METHOD AND KIT FOR EXTRACTING DNA FROM COMPLEX SAMPLES

Abstract

The invention relates to a method and a kit for extracting DNA from biological samples after optional homogenization and lysis of the samples, comprising the steps: a) binding the DNA to a solid phase using a binding buffer, b) repeatedly washing the DNA bound to the particles using a washing buffer, c) drying the particles, and d) elution of the DNA, wherein neither the binding nor the washing buffer comprises flammable aliphatic alcoholic components nor chaotropic salts, and is characterized in that both the binding and the washing buffer comprise a combination of water-soluble polymers, preferably PEG, and water-soluble salts as active components.

Description

Technical background Ultra-high-throughput DNA extraction from plant seeds is a crucial step in plant breeding, but it presents several challenges. The difficulties in this process lie primarily in the need to ensure high speed, precision, and reproducibility when extracting large quantities of genetic material. One of the key challenges lies in adapting traditional extraction methods to ultra-high throughput. Conventional DNA extraction techniques, typically designed for smaller sample sets, must be optimized and automated for mass throughput. Transferring these techniques to a large number of samples often requires redesign and adaptation to ensure efficient and reliable extraction. The speed of DNA extraction is the most critical step, as the possible sample throughput is defined by the speed. The quality of the extracted DNA presents another challenge. High-throughput DNA extraction carries the risk of contamination, which can affect the results of genetic analyses. Therefore, the extraction methods used must ensure that the obtained DNA is of high quality and free of contaminants to enable accurate genetic analyses. This is becoming increasingly important, as the analytical methods employed are often multiplex applications that place high demands on DNA quality. Cost is also a crucial aspect. One of the key cost factors is the consumption of reagents during the extraction process. Ultra-high-throughput DNA extraction often requires large quantities of reagents to ensure efficient and high-quality extraction. Therefore, reagent costs can be substantial. For this reason, very simple methods exist (“quick and dirty” methods), such as boiling the homogenized samples in a NaOH solution and subsequent neutralization with an acid. Such a method is inexpensive. It is important to note that these methods are often considered less accurate and less selective than specialized DNA extraction techniques. They can lead to higher levels of impurities and lower DNA quality. Therefore, they are more suitable for rapid, preliminary analyses or in cases where DNA purity is not critical. Overall, it is crucial that plant breeders adopt a holistic approach when implementing ultra-high-throughput DNA extraction technologies. Balancing speed, automation, quality, versatility, and financial efficiency, combined with the desired analytical method, is essential to ensuring the success of breeding programs and driving innovative solutions for agriculture. Automated high-throughput DNA extraction is typically performed using methods that bind DNA to magnetic or paramagnetic particles. These methods are based on the well-established principle of DNA binding to silicate or silica-modified surfaces in the presence of high concentrations of chaotropic salts, or in the presence of high concentrations of chaotropic salts and ethanol or isopropanol. The DNA bound to the particles is subsequently washed with ethanol- or isopropanol-containing wash buffers. After drying the particles, water or a low-salt buffer is added to desorb the DNA. State of the art Based on these well-known extraction reagents, a number of commercially available products are offered for the high-throughput extraction of DNA from plant materials. These include, among others, the MagSi-DNA Plant CLS kit (Magtivio BV), the NucleoMag Plant kit (Macherey-Nagel GmbH), and the Wizard Magnetic 96 DNA Plant System kit (Promega GmbH). All of these products are based on the binding of DNA to magnetic particles in the presence of buffers containing chaotropic salts and an alcoholic component. All kits also share the characteristic that the wash buffers are flammable alcoholic buffers and the buffers contain chaotropic salts. After homogenization / mechanical destruction of the plant samples, a lysis buffer is added, followed by sample lysis. After centrifugation, the clean supernatant is used for automated extraction. The extraction steps performed using these reagents are: 1. binding the DNA to the particles, 2. multiple washings of the DNA bound to the particles, 3. drying of the particles to remove alcoholic residues, and 4. elution of the DNA. Implementing the process steps into an automated workflow is relatively easy. It must be considered, however, that the reagents used, based on flammable components, pose an enormous risk. The required quantities of flammable wash buffers and the resulting waste of flammable liquids represent a serious problem in the field of high-throughput extraction. In some cases, several hundred liters of flammable liquids must be kept on hand, and additional waste is generated. This necessitates very expensive safety systems, which in turn drastically increases costs. Interestingly, the literature describes a number of different approaches that allow nucleic acids to be bound to mineral solid phases known to those skilled in the art without alcoholic buffers, and there are also methods that use wash buffers containing no alcoholic components. For example, patents US 5,705,628 and US 5,898,071 disclose methods for separating polynucleotides from a solution by binding the nucleic acids to functionalized magnetic particles using polyethylene glycol in combination with various salts, preferably sodium chloride. Washing the bound nucleic acids can be carried out with an alcoholic wash buffer. Wash buffers that represent a combination of different salts are also used. However, these methods do not demonstrate any application for the extraction of genomic DNA from plant samples or, more generally, DNA from complex biological samples.For the purposes of this invention, the term "complex samples" refers to biological materials that contain, among other things, plant parts. It is known that plant samples, in particular, require very efficient washing steps due to their composition. Furthermore, the composition of the preferred binding buffer shows a high concentration of sodium chloride (2.5 M). EP 2163621 discloses solvent-based washing buffers, which may eliminate the need for alcoholic buffers. However, the binding of nucleic acids to the magnetic particles occurs using solutions exhibiting chaotropic and / or high-salinity conditions. This results in chemical waste that is hazardous to the environment. WO 2015 / 188994 describes washing buffers containing a combination of polyethylene glycol and various salts. Chaotropic salts or sodium chloride are suitable as salts. A silica membrane is used as the solid phase. The use of magnetic or paramagnetic particles with functionalized surfaces is not disclosed. While