Methods for preserving nuclear nuclei

Abstract

To provide methods and kits which can be used to store nuclei at ambient temperature with advantage of: reducing equipment capital and running expenditure; and simplifying workflows.SOLUTION: According to a first aspect of the present invention, there is provided a method for storing cell nuclei including: i) selectively lysing cytoplasmic membranes and a small proportion of the nuclear membranes present in a cellular sample to leave a large proportion of the cell nuclei intact; ii) collecting the cellular sample on a solid support; iii) washing the solid support; and iv) storing the intact nuclei on the solid support at ambient temperature.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid preservation, and particularly to the long-term preservation of nuclei and the recovery of nucleic acids. The present invention provides methods and kits that can be used to capture nuclei, store them at ambient temperature, and isolate nucleic acids by passive washing with a washing buffer. The present invention is applicable to the long-term preservation and easy processing of nucleic acids, and is particularly useful for genotyping, diagnosis, and forensic investigations.

Background Art

[0002] Prior to extracting and isolating DNA or RNA in a form suitable for use in genetic analysis such as polymerase chain reaction (PCR), long-term storage, transportation, and storage of nucleic acids on filter paper or chemically modified matrices are well-known techniques for preserving genetic material. Thus, European Patent Application Publication No. 1563091 (Smith et al., Whatman) relates to methods for preserving nucleic acids from samples such as cells or cell lysates. The nucleic acids are isolated and stored for long periods at room temperature and humidity on a wide variety of filters and other types of solid supports or solid-phase media. This document also describes methods for storing nucleic acid-containing samples on a wide range of solid support matrices within tubes, columns, or multiwell plates.

[0003] International Publication No. 9003959 (Burgoyne) describes a cellulose-based solid support for storing DNA (including bound DNA), comprising a solid matrix having a compound or composition that prevents degradation of the DNA incorporated within or adsorbed onto the matrix. This document also discloses methods for storing DNA using a solid medium, methods for recovering DNA, and methods for using DNA in situ.

[0004] U.S. Patent No. 5705345 (Lundin et al.) describes a method for preparing nucleic acids by lysing a cell-containing sample to release nucleic acids, and then treating the sample with cyclodextrin to neutralize the extractant. The advantage of this method is that it eliminates the need for a separation step required to remove the lysis reagent.

[0005] UK Patent Application Publication No. 2346370 (Cambridge Molecular Technologies Ltd) describes applying a nucleic acid-containing cell sample to a filter. A method is disclosed that includes lysing cells, and then retaining nucleic acids on a filter while removing contaminants.

[0006] International Publication No. 9618731 (Deggerdal) describes a nucleic acid isolation method comprising the steps of conjugating a sample to a solid support, contacting the sample with a washing agent, and subsequently isolating the nucleic acid.

[0007] International Publication No. 0053807 (Smith) discloses a medium for storing and dissolving samples containing genetic material, which is capable of elution and analysis. This medium is coated with a dissolving reagent and optionally with a weak base, a chelating agent, a surfactant, or uric acid.

[0008] Ambient storage of biological samples is seen as an excellent alternative to cryogenic storage. FTA(trademark) (GE Healthcare) can be used to store DNA in small sample sizes of approximately 100-200 ng. However, there is a growing need to store larger sample volumes at ambient temperature. Several international prospective studies are recruiting thousands of participants to investigate the association between living environment, lifestyle, and the genetic features of disease development. Examples include the EPIC prospective study on diet and cancer, and the Canadian Partnership for Tomorrow Project. These studies rely on detailed analysis of participants at the time of disease onset and subsequent DNA analysis after diagnosis of a major fatal disease at some point in the future. When conducting large-scale cohort studies, a moderate blood volume of 4 ml or more is required to genetically analyze samples using advanced modern techniques. Ideally, the sample should contain material that allows for further investigation of other important molecules (e.g., long non-coding RNA). Therefore, there is a need for means of storing large quantities of cell nuclei containing DNA, RNA, and numerous other proteins at ambient temperature for current and future analytical studies.

[0009] Previous nuclear capture devices included Nuclitip (GE Healthcare; U.S. Patent No. 5,447,864, described by Kenrick et al.). This device consists of a microfilament weave in the tip of a pipette that handles up to 10 ml of fresh blood. The blood undergoes controlled lysis; the cell membrane is lysed, leaving most of the nuclear membrane intact. The sample is filtered before entering the tip, and a planar-processed membrane is placed outside the Nuclitip pipette tip to completely cover the tip opening, so that the DNA and nuclei present in the sample bind to the filter. The pipette tip is then washed with phosphate-buffered saline (PBS) to remove any contaminants. U.S. Patent No. 5,447,864 describes a method for separating the cellular components of a cell using the nuclitip method and the possibility of preserving nuclei for extended periods on a membrane below -20°C, and for shorter periods when maintained at 4°C. However, this literature neither discloses nor suggests the long-term preservation of cell nuclei at ambient temperature. In addition, this literature suggests using standard nucleic acid extraction techniques (including the use of surfactants) to dissolve the nuclear membrane.

