Method, means and kit for extracting high-molecular weight DNA

Abstract

The invention relates to a method and a kit for extracting high-molecular weight DNA from a biological sample, in which the mixture is brought into contact with a binding buffer after the biological sample has optionally been lysed, wherein the DNA is bound to a solid phase and the DNA is subsequently purified, characterised in that the solid phase consists of a magnetic plastics material which is produced by a 3D printing method.

Description

Method, means and kit for the extraction of high molecular weight DNA Description Technical field

[0001] The invention relates to a simple means for extracting high molecular weight DNA. State of the art

[0002] Next-generation sequencing (NGS) technology is becoming increasingly important in the natural sciences, medicine, and other related fields. The goal of comprehensive genome sequencing is not only to identify specific target sequences (pathogen detection, mutation detection, etc.), but also to elucidate the complete sequence of an organism. Logically, when using these new sequencing technologies (e.g., nanopore sequencing; Oxford Nanopore Technologies) to address such questions, the length of the sequenced genome segments is of primary importance: the larger the sequencing read lengths, the easier and more resource-efficient the bioinformatics analysis of the generated sequences and their assignment to the entire genome.

[0003] There are currently several sequencing platforms on the market with varying capabilities for capturing longer DNA fragments. For example, the Nanopore sequencing platform from Oxford Nanopore Technologies allows for sequencing of megabase-length fragments without pre-amplification, provided high-quality, high-molecular-weight DNA is used (Ultra-Long DNA Sequencing Kit SQK-ULK001).

[0004] In this context, the extraction of high molecular weight DNA (HMW DNA) from various biological sample materials (e.g., blood, eukaryotic cells, plants, tissue) has gained significant interest in recent years, as HMW DNA is the essential basis for long-read sequencing. Long reads enable bridging of repetitive sequences, uniform coverage of GC-rich regions, and Complete sequencing of specific target regions. The present article, "Method of the Year: Long-Read Sequencing," published in Nature on January 12, 2023, clearly defines the advantages.

[0005] Those skilled in the art are aware that a variety of technologies and products based on them exist for DNA extraction. However, methods for extracting high-molecular-weight DNA of good quality are scarce. This applies to both manual extractions and automated methods.

[0006] Using a technology developed by Circulomics, it is possible to isolate high-molecular-weight DNA. This method is available commercially from Pacbio (Nanobind CBB kit (SKU 102-301-900).

[0007] The core of this technology is a so-called "nanodisc." The nanodisc is a magnetic nanomembrane that binds the DNA to be isolated. The chemistry underlying DNA binding involves the use of buffers containing chaotropic salts, thus representing the state of the art. This also applies to other extraction reagents such as wash buffers and eluents. The nanodisc is crucial for the extraction of high-molecular-weight DNA.

[0008] The production of this nanodisc follows a very complex multi-step process and is described in the published application (US 2020 / 0331004 Al).

[0009] This multi-stage process consists of the following individual steps: The starting material is a plastic core with a size of 10-100 pm. Using an electron beam evaporator, thin silicon dioxide and iron layers (20-100 nm) are alternately deposited in multi-stage processes. This multiply-deposited component must then be heat-shrunk at 300°C. This is where nanomembranes form, which are used for the upcoming nucleic acid extraction. Various combinations of individual intermediate layers, as well as the size of these layers, are intended to increase and influence yields and purities depending on the application. With the iron layer, the main focus is on achieving sufficient magnetic force to overcome viscous forces, surface tension, and buoyancy. It is also biologically and chemically vulnerable if the outer silicon dioxide layer is exposed to mechanical stress. This can lead to degradation and corrosion, which can be detrimental to extraction.

[0010] The description reveals that the production of such a nanomembrane is very complex and time-consuming and therefore ultimately very expensive.

