New technology for preserving DNA (deoxyribonucleic acid) sample at room temperature

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a novel technology for preserving a DNA sample at room temperature. The technology selects calcium sulfate dihydrate as a stabilizer for DNA sample preservation, the preparation process is simple and easy to operate, the source is convenient, the cost is low, and the preservation effect is good. It is found that a DNA sample is mixed with a calcium sulfate aqueous solution, the content of DNA / CaSO4 is not higher than 25% (w / w), a solid mixture is prepared through a freeze drying technology, stable preservation of the DNA sample at the room temperature (10-40 DEG C, preferably 20-25 DEG C) can be achieved, the total loss caused by degradation of the DNA sample at the high temperature of 70-95 DEG C is reduced, the DNA degradation rate is improved, and the DNA sample can be stored stably at the room temperature (10-40 DEG C, preferably 20-25 DEG C). The STR sequence of a human genome DNA sample is stabilized; non-accelerated experiments show that the technology can maintain the effective stability of a DNA sample at room temperature for not less than 130 days.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a stabilizer for improving DNA stability and application thereof. Background Art

[0002] Deoxyribonucleic acid (DNA) is an important carrier of biological genetic information, including natural DNA and artificially synthesized DNA.

[0003] The continuous advancement of basic DNA research and the continued expansion of applied research have led to a sharp increase in the demand for the preservation of DNA biological samples. For example, exploration in areas such as pathogen detection, individual identification, disease diagnosis, drug development, synthetic biology, DNA data storage, and biomimetic material design all require the long-term, large-scale, stable, and convenient preservation of DNA samples in vitro.

[0004] To maintain the integrity of DNA structure, its physical and chemical properties, and the stability of its biological activity, DNA samples are typically stored at temperatures no higher than -20°C to protect them from adverse physical, chemical, and biological influences. However, this cryogenic storage strategy has numerous drawbacks, including reliance on specialized cryogenic equipment, significant space requirements, high costs, transportation difficulties, and difficulty for individuals to implement.

[0005] Therefore, it is necessary to explore new DNA sample preservation technologies to ensure the integrity of DNA structure and stable activity, simplify operations, reduce energy consumption, lower costs, and extend the preservation period to meet the DNA sample preservation needs of scientific research and industrial applications. Summary of the Invention

[0006] The present invention proposes a new technology for preserving DNA samples at room temperature, which has high preservation throughput, low cost, and simple operation, and can achieve long-term stable preservation of DNA samples.

[0007] The present invention aims to provide a new room-temperature storage method for DNA samples to address the problems faced by long-term storage of DNA samples, such as reliance on specialized cryogenic equipment, large space requirements, high costs, and difficulty in transportation. This method is simple to operate, low in cost, and flexible in space requirements, enabling long-term stable storage of samples at room temperature.

[0008] The room temperature storage technology of the DNA sample of the present invention includes mixing the DNA sample with a calcium sulfate solution of appropriate concentration, drying the mixture to prepare a solid DNA sample that can be stored at room temperature, and then placing the sample in a sealed container for storage at room temperature.

[0009] The present invention places the solid-state DNA sample in a container and seals it, without the nitrogen filling and deoxygenation steps.

[0010] In some embodiments, the concentration of the aqueous calcium sulfate solution is no higher than 2.08 mg / mL at 25°C, preferably 1 mg / mL.

[0011] In some embodiments, the DNA is double-stranded DNA.

[0012] In some embodiments, the DNA can be obtained by artificial synthesis or extraction, including but not limited to all extraction methods known in the art.

[0013] In some embodiments, the form of the DNA sample includes one or more of a dried solid state, dissolved in TE buffer, or dissolved in enzyme-free sterile deionized water.

[0014] In some embodiments, the room temperature may be 10°C to 40°C, preferably 20°C to 25°C;

[0015] The preservation solution of the present invention has a wider applicable temperature range and can be used to preserve DNA samples in different temperature environments not higher than 50° C., thus having more important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Calcium sulfate preservation solution reduces DNA degradation loss due to high temperature (A. Agarose gel electrophoresis; B. Qubit quantitative detection results)

[0017] Figure 2 : Calcium sulfate preservation solution improves DNA degradation rate

[0018] Figure 3 : Stabilization effect of calcium sulfate stabilizer on STR sequences in DNA samples (A. Sequencing results of DNA samples stored at -20°C; B. Sequencing results of DNA samples in the group without stabilizer; C. Sequencing results of DNA samples in the group with calcium sulfate stabilizer)

[0019] Figure 4 : DNA loading limit of calcium sulfate stabilization (A. Concentration test results; B. Sample degradation rate test results)

[0020] Figure 5 Calcium sulfate maintains DNA stable at room temperature for at least 130 days DETAILED DESCRIPTION

[0021] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

[0022] The chemicals described in the following examples were from China National Pharmaceutical Group Co., Ltd. (Sinopharm Group) or Sigma-Aldrich.