the process does not use alcoholic components for the washing buffers, the step of binding the nucleic acid to the silica membrane is carried out using buffers with high alcohol concentrations and high concentrations of chaotropic salts. Ultimately, all these methods show that they are either unsuitable for isolating nucleic acids from a complex biological sample or that they rely on the use of alcoholic and chaotropic buffers or high-salt buffers for the binding step. Object of the invention The object of the invention is to eliminate the disadvantages of the solutions described in the prior art. Solution to the task The problem was solved – in accordance with the features of the patent claims. According to the invention, it has been possible to provide an extraction process that can be carried out without flammable buffers and that also does not rely on the use of chaotropic buffers or buffers with high salt concentrations. Furthermore, the process is very fast. The cost per DNA extracted has been significantly reduced. Surprisingly, it turns out that such a solution is possible using reagents known to the expert when they are combined in a new constellation. For the lysis of plant material, an existing lysis buffer (Lysis Solution CBV; IST Innuscreen GmbH) was used. According to the objective, the binding of DNA to paramagnetic particles (Mag Suspension F; IST Innuscreen GmbH) was to be carried out using a binding buffer that did not contain any alcoholic components and / or chaotropic salts. The subsequent washing steps were then also to be performed with wash buffers that did not contain any alcoholic components and / or chaotropic salts. This would allow the entire extraction protocol to be carried out without flammable alcoholic liquids. For the purposes of this invention, flammable alcoholic liquids are defined as alcohols that are liquid at room temperature and miscible with water in unlimited quantities. Examples include methanol, ethanol, n-propanol, and isopropanol. The solution according to the invention is based on the use of a mixture of water-soluble polymers, preferably water-soluble polyethers such as polyethylene glycol (PEG), and one or more monovalent, divalent, or multivalent salts as components of a binding buffer and as a component of washing buffers used. Water-soluble polymers are known to those skilled in the art. Examples include polyacrylamide (PAM), also as a copolymer with anionic or cationic monomers, polyacrylic acid homopolymer and its sodium salt (P-AA), acrylic acid / maleic acid copolymer and their sodium salts (P-AA / MA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and polyquaternium (PQ). Preferably, PEG with a molecular weight of 4,000 to 20,000 is used in the invention. Magnesium chloride and sodium chloride, or mixtures of these two, are preferably used as salts, with concentrations between 50 mM and 2.5 M, preferably between 100 mM and 900 mM. Furthermore, the binding buffer can also contain a detergent, preferably a polysorbate. Surprisingly, such buffers, acting as both binding buffers and wash buffers for the extraction of DNA from plant samples using magnetic or paramagnetic particles with various functionalizations, have not previously been used for the extraction of DNA from complex samples. However, these simple and harmless buffer compositions allow the extraction process to be carried out entirely without flammable alcoholic buffers. This represents a significant advantage for high-throughput DNA extraction from plant samples.This eliminates the need for various safety measures to prevent fires or explosions caused by large quantities of combustible waste. Furthermore, experimental results show that very high quantities of DNA can be isolated using the buffers according to the invention (see Fig. 2). The DNA extraction yields are at least as high as those achieved with conventional buffers containing alcoholic components. The composition according to the invention offers another significant advantage. The buffer used for DNA binding (binding buffer) can simultaneously be used as a wash buffer, or the wash buffer can have the same composition as the binding buffer, but be a diluted version of the binding buffer.This significantly reduces the complexity of a kit for high-throughput DNA extraction from plant samples, which can lead to substantial cost savings. Ultimately, the automated implementation of the extraction protocol is also very simple. This allows for a very high sample DNA output per unit of time. The means according to the invention can be components of a kit for carrying out DNA extractions from plant samples, but possibly also from other complex biological samples. The present invention is illustrated by means of an example. Example of implementation 1. Extraction of DNA from parsley leaves. Comparison of the agent according to the invention with extraction reagents containing alcohol. The sample material was homogenized in a Bioraba bag. Lysis was performed using a commercially available lysis buffer (Lysis Solution CBV; IST Innuscreen GmbH). Proteinase K was added to the lysis buffer. Lysis was carried out for 15 minutes at 65°C in a thermoshaker. The lysis mixture was then centrifuged for 2 minutes. 200 µl of the supernatant were used for extraction. Sample 1 was processed using "classical" extraction reagents with alcoholic components. 200 µl of Binding Solution SBS (IST Innuscreen GmbH) was added. This buffer contains 70% isopropanol. Sample 2 was processed with the binding buffer according to the invention, which contains no alcoholic components. This buffer contains 22% PEG 6000 and 0.4 M magnesium chloride. After the addition of the binding buffers, paramagnetic particles were added to the sample (MAG Suspension F; IST Innuscreen GmbH).An alcoholic wash buffer (Washing Solution A; IST Innuscreen GmbH) was used for sample 1. For sample 2, the inventive wash buffer without an alcoholic component was used. This is a dilution of the binding buffer used (7.5% PEG 6000 and 0.1 M magnesium chloride). After the two washing steps, the particles with the bound DNA were briefly rinsed with a Tris buffer to remove any remaining wash buffer. This step can also be omitted. The DNA was then eluted with water. The extraction was performed automatically using the Pureprep Maxi magnetic particle processor (IST Innuscreen GmbH). The following protocol was used for the automated extraction: Binding step: 2.5 minutes; Wash step 1: 18 seconds; Wash step 2: 18 seconds; Wash buffer removal: 0; Elution step: 2.5 minutes. After extraction, the DNA was analyzed using the tape station (Agilent). As the data show, a higher DNA yield could be achieved with the agent according to the invention (binding buffer and wash buffer without alcoholic component) than with classical buffers containing alcoholic components. Fig. 1 shows the analysis of DNA on the tape station (Agilent) using "classical" extraction reagents with alcoholic components. The DNA yield was 3.3 µg. Fig. 2 shows the analysis of DNA on the tape station (Agilent) using the binding buffer according to the invention without alcoholic components. The DNA yield was 4.5 µg. QUOTES INCLUDED IN THE DESCRIPTION This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature US 5,705,628