[0011] The actual extraction process is relatively simple. After sample lysis, a binding buffer is added to the lysate, and the mixture is brought into contact with a nanodisc. The chaotropic salts contained in the buffer mediate the binding of the DNA to the surface of the nanodisc. This occurs by incubating the sample vessel in a rotating shaker. After the DNA binds to the surface of the nanodisc, mandatory washing steps follow. Finally, the disc is dried, and after adding an eluent, the DNA is detached from the nanodisc. The nanodisc is processed using a magnet (using a magnetic stand). This method enables the isolation of high-molecular-weight DNA, as the architecture of this nanodisc allows for very gentle handling of the DNA and essentially eliminates shear forces. Patent literature

[0012] European patent EP 3 286 325 B1 (also in US patent No. 10,934,540 B2) disclosed that plastic materials with rough surfaces enable the binding of nucleic acids. It also described that the rough surfaces can be produced using 3D printing processes. A magnetic composite material, commercially available under the name "TECACOMP®," was also mentioned there. 3D printing with this magnetic composite material was not described. Likewise, these patents did not provide any information about the length of the bound nucleic acids. No significant differences were found regarding the effectiveness of nucleic acid binding with the various rough plastics produced.

[0013] A method for extracting high molecular weight DNA from a biological sample was described in the publication DE 10 2022 115 445 A1. The binding of the High-molecular-weight DNA is attached to a solid phase using a combination of a polyether (e.g., polyethylene glycol) and a salt, and in the absence of monohydric alcohols. The solid phase can have a rough surface and be made of a magnetic plastic. A 3D printing process was not mentioned there.

[0014] The prior art also includes the publication WO 2021 / 055084 A1. This shows in Figure 3, page 17, paragraph

[0075] , the structure of a magnetic particle, functionalized, for example, with a polymer surface, as shown in Figure 2, bottom right, for the isolation of long DNA fragments.

[0015] In the published patent application WO 2021 / 119425 A1, in Figure 5 after page 7, paragraph

[0024] , the improvement in the isolation of long DNA fragments by adding Short Read Eliminator reagents to Qiagen Puregene Kit reagents, comprising polyvinylpyrrolidone according to claim 29, is investigated. Object of the invention

[0016] The object of the invention was to provide an alternative to the Nanaodisc described above in a method for extracting high molecular weight DNA. Solution to the task

[0017] The problem was solved according to the features of the patent claims. According to the invention, it has been possible to provide a means that possesses the advantages of the nanodisc, allowing it to be used in a simple extraction process for the extraction of high-molecular-weight DNA – both in a manual and an automated process. The production of such a means is simple and inexpensive, thus differing significantly from the production of the nanodisc described in the prior art.

[0018] Surprisingly, it has been found that an alternative solid phase (disc), which is analogous to the nanodisc for a gentle extraction of high molecular weight DNA used, can be provided simply, quickly, and very inexpensively using 3D printing technology. This makes it possible to produce an alternative solid phase for the adsorption of high-molecular-weight DNA simply, quickly, flexibly, and extremely inexpensively. This technology allows flexible disk printing—flexible in terms of size, architecture, and material. A material, preferably a composite material consisting of a plastic and a magnetic (preferably paramagnetic) component, can be used for 3D printing. Such a material is commercially available, for example, under the name "TECACOMP PP."

[0019] This gives the 3D-printed plastic disc a paramagnetic property—as is also the case with the nanodisc. Production is also resource-efficient, as the material used for printing can be made from waste plastic. The disc printed in this way also does not require a coating with a DNA-binding mineral material. It has been shown that the plastic disc is sufficient to adsorb high-molecular-weight DNA from a sample. The extraction process is simple and quick to perform.

