JP7608697B2 - Sample preparation method for DNA sampling and suitable container therefor - Google Patents

Description

This article relates to DNA extraction techniques, particularly those that can be applied when collecting samples from the field.

Patent Document 1 (Utility Model Registration No. 3211475) discloses an invention relating to a tissue disruption tube (see Figure 7) that includes a tube body with an opening, a bottom wall, and a cylindrical side wall, and a lid member that closes the opening in an openable and closable manner, and is surrounded by the inner surfaces of the bottom wall and side wall of the tube body and the lid member to form a disruption chamber that can contain tissue as a specimen, a liquid that elutes genetic material, and hard spheres that disrupt the tissue.

Patent Document 2 (JP 2018-148847 A) proposes a DNA sample collection device that can suppress the proliferation of germs during transportation and storage and also prevents DNA samples from being damaged during transportation, and that includes a container, a lid that seals the internal space of the container, a rod body attached to the lid and having a collection portion at its tip, and a desiccant filled in the tip of the internal space, where the desiccant is fixed to the tip of the internal space by being blocked from the base end with a covering member, the covering member having a two-layer structure consisting of a piece of paper on the tip side and a gasket on the base end side, the gasket being fixed to the container by pressing the corners of the paper piece against the inner surface of the container, and the gasket having an air hole.
Patent Document 3 (JP Patent Publication No. 2020-505048) proposes an invention relating to a bead crushing system that includes a sample tube, beads, and a dried blocking agent, and is used to extract nucleic acids from cells that contain nucleic acids.

Utility Model Registration No. 3211475 JP 2018-148847 A Patent Publication No. 2020-505048

Plant samples collected in the field are stored and dried, and when it is time to disrupt the cells, the biological tissue is transferred to a container used in a bead-type cell disrupter, where zirconia beads are inserted to disrupt the cells, which creates a risk of sample contamination. When disrupting cells, the biological tissue needs to be transferred from one sealed container to another and labeled, which creates a risk of mix-ups and misidentification.
The present invention aims to develop a technology that can reduce the labor required from collection to crushing.

1. A method for preparing a sample for DNA sampling, comprising storing a collected biological tissue sample in a container containing a desiccant/disintegrator, drying the sample, and then disintegrating the dried sample using the container.
2. Prepare a container containing a granular desiccant/disintegrator and place the collected biological tissue sample in the container;
Recording sample collection data on the container;
The collected samples are dried and stored.
2. The method for preparing a sample for DNA sampling according to 1., characterized in that the cells are disrupted using a cell disrupter using a container containing the dried collected sample.
3. The method for preparing a sample for DNA sampling according to 1. or 2., wherein the desiccant/disintegrating material is silica gel granules.
4. The method for preparing a sample for DNA sampling according to any one of 1. to 3., wherein the collected sample is a wild organism, a cultivated organism, or a captive organism.
5. A container containing silica gel granules for storing, drying and crushing biological tissue samples.
6. A sample of collected biological tissue stored in the container described in 5. for DNA analysis.

1. The sample can be collected, dried, and crushed in the same container, which allows DNA to be extracted quickly, easily, and inexpensively. The sample is suitable for biological tissues with cell walls, such as filamentous fungi, basidiomycetes, and plants.
There is no need to replace collected samples or to repeatedly label or record samples, so there is no need to generate labor or errors, and sampling for DNA analysis can be carried out consistently.
2. When disrupting cells, there is no need to transfer biological tissue from one sealed container to another or to label it again, which reduces the work time.
Since a granular desiccant such as silica gel is placed in the container beforehand, there is no need to open the lid until crushing, so biological tissues can be stably dried and stored in the container even if DNA extraction cannot be performed immediately.
By not transferring samples during the homogenization process, the risk of sample contamination, which is the most important issue in experiments using DNA, can be reduced. It is also possible to add silica gel particles and other beads as auxiliary homogenization materials, and it is also possible to add homogenization beads during the homogenization process.
3. Since the granular desiccant used for drying is used for crushing, stainless steel beads, zirconia beads, etc. that were previously used are no longer necessary, which reduces material costs.
4. This invention is suitable for investigating the distribution of varieties by region and for investigating cultivated varieties. In greening reclaimed land and collapsed land, it is recommended to use native species of the land and prevent the use of foreign species from other regions. This invention is suitable for identifying native species and identifying greening materials owned by landscape gardeners. It is also suitable for DNA analysis required by the Seed and Seedlings Act to identify registered varieties and infringing varieties.
5. They may be stored to confirm initial vegetation materials or as basic samples for registered varieties.
In particular, because collection and storage can be done in a single container, the sealed container can guarantee identity, making it suitable as identification material for registered varieties. Cultivated varieties may mutate over repeated generations, and identity with the plant body at the time of registration can be an issue in disputes over infringement under the Seed and Seedlings Act, so DNA comparison can be used as an effective means of resolving the issue.