[0013] US 5,898,071

[0013] EP 2163621

[0014] WO 2015 / 188994

[0015]

Claims

Method for extracting DNA from biological samples after possible homogenization and lysis of the samples, comprising the steps: a) binding the DNA to a solid phase using a binding buffer; b) repeatedly washing the DNA bound to the particles using a washing buffer; c) drying the particles; and d) elution of the DNA, wherein neither the binding nor the washing buffer comprises flammable aliphatic alcoholic components nor chaotropic salts. The method is characterized by the fact that both the binding and the washing buffer have a combination of water-soluble polymers and water-soluble salts as active components. The method according to claim 1, characterized in that the water-soluble polymers are water-soluble polyethers, preferably PEG. Method according to claim 2, characterized in that PEG with a molar mass between 4,000 and 20,000, preferably PEG 6,000, is used. Method according to any one of claims 1 to 3, characterized in that alkali or alkaline earth salts, preferably sodium chloride or magnesium chloride, are used as water-soluble salts. The method according to claim 4, characterized in that the concentration of the water-soluble alkali or alkaline earth salts is between 50 mM and 2.5 M, preferably between 100 mM and 900 mM. Method according to one of claims 1 to 5, characterized in that the same water-soluble polymer is used for both the binding buffer and the washing buffer. The method according to claim 6, characterized in that the same water-soluble polymer is used for the washing buffer as for the binding buffer, but in diluted form, preferably 2-10 times dilution, particularly preferably 3-5 times dilution. Method according to claim 6, characterized in that the salts for the washing buffer are present in a diluted form compared to the binding buffer, preferably 2-10 times dilution, particularly preferably 3-5 times dilution. Method according to any one of claims 1 to 8, characterized in that magnetic particles, preferably para-magnetic particles, are used as the solid phase. Method according to any one of claims 1 to 9, characterized in that the binding buffer additionally contains proteinase K and / or a detergent, preferably polysorbate. Method according to one of claims 1 to 10, characterized in that the washing buffer is a diluted variant of the binding buffer. Method according to one of claims 1 to 11, characterized in that it runs as an automated workflow. Kit for carrying out the method according to one of claims 1 to 12, comprising at least one binding buffer, one washing buffer according to one of claims 1 to 11 and corresponding equipment. Use of the method according to any one of claims 1 to 12 or of the kit according to claim 13 for the extraction of DNA from complex biological samples, in particular from plant components. Use according to claim 14 for the extraction of DNA from plant seeds.

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