[0020] This is done by lysing the biological sample in a reaction vessel with a lysis buffer (e.g. Lysis Solution CBV; IST Innuscreen GmbH), then adding the disc according to the invention and a binding buffer (e.g. Buffer HI and Buffer H2; IST Innuscreen GmbH) to the sample. The vessel is then carefully inverted by 180° several times. This causes the DNA contained in the sample to adsorb onto the disc according to the invention. The vessel is placed in a magnetic rack, and the disc is fixed to the wall of the vessel by the magnet. The supernatant is poured off, and the disc is rinsed twice with a washing buffer (e.g. Washing Solution MS or 80% ethanol) by swirling the vessel. The washing buffer is poured off after each washing step. After removing the washing buffer, ddH2O (double distilled water) is added to the vessel, and the DNA is detached from the disc and dissolved while gently shaking.This method allows for the very gentle isolation of high-molecular-weight DNA using the agent according to the invention. The method is very simple to perform and can also be used in an automated manner. It uses conventional devices designed for handling magnetic particles (e.g., the KingFisher device platform). The agent according to the invention is based on the "walk-away process" known to those skilled in the art. from the cavity containing the lysate and the binding buffer (where the DNA binds to the disc), through the cavities with washing buffers (where the DNA on the disc is washed) to the cavity with the ddH2O (where the DNA is detached).

[0021] The production of the paramagnetic disc using 3D printing technology represents a surprisingly significant advance over the technology required for the nanodisc from Circulomics. What was particularly surprising was that the 3D printing process with the magnetic materials resulted in the desired properties, even though this had not previously been observed by simply roughening the magnetic materials. Apparently, the manufacturing process (3D printing) plays a decisive role in the success and gives the magnetic material its surprising properties.

[0022] This provides a simple tool that can be used excellently for the extraction of high-molecular-weight DNA. Another particularly advantageous feature is that the disc according to the invention can be manufactured in any size. Furthermore, the tool according to the invention is not limited to the format of a disc. In principle, any geometric shape can be manufactured and used.

[0023] The invention is described in more detail below using two exemplary embodiments. Example 1: Preparation of a disc measuring 1 mm x 0.8 mm and its use for the extraction of high molecular weight DNA from 5 ml of blood

[0024] A paramagnetic plastic material (TECACOMP PP) was used to manufacture the disc. It was produced on a 3D printer (Kobra Plus; Anycubic). The disc dimensions were 1 mm x 0.8 mm.

[0025] For the extraction of high molecular weight DNA, 5 ml of a whole blood sample was used. This was transferred into a 15 ml reaction tube. Lysis of the erythrocytes was performed using Ery Lysis Solution A and Ery Lysis Solution B (IST Innuscreen GmbH). After lysis of the erythrocytes, the resulting cell pellet was nucleated blood cells, which was located at the bottom of the 15 ml reaction vessel, were mixed with a lysis buffer and proteinase K (Lysis Solution CBV; IST Innuscreen GmbH) and incubated in a thermomixer for 20 min at 55°C. After lysis of the cells, the disc according to the invention was added to the 15 ml reaction vessel. This was followed by the addition of the binding buffers (Buffer HI and Buffer H2; IST Innuscreen GmbH). The vessel was then carefully inverted 30 times by 180°. During this step, the high-molecular-weight DNA adsorbs to the surface of the disc. The vessel was then placed in a magnetic rack to fix the disc, and the lysis mixture was poured out. A washing buffer (Washing Solution MS; IST Innuscreen GmbH) was then added. The vessel was left on the magnetic rack, inverted three times, and the washing buffer was poured off. The washing step was repeated one more time.Next, 800 μl of another washing solution (Washing Solution ER; IST Innuscreen GmbH) was added. The tube was inverted twice, and the solution was decanted. The DNA on the disc was dissolved after adding 1000 μl of ddH2O, which was then incubated for approximately 30 minutes at 300 rpm and 37°C. The high-molecular-weight DNA was transferred to a new tube using a wide-bore pipette tip, and the DNA was carefully pipetted up and down several times. The DNA was then analyzed on a Tapestation (Agilent). As the data show, the DNA is of high molecular weight with a very high yield. The procedure is simple and very rapid, and the required reaction plastics are very minimal.Both the lysis of the erythrocytes and the subsequent lysis of the nucleated blood cells, as well as the binding of the released DNA to the disc according to the invention and the final dissolution of the DNA, take place in the same 15 ml reaction vessel. This represents an advantage over conventional methods for extracting high-molecular-weight DNA from blood samples.