1A to 1C are diagrams showing example steps of a sample preparation method for DNA sampling. FIG. FIG. A diagram showing an example of silica gel granules as a desiccant and crushing agent Figure showing the results of DNA extraction test Conventional process diagram FIG. 1 is a diagram showing an example of a conventional crushing tube described in Patent Document 1. The illustrated reference numerals are those in Patent Document 1.

The present invention relates to a method for drying and disrupting cells, which uses a granular desiccant to dry and preserve biological tissue and then disrupt it when preserving and disrupting biological tissue having cell walls, such as filamentous fungi, basidiomycetes, and plants, for the purpose of DNA extraction, etc. The present invention is suitable for collecting and analyzing samples required for investigating the distribution of species and varieties in nature and for identifying varieties cultivated in fields.
The present invention relates to a method for preparing samples for DNA sampling, which comprises storing a collected biological tissue sample in a container containing a desiccant/disintegrator, drying the sample, and then disintegrating the dried sample using the container. The present invention also relates to a container for storing, drying, and disintegrating collected biological tissue samples, which contains silica gel granules used in the method.
The present invention develops a desiccant that can be used as a homogenizer, and allows the drying and preservation of samples collected outdoors, followed by a series of homogenization processes, to be carried out in the same container, enabling DNA to be extracted from a large number of specimens quickly and easily. Furthermore, the management of sample data, such as copying data, can be completed in one go, reducing the risk of misprinting and mix-ups, saving labor and improving the reliability of data.

A general description of how DNA is sampled from a sample is provided.
Although DNA is relatively stable, in dead cells it is decomposed and fragmented rapidly by DNA degrading enzymes (nucleases) present in biological tissues. Therefore, in order to extract high-quality DNA, it is necessary to use living biological tissues or to preserve the tissue by suppressing the activity of DNA degrading enzymes until the DNA extraction. When collecting DNA from outdoors, such as from green plants, it is difficult to immediately perform DNA extraction work, so the biological tissue is usually preserved by suppressing the activity of DNA degrading enzymes. Methods for preserving the tissue by suppressing the activity of DNA degrading enzymes include drying, freezing, and preservation in ethanol. When using outdoor samples, freezing is difficult, so drying and preservation in ethanol are mainly used. It is common to preserve plants by drying and animals by ethanol.
The next step in extracting DNA from the cell samples brought back to the laboratory in a dry state is to crush the dried biological tissue. DNA in cells is bound to proteins (histones) and is surrounded by the nuclear membrane and cell membrane. In addition, plants and algae have cell walls that are mainly composed of cellulose and lignin on the outside of the cell membrane, so when extracting DNA, it is first necessary to crush the biological tissue and physically destroy the cell walls. One method of crushing biological tissue is to use liquid nitrogen and a mortar. Another method is to crush cells using a bead-type cell crusher that can process large amounts of samples. Cell crushing using a bead-type cell crusher is a method in which a container containing biological tissue and beads is shaken at high speed, and the biological tissue and beads collide with each other to crush the biological cells.

FIG. 6 shows the conventional process. In the first step, materials such as leaves, stems, and roots are collected during field surveys in mountains, etc., and stored in a container such as a collection bag. The collection site, date, time, part of the material collected, species, etc. are recorded in the field notebook. In the second step, all or part of the collected materials are transferred to an airtight container, dried, and stored. In the third step, the parts necessary for DNA analysis are selected from the stored materials and placed in a crushing container, cell walls are crushed, and DNA is extracted using PCR or the like, and samples for analysis are prepared. In this series of steps, it is important to record and maintain the consistency of the data. In surveys of variation within the same species or surveys of cultivated varieties, there are many cases where it is difficult to distinguish between varieties based on the appearance of leaves, etc., and crushed materials make it difficult to distinguish between varieties, so recording is important for accurate analysis. Handling a large number of samples requires carefulness and time to record data.
According to the present invention, the series of steps from sample collection to crushing, which are necessary as preparatory steps prior to sampling of DNA, can be completed in one container, and recording only needs to be done once.