[0010] Therefore, an improved and simplified method is needed for capturing and storing large quantities of nucleic acids (including DNA and RNA) at ambient temperature. The present invention addresses this problem and provides a method and kit that can be used to store and isolate nucleic acids from solid supports, particularly cellulosic supports, in a single step. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 2004 / 033470 [Overview of the project] [Problems that the invention aims to solve]

[0012] According to a first aspect of the present invention, a method for preserving a cell nucleus, i) Selectively lyse a portion of the cytoplasmic membrane and nuclear membrane present in the cell sample, leaving the majority of the cell nucleus intact. ii) Collecting the cell sample onto a solid support, iii) Cleaning the solid support, and iv) Preserving intact nuclei on a solid support at ambient temperature. A method including this is provided. [Means for solving the problem]

[0013] Intact nuclei can be stored at ambient temperature for 1 day, 1 week, 1 month, 2 months, 6 months, 12 months, 1 year, 2 years, 5 years, 10 years, 15 years, or 20 years.

[0014] In one embodiment, the intact nucleus contains nucleic acids such as DNA or RNA.

[0015] In another embodiment, the method further includes immersing the solid support in an organic solvent after step iii) and before the storage step iv). Suitable organic solvents include, but are not limited to, alcohols such as ethanol or isopropanol.

[0016] In a further embodiment, the solid support is air-dried before step iv).

[0017] In one embodiment, a dissolving reagent is added to a solid support, which is then dried in situ to preserve the nucleic acid.

[0018] In another embodiment, the dissolving agent includes an anionic surfactant or a detergent. Examples of anionic surfactants include sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate, sodium laureth sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, and sodium stearate.

[0019] In a further aspect, the cell sample has a volume of more than 1 ml. Optionally, the cell sample can have a volume of more than 5 ml. Optionally, the cell sample can have a volume of more than 10 ml.

[0020] In one aspect, the solid support is a permeable membrane.

[0021] In another aspect, the solid support is selected from the group consisting of a polyester membrane, a polyamide membrane, a polycarbonate membrane, and a cellulose membrane.

[0022] In a further aspect, the solid support is a polyester membrane.

[0023] In one aspect, the solid support is attached to a pipette tip. In this aspect, the solid support can be a polyester membrane.

[0024] In another aspect, the pipette tip is a Nuclitip.

[0025] In a further aspect, the method includes a further step v) of recovering nucleic acid from the nuclei, for example, by lysing the nuclei on the membrane.

[0026] In one aspect, the membrane is subject to passive washing with a washing buffer.

[0027] In another aspect, the nucleic acid is recovered by centrifugation.

[0028] In a further aspect, step iii) is carried out by flowing a washing solution over the solid support.

[0029] According to a second aspect of the present invention, there is provided a kit for storing cell nuclei at ambient temperature, the kit comprising a solid support and instructions for performing the method described herein. The kit may optionally include a lysis reagent for lysing the cytoplasmic membrane and the nuclear membrane.

Brief Description of the Drawings

[0030] [Figure 1] N1 and N2 are high molecular weight DNA recovered from Nuclitip by short-time spinning in a microcentrifuge, M is a DNA molecular weight marker, and S1 and S2 are solutions removed from the respective Nuclitip membranes after protease digestion and before gentle washing. It should be noted that very small amounts of nucleic acid (fluorescence of ethidium bromide) are observed in S1 and S2, demonstrating that high molecular weight DNA was retained on the membrane during digestion. Gentle washing is effective and does not remove retained DNA. Short-time high-speed spinning in a microcentrifuge is effective in removing the viscous high molecular weight DNA shown in lanes N1 and N2. [Figure 2] Figure 2 shows the results for both N1 and N2 DNA samples treated using the nuclitip method described in the present invention. The photographs show uncut DNA, Roche control DNA digested with Hind III and BamH1, and the N1 and N2 samples. The results shown demonstrate that enriched nuclei can be stored at room temperature without damaging the genomic sample. [Modes for carrying out the invention]

[0031] definition To provide a clearer and more concise description and reference to the subject matter described in the claims, the following definitions are provided for the specific terms used in the following description and the attached claims. Throughout this specification, examples of specific terms should be considered non-limiting examples.

[0032] As used herein, the term “preservation” is used to describe the process of maintaining or preserving nucleic acids in a stable state.

[0033] The term "dissolution" is used herein to describe the process of rupturing, denature, or puncturing a structure such as a cell membrane or small cell membrane, including the nuclear membrane.