[0023] In this example, human genomic DNA (about 15,000 bp in length) extracted from HEK 293T cells was used as a model to study the effect of a new preservation technology on the preservation of DNA samples.

[0024] Example 1. Preparation of DNA Preservation Samples

[0025] 1. Preparation of Preservative Solution

[0026] Calcium sulfate dihydrate (calcium sulfate dihydrate, molecular formula CaSO4·2H2O, CAS No. 10101-41-4) was accurately weighed and fully dissolved in sterile, enzyme-free deionized water to prepare a calcium sulfate aqueous solution with a concentration not higher than 2.08 mg / mL and stored at room temperature.

[0027] 2. DNA-Calcium Sulfate Freeze-Drying Sample Preparation

[0028] DNA (solid state, or dissolved in TE buffer, or dissolved in sterile, enzyme-free deionized water) was added to the above-mentioned calcium sulfate aqueous solution to make the DNA / CaSO4 content 5-25% (w / w), and a solid mixture was prepared by freeze-drying technology. The freeze-drying conditions were: -40°C for 6 hours, -30°C for 6 hours, -10°C for 30 minutes, 10°C for 30 minutes, and 25°C for 24 hours.

[0029] 3. DNA sample release and recovery

[0030] Dissolve the freeze-dried sample in TE buffer or sterile enzyme-free deionized water, pipette and mix, let stand at room temperature for 30 minutes, and purify the DNA using an adsorption column method or magnetic bead method to recover the preserved sample.

[0031] Example 2. Calcium sulfate stabilizer reduces high temperature degradation loss of DNA samples

[0032] DNA-calcium sulfate freeze-dried complexes were prepared with a DNA / CaSO4 content of 5% (w / w). The complexes were accelerated at 95°C for 36 hours, followed by centrifugation at 12,000 rpm for 10 minutes at room temperature. Dissolved in 60 μL of TE buffer, the sample was purified, and the target fragment content was observed by agarose gel electrophoresis. The Qubit fluorescence quantitative method was used for accurate content determination.

[0033] Agarose gel electrophoresis results showed that ( Figure 1A): After 36 hours of accelerated culture at 95°C, the DNA was severely degraded and the 15,000 bp target fragment could not be detected. However, after adding calcium sulfate as a stabilizer, the degradation reaction was effectively alleviated and the 15,000 bp target fragment could still be clearly detected.

[0034] The results of content determination showed that ( Figure 1 B): After 36 hours of accelerated storage at 95°C, the total amount of DNA sample lost about 90%. After using calcium sulfate as a stabilizer, the total amount of sample loss was greatly improved, with only 30-40% of the original total amount lost, indicating that calcium sulfate as a storage stabilizer significantly reduced the degradation loss of DNA samples.

[0035] Example 3. Calcium sulfate stabilizer improves the degradation rate of DNA samples

[0036] Prepare DNA-calcium sulfate freeze-dried complex with a DNA / CaSO4 content of 5% (w / w) and accelerate at 95°C for 36 hours, then centrifuge at 12000 rpm for 10 minutes, add 60 μl TE buffer to dissolve, and use Quantifiler TM Degradation rate was detected using Trio DNA Quantification reagent.

[0037] The results show that ( Figure 2 ): After 36 hours of accelerated temperature at 95°C, the DNA samples were severely degraded, with the degradation rate increasing by about 4 times; after using calcium sulfate as a stabilizer, the degradation rate was only slightly higher than that of the normal group, indicating that only a small number of samples may have been degraded.

[0038] The principle of using this method to detect fragment degradation rates is as follows: Real-time fluorescence quantitative PCR experiments are used to quantify the presence of multiple copies of a target gene with two amplicons of different sizes. As the degree of degradation increases, the longer target amplicons tend to decrease disproportionately. Therefore, by comparing the concentrations of the large autosomal target (LA, 80 bp) and the small autosomal target (SA, 214 bp), the degradation index (DI) of the sample is calculated using the formula: DI = (SA DNA concentration) / (LA DNA concentration). This indicates the potential degradation of the DNA sample. For undegraded DNA, the amplification efficiencies of LA and SA are similar, and the DI is close to 1. For degraded DNA, the concentration of LA is significantly lower than that of SA due to fragment fragmentation, and the DI is greater than 1. Higher DI values ​​indicate more severe degradation.