An example of the steps of a sample preparation method for DNA sampling is shown in FIG.
This process is a method for extracting DNA (nucleic acid) from biological tissues, in which the processes of collecting, storing, drying, and crushing biological cells are carried out using a single container and a single type of granular desiccant, without opening the container between the series of processes, and the granular desiccant is used as a crushing material.

In the first step, samples collected outdoors, such as in the field or on cultivated land, are stored on the spot in a container containing a desiccant/crushing material, and data is displayed on the container.
The containers are sealed with a granular desiccant/crushing agent already inside, and are then brought to the collection site. At this time, barcodes or two-dimensional barcodes are attached to the containers to ensure sample traceability, reducing the risk of mixing up samples and making information management easier.
At the sampling site, the biological tissue is cut into pieces that will fit into a container, and then placed in a container containing a desiccant and crushing agent, which is then sealed. A record sheet for identifying the sample is attached to a visible location, such as the side of the container or the top of the lid. Identification information, such as the collection date, collection location, species name, and sample number, is recorded on the record sheet.
Although it depends on the amount of sample, it will dry in about 6 hours and the activity of DNA degrading enzymes will be suppressed.
The samples are dried in the container and stored until they are crushed. They can be stored at room temperature in a dark place for several years or more.

The second step is a step of crushing the sample using a container containing the sample.
The dried and stored container is then subjected to shaking and disruption in a bead-type cell disrupter at 2000-5500 rpm for 90-120 seconds. Any shaking and disrupting machine in practical use can be used. In the present invention, silica gel particles having a desiccant function are used as the disruption beads. In the present invention, conventional beads can also be mixed in as auxiliary disruption beads.
Conventionally, mineral beads, ceramic beads, glass beads, metal beads, zirconium beads, zircon beads, zirconia beads, etc. have been used as crushing beads.

The third step is a DNA analysis step, but the present invention is limited to the second step. PCR is applied, and DNA is analyzed using electrophoresis or the like, and the analysis method is a conventional method.
For example, DNA is extracted using the CTAB method, glass adsorption method, etc. To briefly explain the application of the CTAB method as an example, (1) the disrupted sample is added to a CTAB solution to dissolve the cells, (2) lipids are dissolved and proteins are denatured using chloroform and isoamyl alcohol, (3) the upper layer containing DNA is collected (the lower layer is separated because of the presence of an organic layer and a silica gel layer), and (4) the DNA is collected by performing ethanol precipitation.

Next, the components of the present invention will be described.
<Biological tissue samples>
The biological tissue is suitable for biological tissues having cell walls, such as filamentous fungi, basidiomycetes, and plants. It is suitable for targets that require cell wall disruption to prepare for DNA sampling. In particular, plants are suitable, and among plants, leaves are particularly suitable.
It is suitable for samples collected in the field and for cultivars for variety identification. It can also be stored for a long time, making it suitable as a basic material for preservation samples, and when necessary, DNA can be analyzed and compared. For example, it can be used to identify registered varieties and compare them with suspected infringing plants.

<Container>
The container must be capable of maintaining a dry, sealed condition. If an existing bead-type cell disrupter is used, the container must be compatible with the bead-type cell disrupter.
The container is a tube with an opening, a bottom wall, and a cylindrical side wall, has a gasket that can seal in granular desiccant, is resistant to damage during crushing operations, and can be sterilized.
An example of the container 1 is shown in Fig. 2. It is composed of a container body 11 and a lid 12 provided with a packing 13 so as to be airtight, and it is advisable to attach an ID code such as a barcode 42 to the container body 11. A particulate desiccant/crushing material 3 is placed inside the container body 11 beforehand. Zirconium beads or the like can also be mixed in as auxiliary crushing beads.
When samples are to be stored for a long period of time, a packing material and a lid structure that can provide airtightness for a long period of time are used.