[0034] As used herein, the term “cell sample” is used to refer to a liquid sample containing cellular material. Cellular material may originate from any suitable eukaryote containing a nucleus, such as humans, animals, plants, birds, insects, and fish. Cellular material may be, for example, blood, saliva, urine, or plasma.

[0035] As used herein, the term "ambient temperature" means a temperature within the range of 10°C to 30°C, preferably 15°C to 26°C, and more preferably 18°C ​​to 23°C.

[0036] As used herein, the term “solid support” includes, but is not limited to, cellulose-based products, cellulose, cellulose acetate, glass fibers, polyester, polyamide, and polycarbonate, or any combination thereof. The solid support of the present invention may be porous.

[0037] As used herein, "nucleic acids" refers to RNA (e.g., mRNA) and DNA (e.g., This refers to all forms of DNA or RNA analogs, or mixtures thereof, created using genomic DNA, recombinant RNA and DNA molecules, or nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded. Chemicals and materials used The following is a list of chemical substances and their suppliers: Human whole blood collected in an EDTA tube (Tissue Solutions Ltd).

[0038] Sucrose / Triton erythrocyte lysis buffer (100 ml) was prepared by mixing the following ingredients, and then diluted to 100 ml with sterile distilled water: - Sucrose (Sigma, S7903) - 11.0g -10mM Tris pH 8.0 (1M stock solution at 10ml / L) (Sigma, T3038) - 1ml -5mM MgCl2(1.02g / L MgCl2 6H2O)(Merck,1.05833)-100mg -1%(w / v)Triton x-100(10g / L)(Sigma,T9284)-1g; Phosphate-buffered saline (PAA, H15-002); Nuclitip (GE Healthcare); Illustration Tissue and Cells GenomicPrep mini spin kit (GE Healthcare, 28-9042-74); Tris EDTA buffer: 10mM Tris pH8.0 and 1.0mM EDTA (Sigma, T9285-100ml); 0.8% agarose / TAE gel (Affymetrix, part #75817); ethidium bromide (Sigma, E1510); control genomic DNA (Roche, 11691112001); Hind III and Bam H1 restriction endonucleases and their respective restriction buffers (New England Biolabs, R0104T and R0136T); The following experimental results and examples are shown as examples, not limitations: Experimental results DNA measurement from nuclitip using gel electrophoresis.

[0039] Two ml of blood was collected and added to an equal volume of red blood cell lysis buffer. The sample was swirled and mixed briefly, then left on the bench for three minutes. Samples were collected using multiple nuclitips to capture nuclei. Blood was collected until occlusion of the polyester membrane became apparent, by delaying the aspiration and discharge of lysed blood as it was pumped at all points throughout the membrane.

[0040] The tip was sequentially washed with fresh phosphate-buffered saline by pressing down the pipette plunger and pumping the liquid into every part of the membrane until the membrane was substantially free of heme.

[0041] The washing solution was drained, the tip was removed from the pipette, and the remaining liquid was removed by briefly spinning it for 10 seconds in a benchtop microcentrifuge (1200 rpm, radius approximately 7 cm). The tip was then placed in a desiccator cabinet overnight for further drying.

[0042] The following day, three dried tips containing nuclei were stored directly in 1.5 ml microcentrifuge tubes, and the other three tips were treated with 100 μl of anhydrous ethanol and pipetted directly onto the membrane. The tubes were capped and stored in a laboratory cabinet at ambient temperature (approximately 15°C to 25°C) for 60 days. Sample processing after long-term storage of nuclitip.

[0043] Ethanol was removed from the ethanol-soaked tip by decanting and pulse spinning to dry the film.

[0044] Genomic DNA was lysed, digested, and removed from dried nuclei trapped on the membranes of both ethanol-treated and dried chips using lysis solution I from the Illustra Tissue and Cells GenomicPrep mini spin kit.

[0045] Lyophilized proteinase K (3 mg) was dissolved in 1.5 ml of sterile distilled water to prepare a 20 mg / ml protease solution, and 20 µl of this solution was added to 100 µl of lysis buffer 1. 50 µl of protease containing the lysis buffer was pipetteed directly onto a dry Nuclitip film. The tube was then closed and placed in a 55°C water bath for 30 minutes.

[0046] Next, open the tube and add approximately 300 μl of TE -1 The buffer was added to the bottom of the tube slide and inside the tip barrel to wash away the lysis buffer and digested peptide. The tube was left on the bench for 2 minutes, then the washing solution was decanted. This process was repeated twice, and then 50 μl of TE was added. -1 Buffer solution was added to each tube, and the tubes were spun in a microcentrifuge at maximum speed (12,000 rpm) for 1 minute to collect genomic DNA.

