

PCR in a water-in-oil emulsion was previously proposed due to the fact that with conventional PCR there is some selectivity for the accumulation of certain amplicons, which is eliminated or minimized during emulsion PCR [Nakano et al., 2003; Musyanovych et al., 2005; Williams et al., 2006]. Moreover, in the papers cited by the authors, either after several cycles or at the end of the reaction, the emulsion was separated by centrifugation into two phases - oil and water, thereby leading to the mixing of amplicons from all microdroplets, where their independent amplification reactions had gone before. There is also a known method called BEAMing [Diehl et al., 2006], designed to ensure that magnetic particles coated with streptavidin get into all or only part of the microdroplets of an aqueous solution containing all the necessary ingredients for PCR, when the emulsion is formed. Due to the fact that a biotinylated primer binds to these magnetic particles, solid-phase amplification of DNA occurs, where, due to steric restrictions, the size of the amplified fragment is of great importance, and it has been shown that the multiplication efficiency of DNA molecules with sizes of 200 nucleotide pairs and 2700 nucleotide pairs is only 30 % and 5% of that for a fragment with a length of 110 nucleotide pairs, respectively [Diehl et al., 2006].

In the future, emulsion PCR improved noticeably, and if in the early stages of the existence of this method of amplification, emulsions were prepared by simply shaking the tubes with the water and oil phases on a shaker, then later special chip devices Y were used to mix the oil and water flows supplied by syringe pumps - and / or T-types. A new task of emulsion PCR was to ensure that this reaction is carried out in a digital format with the aim of high-precision counting of the number of desired copies of DNA or RNA molecules initially present in the analyzed sample. So, by QuantaLife, Inc. A new technology for quantitative (digital) PCR amplification, called Droplet Digital PCR, or ddPCR for short, has been proposed. Based on this technology, Bio-Rad Laboratories began to produce a mono-droplet DNA thermocycler model QX-100, where it is possible to analyze 8 samples at the same time, but each sample is amplified in 20 thousand reactors with a volume of 1 nanoliter each [Hindson et al., 2011; Pinheiro et al., 2012].

A very important point when conducting single-phase emulsion PCR is to prevent the merging of the drops of the aqueous phase in which amplification occurred. One of the technical solutions to achieve this is to ensure solidification after completion of the PCR of the aqueous phase, which is achieved due to the fact that in the aqueous phase, for example, fusible agarose is present, which is at the stages of denaturation, annealing and elongation in the molten state and does not interfere with the course of PCR [Leng et al., 2010; Zhu et al., 2012]. After completion of PCR and cooling of the reaction mixture, the mono-droplets contained in the water-in-oil emulsion become gel mono-balls, unable to merge with each other.

Closest to our proposed method of handling single DNA or RNA molecules using digital PCR in a two-phase emulsion “B-M-B” is a known method of propagation of nucleic acids in a cell-free system in the form of so-called molecular n olimerase colonies (polonium), when the action of thermostable DNA polymerase in a polyacrylamide gel using PCR amplifies DNA fragments or RNA [Chetverin, Chetverina, 1995; 1998; Chetverin, Chetverma, 1997; 1999]. However, this method is not technologically advanced due to the multi-stage preparation of a layer of polyacrylamide gel containing in random places single nucleic acid molecules suitable for reproduction.

Another serious drawback is the obviously lower coefficient of reproduction of DNA molecules in the gel than during PCR in solution due to less diffusion and other steric restrictions imposed by the cellular or other structure of the gel, which also increase in direct proportion to the amplification of longer DNA fragments. In this approach, when forming a medium for amplification of individual nucleic acid molecules, microfluidic technology is not used, which allows changing the diameter (volume of drops) and actually the number of reaction vessels (mono-droplets of water) by changing (choosing) the diameters of the channels and the velocity of the fluids (aqueous and oil phases) phase) for a specific fluid volume selected for the experiment.

The essence of this invention lies in the fact that the work with single molecules of DNA or RNA and their amplification by PCR can be carried out in separate independent reaction vessels formed when creating a double emulsion "B-M-B", the main difference from which is currently used is the presence of an external hardening (polymerizing) phase, formed from monomers of a polyacrylamide gel or other substance that meets the necessary requirements in the form of transparency of the medium, its inertness for DNA polymerases, and others Nucleic exchange enzymes and the ability to withstand temperature fluctuations in the desired range during PCR. As is known, PCR usually requires a cyclical change in temperature, because in order for a new cycle to begin, DNA denaturation (at a temperature of about 95 ° C) must occur, followed by annealing of the primers (most often at temperatures from 50 to 60 ° C) and elongation (most commonly used temperatures are 72-75 ° C).