<Desiccant and crushing agent>
Granular desiccants are spherical materials with a certain strength and water absorption effect. Examples include silica gel, decyl clay, and synthetic zeolite. Granular desiccants are suitable for heat sterilization. For example, by heating at 160-200°C for 30 minutes to 2 hours, microorganisms, enzymes such as DNA degrading enzymes, and proteins are thermally denatured, resulting in dry heat sterilization.
Silica gel can dry most liquids and gases, including water, and is made of silicon dioxide in the form of transparent beads. It does not melt or swell even when it absorbs moisture. The raw material, silicon dioxide, is a highly safe substance and is often used as a food desiccant. Silica gel with indicator functions, colored with cobalt chloride, which changes color when it absorbs moisture, is also available on the market, but this colors the buffer during DNA extraction, reducing the visibility of the sample, so pure silica gel without coloring agents is more suitable as a desiccant and crushing material.
The conditions for granular silica gel that can obtain both drying and crushing effects are that the diameter is 2.0 mm to 10.0 mm and the amount is 0.5 to 60 times the amount of the biological tissue.
Deciclay is a desiccant made from natural clay minerals such as bentonite. Its moisture absorption performance does not change much with the humidity of the outside air, and its size and shape do not change either. It also has excellent chemical resistance, and can absorb not only moisture but also odorous components. It has higher moisture absorption at low humidity than silica gel.
Synthetic zeolite is a desiccant that comes in sheet or tablet form.

<Crushing device>
As the homogenizer, a rotary shaking homogenizer that is in practical use can be used. For example, there is an example disclosed in JP-A-2000-023660. In addition, there is a bead type cell homogenizer manufactured by Tommy Kogyo Co., Ltd.

<About collection and sealing operations>
Figure 3 shows an example of collecting samples using a container containing a desiccant/crushing material.
For example, leaves of Camellia japonica are collected at a sampling point in the field and cut to a size that will fit into container 1 to provide collected sample 2. Collected sample 2 is then placed into container body 11 containing desiccant/crusher 3, and lid 12 is immediately tightened. Lid 12 is equipped with packing 13 to provide a tight seal. Record sheets 41 for identifying samples are attached to visible locations such as the side of container body 11 and the top of lid 12. Identification information such as collection date, collection location, species name, and sample number are recorded on the record sheet.
The container 1 is provided with a bar code, and is identified by an individual container ID and linked to the contents recorded on the recording sheet, so that the sample only needs to be managed once.

<Test Example>
A crushing test was carried out using silica gel particles, and a test was carried out to confirm the DNA extraction efficiency.
Lilium longiflorum leaves, which have a relatively hard cuticle, were used and crushed using silica gel particles of φ1.5 mm, φ2.0 mm, and φ4.0 mm. The leaves were crushed using a bead-type cell crusher (Tomy Industries MS-100R) at a rotation speed of 5,500 rpm, and a DNA extraction efficiency test was performed. An example of silica gel particles is shown in Figure 4.
Zirconia particles having a diameter of 2.0 mm were used as crushing beads for comparison.
The results of the DNA extraction test are shown in FIG.
DNA could be extracted from silica gel particles of any size. It was confirmed that the φ2.0 mm and φ4.0 mm silica gel particles were capable of extracting DNA equal to or greater than that from φ2.0 mm zirconia particles.

1 Container 11 Container body 12 Lid 13 Gasket 2 Sample 3 Desiccant/crusher 31 Silica gel granules 4 Sample data 41 Recording sheet 42 Barcode

Claims (5)

A method for preparing a sample for DNA sampling, comprising storing a collected biological tissue sample in a container containing a granular desiccant/crushing material, drying the sample, and then crushing the dried sample using the container. A container containing a granular desiccant and crushing agent is prepared, and the collected biological tissue sample is placed in the container;
Recording sample collection data on the container;
The collected samples are dried and stored.
2. The method for preparing a sample for DNA sampling according to claim 1, further comprising the step of disrupting cells with a cell disrupter using a container containing the dried collected sample. 3. The method for preparing a sample for DNA sampling according to claim 1, wherein the granular desiccant/disintegrating material is silica gel granules. A sample preparation method for DNA sampling according to any one of claims 1 to 3, characterized in that the collected sample is a wild organism, a cultivated organism, or a captive organism. A container for storing, drying and crushing biological tissue samples containing silica gel granules as a desiccant and crushing material .