[0047] The DNA solution recovered from each device was a highly viscous, colorless, transparent solution of approximately 70 μl. The solution on the upper side of the digested membrane was aspirated and held before adding the first washing solution. This solution was electrophoresed on a 0.8% agarose / TAE gel along with the recovered gDNA samples and stained with ethidium bromide (Figure 1). For samples S1 and S2, only a small amount of nucleic acid staining was detected, indicating that a small amount of nucleic acid was released from the membrane during proteolysis. After gentle washing, purified high molecular weight DNA could be recovered by short-time high-speed spinning in a microcentrifuge. Therefore, for N1 and N2, the described method was effective in collecting the majority of nucleic acid from the membrane, as indicated by the strong fluorescence of ethidium bromide on the gel.

[0048] To determine the concentration and quality of the DNA, small samples were taken from each tube after prolonged vortexing (to reduce viscosity) and read using a NanoVue Plus spectrophotometer. The results are shown in Table 1 below, demonstrating the high quality and concentration of the nucleic acids present. The genomic DNA concentration in N1 was 134 ng / ul, and the genomic DNA concentration in N2 was 86 ng / ul.

[0049] [Table 1] Enzymatic digestion of genomic DNA recovered from dried nuclei.

[0050] In a total volume of 20 µl, 10 µl each of samples N1 (1.5 µg) and N2 (1 µg), as well as the control sample Roche DNA (2 µg), were digested with either 40 units of BamH1 or Hind III endonuclease and stored at 37°C for 3 hours. See Figure 2. As can be seen, fragmented and unfragmented DNA were electrophoresed against DNA markers of known molecular weight on a 0.8% agarose / TAE gel and stained with ethidium bromide. The results shown demonstrate that enriched nuclei can be stored at room temperature without damaging the genomic sample. The developed method makes it possible to produce high-quality high molecular weight genomic DNA with concentrations greater than 50 ng / µl and an A260:A280 absorbance ratio of approximately 1.8, suitable for downstream operations such as restriction endonuclease digestion.

[0051] While preferred exemplary embodiments of the present invention have been described, those skilled in the art will recognize that the present invention can be carried out in ways other than those described, which are provided for illustrative purposes only and not as limitations. The present invention is limited only by the following claims.

Claims

1. A method for preserving the cell nucleus, i) Selectively lyse a portion of the cytoplasmic membrane and nuclear membrane present in the cell sample, leaving the majority of the cell nucleus intact. ii) Collecting the cell sample onto a solid support, iii) Cleaning the solid support, and iv) Preserve intact nuclei on a solid support at a temperature of 10°C to 30°C. A method that includes a fixative and does not include the step of fixing the cell nucleus by adding a fixative.

2. The method according to claim 1, wherein the intact nucleus contains nucleic acid.

3. The method according to claim 1 or 2, wherein the solid support is air-dried before step iv).

4. The method according to claim 1 or 2, comprising adding a dissolving reagent to a solid support and subsequently drying it in situ to preserve the nucleic acid.

5. The method according to claim 4, wherein the dissolving reagent comprises an anionic surfactant or a detergent.

6. The method according to claim 5, wherein the anionic surfactant is sodium dodecyl sulfate (SDS).

7. The method according to any one of claims 1 to 6, wherein the cell sample has a volume of more than 1 ml.

8. The method according to any one of claims 1 to 7, wherein the cell sample has a volume of more than 5 ml.

9. The method according to any one of claims 1 to 8, wherein the cell sample has a volume of more than 10 ml.

10. The method according to any one of claims 1 to 9, wherein the solid support is a permeable film.

11. The method according to any one of claims 1 to 10, wherein the solid support is selected from the group consisting of a polyester film, a polyamide film, a polycarbonate film, and a cellulose film.

12. The method according to any one of claims 1 to 11, wherein the solid support is a polyester film.

13. The method according to any one of claims 1 to 12, wherein a solid support is attached to a pipette tip.

14. The method according to claim 13, wherein the solid support is a polyester film.

15. The method according to any one of claims 1 to 14, further comprising step v) recovering nucleic acids from the nucleus.

16. The method according to claim 15, wherein nucleic acids are recovered by lysing nuclei on a membrane.

17. The method according to claim 16, wherein the membrane undergoes passive washing with a washing buffer.

18. The method according to any one of claims 14 to 17, wherein nucleic acids are recovered by centrifugation.

19. The method according to any one of claims 1 to 18, wherein step iii) is carried out by flowing a cleaning solution onto a solid support.

20. A kit for use in the method according to any one of claims 1 to 19, comprising a solid support and instructions for performing the method according to any one of claims 1 to 19.

21. The kit according to claim 20, further comprising a dissolving reagent.

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