[0039] Example 4. Stabilization of STR sites in DNA samples by calcium sulfate

[0040] Prepare DNA-calcium sulfate freeze-dried complex with DNA / CaSO4 content of 5% (w / w), accelerate at 95℃ for 36 hours, then centrifuge at 12000rpm for 10min, add 60ul TE buffer to dissolve, and use DNA identification system 30A performs STR locus sequencing analysis.

[0041] The results show that ( Figure 3 ): After 36 hours of accelerated storage at 95°C, the human genomic DNA sample used was severely degraded and damaged, and the sequencing response values ​​of 27 out of 31 STR loci were significantly reduced. However, after adding calcium sulfate as a preservation stabilizer, the number of STR loci detected in the sequencing map of the sample and the sequencing response value were improved. This shows that the use of calcium sulfate as a preservation stabilizer can effectively maintain the sequence integrity of human genomic DNA and can meet the needs of subsequent individual identity identification applications based on STR loci.

[0042] Example 5. Exploration of the DNA sample loading limit for calcium sulfate stabilization

[0043] Prepare DNA-calcium sulfate freeze-dried complexes of different masses to achieve a DNA / CaSO4 content of 0-25% (w / w). Accelerate at 95°C for 36 hours, then reconstitute the sample with TE buffer and use Quantifiler TM Trio DNA Quantification reagent (Applied biosystems) was used to detect the precise content and fragment degradation rate.

[0044] The concentration test results showed that ( Figure 4 A): After 36 hours of accelerated DNA loading at 95°C, the total amount of DNA in the calcium sulfate group was still significantly higher than that in the group without stabilizer when the DNA loading reached 20% (w / w). Figure 4 B) After 36 hours of accelerated incubation at 95°C, the DNA degradation rate in the calcium sulfate stabilizer group was significantly lower than that in the unstabilized group, even at a DNA loading of 25% (w / w). Based on this data, the use of calcium sulfate as a DNA stabilizer should limit the DNA loading to at least 25% (w / w) to effectively mitigate sample degradation.

[0045] Example 6. Calcium sulfate can maintain DNA stability at room temperature for at least 130 days

[0046] In order to observe the effect of the new technology proposed in this invention on the preservation of DNA samples under real room temperature storage conditions, DNA-calcium sulfate freeze-dried complexes of different masses were prepared to adjust the DNA / CaSO4 content to 0-25% (w / w). The complexes were stored at 20-25°C (20-30% RH) for 130 days. The samples were then re-dissolved in TE buffer and subjected to agarose gel electrophoresis. The results showed that ( Figure 5 ): After 130 days of storage at room temperature, the target band (about 15,000bp) of the DNA sample without stabilizer (DNA content 100%) weakened, indicating that the sample content was reduced: After adding different amounts of calcium sulfate as a storage stabilizer, the target band of the DNA sample was basically the same as that of the -20℃ low-temperature storage group, suggesting that calcium sulfate can slow down the degradation reaction of DNA samples at room temperature.

Claims

1. A new technology for room temperature storage of DNA samples.

2. The room temperature storage technology according to claim 1, wherein DNA is mixed with a calcium sulfate dihydrate aqueous solution and dried to prepare a DNA solid sample that can be stored at room temperature. The solid sample is placed in a container, sealed, and placed in an environment with a temperature not higher than room temperature.

3. The room temperature storage technology according to claim 1, wherein The DNA includes one or more of a dry solid state, a solution in a TE buffer, or a solution in enzyme-free sterile deionized water. The DNA refers to double-stranded DNA and can be obtained by artificial synthesis or extraction, including but not limited to all known extraction methods in the art.

4. The room temperature storage technology according to claim 1, wherein The concentration of the calcium sulfate dihydrate aqueous solution is not higher than 2.08 mg / mL at 25°C.

5. The room temperature storage technology according to claim 1, wherein The room temperature is 10°C to 40°C, preferably 20°C to 25°C.

6. The drying as claimed in claim 2 can be freeze drying, vacuum drying or room temperature drying, and the drying temperature is not higher than room temperature.

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