Fig. 1 shows a diagram of a utility model device that allows using three independently controlled syringe pumps and two flowing Y- or T-type microfluidic chips on a microscope slide (or the like) covered with a glass or plexiglass of the same size, separated by a space, formed by a silicone rubber frame of suitable thickness (equal to or slightly greater than the diameter of the outlet channel of the second flow microfluidic chip), form a double emulsion "B-M-B", in which the inner aqueous layer p edstavlyaet a plurality monokapel containing all the necessary ingredients for the PCR, including the intercalating dye SYBR Green I or the like, as well as nucleic acid sample to be analyzed. Instead of intercalating dyes, detection amplification systems can be used with a variety of hybridization probes labeled with fluorochromes. The middle (oil) layer is necessary for spatial separation and elimination of evaporation of water mono-droplets of the inner phase, and the outer water layer is a ready-for-polymerization mixture of polyacrylamide gel monomers, which after filling the space formed between the glass panes and completing the polymerization process becomes a gel matrix and acts sample retainer (mono-droplets) for subsequent detection in them of the results of amplification carried out in a suitable DNA thermal cycler, annom to conduct PCR in situ mode (slides) or in the DNA thermal cycler with a hollow air chamber, heated and cooled by a powerful lamp and fan, respectively, wherein the space may accommodate created mikrofluidny chip for PCR. Among other features of this utility model, it is necessary to indicate the need for the location of the microfluidic chip blank for PCR in the form of an assembled glass structure and a silicone frame with two holes in the latter (one inlet. For supplying a double emulsion located in the lowermost corner of the structure) and one outlet for bleeding air located in the opposite corner of the upper part in a vertically inclined position (to achieve the most complete filling formed by glass and silicone new hollow chamber spacers), as well as pretreatment of the glass surfaces of the chip with γ-methacryloxypropyltrimethoxysilane (or other silanes capable of copolymerization with acrylamide monomers), which allows the formation of reactive groups on the glass that enter into a copolymerization reaction with the polyacrylamide gel monomers to actually gel and phases of the double emulsion "B-M-B". After the gel is polymerized in a microfluidic PCR chip and placed in a suitable DNA thermal cycler, the latter undergoes thermal cycling, the temperature and time parameters of which depend on the characteristics of the amplified nucleic acid fragments, after which this chip is placed on the table of a suitable fluorescence microscope equipped with a digital camera , and the luminescence of the fluorescent dye is recorded, indicating the past amplification and, therefore, the presence of Wash nucleic acid sequences in specific monokaplyah, photodocumentation and digitizing results.

References

1. Chetverin A.B., Chetverina E.V. The method of cloning nucleic acids // RF Patent No. 2, 114, 175. 1998.

2. Chetverin A.B., Chetverina E.V. The method of propagation of nucleic acids, the method of their expression and the environment for their implementation // RF Patent No. 2,048, 522. 1995.

3. Chetverin A.B., Chetverina E.V. Method for amplification and expression of nucleic acids in solid media and its application for nucleic acids cloning and diagnostics // U.S. Patent No. 5,958,698. 1999.

4. Chetverin A.B., Chetverina E.V. Method for amplification of nucleic acids in solid media // U.S. Patent No. 5.616.478. 1997.

5. Diehl F., Li M., He Y., Kinzler K.W., Vogelstein B., Dressman D. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions // Nat. Methods 2006. V. 3. 7. P.551-559.

6. Hindson B.J., Ness K. D., Masquelier D. A., Belgrader P. et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number // Anal. Chem. 2011. V.83. P.8604-8610.

7. Leng X., Zhang W., Wang C., Cui L., Yang C.J. Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR // Lab. Chip. 2010. V.10. P.2841-2843.

8. Musyanovych A., Mailander V., Landfester K. Miniemulsion droplets as single molecule nanoreactors for polymerase chain reaction // Biomacromolecules. 2005.V. 6.4. P. 1824-28.

9. Nakano M. et al. Single-molecule PCR using water-in-oil emulsion // J. Biotechnol. 2003. V. 102. 2. P.117-124.

10. Pinheiro L. B., Coleman V. A., Hindson C. M., Herrmann J., Hindson B. J., Bhat S., Emslie K.R. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification // Anal. Chem. 2012. V.84. P.1003-1011.

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Claims (2)

1. A device for carrying out a digital polymerase chain reaction, characterized in that with the help of two flowing microfluidic chips and three syringe pumps, a double water-oil-water emulsion is created in the chip for PCR, where the polymerized external aqueous phase acts as a gel matrix , it is glued to the glass surfaces of the chip, and contains in a fixed position individual mono-drops of the internal aqueous phase, into which, when forming the emulsion, all the necessary ingredients for PCR are added, including intercalating dye SYBR Green I or the like, as well as an analyzed nucleic acid sample, each monodroplet enclosed in oil shells each, used for their spatial separation and to prevent evaporation during PCR, after which amplification is recorded using a suitable fluorescence microscope , where the luminescence of the corresponding fluorescent dye indicates the presence of the desired nucleic acid sequence in specific mono-drops . 2. The device according to claim 1, characterized in that the registration of the amplification process of nucleic acids is carried out using fluorescently-labeled hybridization probes.

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