[0026] Figure 1 shows the analysis of the genomic DNA. The peak at > 60,000 bp is clearly visible. The yield was 135 pg. Example 2: Production of a disc measuring 6 mm x 0.8 mm and its use for the extraction of high molecular weight DNA from 2.5 ml of blood

[0027] A paramagnetic plastic material (TECACOMP PP) was used to manufacture the discs. They were produced on a 3D printer (Kobra Plus; Anycubic). The disc dimensions were 7 mm x 0.8 mm.

[0028] For the extraction of high-molecular-weight DNA, 2.5 ml of a whole blood sample was used. After lysis of the erythrocytes, the resulting nucleated cells were transferred to a 2.0 ml reaction tube and mixed with a lysis buffer and proteinase K (Lysis Solution CBV; IST Innuscreen GmbH) and incubated in a thermomixer for 20 min at 55°C. After lysis of the cells, the disc according to the invention was added to the 15 ml reaction tube. This was followed by the addition of the binding buffers (Buffer HI and Buffer H2; IST Innuscreen GmbH). The tube was then carefully inverted 30 times by 180°. During this step, the high-molecular-weight DNA adsorbs to the surface of the disc. The tube was then placed in a magnetic rack to fix the disc, and the lysis mixture was poured out. A washing buffer (Washing Solution MS; IST Innuscreen GmbH) was then added. The vessel was left on the magnetic rack and inverted three times and the wash buffer was poured off.The washing step was repeated one more time. 800 μl of another washing solution (Washing Solution ER; IST Innuscreen GmbH) was then added. The tube was inverted twice, and the solution was decanted. The DNA on the disc was dissolved after adding 500 μl of ddH2O. The tube was incubated for approximately 30 minutes at 300 rpm and 37°C. The high-molecular-weight DNA was transferred to a new tube using a wide-bore pipette tip, carefully pipetting up and down several times. The DNA was then analyzed on a Tapestation (Agilent). As the data show, the DNA is high-molecular-weight with a very high yield.

[0029] Figure 2 shows the analysis of the genomic DNA. The peak at > 60,000 bp is clearly visible. The yield was 66 pg. definition High molecular weight DNA:

[0030] DNA with a length of 50,000 bp or greater, in particular 60,000 bp or greater.

Claims

Patent claims 1. A method for extracting high molecular weight DNA from a biological sample, in which - after optionally lysing the biological sample - the mixture is brought into contact with a binding buffer, whereby the DNA is bound to a solid phase and the DNA is subsequently purified, characterized in that the solid phase consists of a magnetic plastic material which is produced by a 3D printing process.

2. Method according to claim 1, characterized in that the magnetic plastic material is a paramagnetic plastic material.

3. Method according to claim 1 or 2, characterized in that the magnetic or paramagnetic plastic material comprises polypropylene.

4. Method according to one of claims 1 to 3, characterized in that the solid phase represents any geometric shape in any dimensions.

5. Method according to one of claims 1 to 4, characterized in that the solid phase has the shape of a disc.

6. Method according to one of claims 1 to 5, characterized in that the plastic material is a composite material.

7. A method for extracting high molecular weight DNA from a biological sample, characterized by the following steps: a) Lysis of the biological sample b) Addition of a binding buffer and addition of a solid phase, which was produced by 3-pressure from a magnetic, preferably paramagnetic plastic material c) Fixation of the material with the adsorbed DNA and pouring off the supernatant d) Addition of a washing buffer e) Add water and dissolve the DNA 8. Automated method according to one of claims 1 to 7, wherein the lysis mixture or the reaction mixture is located in a walk-away extraction device which uses the solid phase according to the invention, to which the high molecular weight DNA is adsorbed by vertical movement of the solid phase within the sample and this solid phase moves according to the walk-away principle into vessels with the washing buffer and finally into the aqueous solution.

9. Kit for a manually performable implementation of the method according to any one of claims 1 to 8, comprising: a) Lysis buffer b) Binding buffer c) Wash buffer d) Magnetic or paramagnetic plastic material produced by a 3D printing process e) ddH2O 10. Use of a magnetic or paramagnetic plastic material produced by a 3D printing process for the extraction of high molecular weight DNA from a biological sample.

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