Preparation and application of kit for efficiently extracting yeast genome DNA

Abstract

The invention relates to preparation and application of a kit for efficiently extracting yeast genome DNA. The kit comprises a lysate prepared from chaotropic salt with the final concentration of 1-5 mol / L, a nonionic surfactant with the mass-volume percent of 0.5%-10%, an anionic surfactant with the mass-volume percent of 0.1%-10%, a buffer solution with the final concentration of 10-100 mmol / L and an ionic chelating agent with the final concentration of 0.1-20 mmol / L, and the lysate is matched with digestive juice, RNase A, a protease K solution, a washing solution 1, a washing solution 2 and an eluent. According to the method, the yeast genome can be effectively extracted without using toxic reagents such as phenol and chloroform, the operation is simple and convenient, and the extracted genome is high in content and good in quality and can be directly used for subsequent experiments.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, and in particular to the preparation and application of a kit for efficiently extracting yeast genomic DNA. Background Art

[0002] Yeast is a single-cell eukaryotic organism. Because of its clear genetic background, easy operation, and rapid growth, it is often used as a eukaryotic cell model for genetics and cytology to study the mechanisms of various life activities of eukaryotic cells. In addition, yeast is also widely used in genetic engineering and food production. Therefore, in order to better study the genetic information of yeast, extracting its genome for the detection of target genes is an important step. However, because the yeast cell wall is tough and thick and rich in polysaccharides, pigments, nucleases and other substances, the current yeast genome extraction methods and related products have problems such as high cost, long time consumption, and unsatisfactory results. Therefore, effectively breaking the yeast cell wall is the key to successfully extracting the yeast genome.

[0003] Existing cell wall breaking methods include liquid nitrogen freeze-thaw, -70℃ freeze-thaw, enzyme digestion, ultrasonic disruption, high salt solution, urea buffer treatment, etc., and methods for extracting and purifying genomic DNA include phenol chloroform extraction, SDS method, centrifugal column method, etc. These methods have their own advantages and disadvantages in terms of efficiency, purity, cost and ease of operation, such as long processing time, relatively high cost, and the need to use highly toxic chemical reagents. For example, Chinese patent CN116769771A discloses a method for preparing yeast genomic DNA templates, which uses liquid nitrogen and an ultrasonic device to repeatedly freeze and thaw samples to obtain yeast genomic DNA. This invention requires the use of liquid nitrogen and an ultrasonic device during the preparation process, which may involve high implementation costs. At the same time, the genomic DNA obtained can only be tested by PCR, and it cannot be ensured that the quality of the extracted genomic DNA template can meet the requirements of applications such as qPCR. Chinese patent CN113265397A discloses a cell lysis solution, a kit and a method for extracting yeast genomes. Although the kit can extract genomes with a higher yield and avoid the use of toxic reagents such as chloroform, the kit contains lysozyme and may require special storage conditions, which will increase the storage cost and usage complexity of the user.

[0004] Based on this, there is an urgent need for an economical and efficient extraction kit for extracting yeast genomic DNA. Summary of the invention

[0005] In view of the defects of the prior art, the present invention designs a kit for efficiently extracting yeast genomic DNA. By optimizing the extraction reagent and lysis method, the extracted yeast nucleic acid has a higher concentration, reduces PCR inhibitors, and has a higher extraction efficiency.

[0006] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions: In a first aspect, the present invention provides a lysis solution for yeast genome extraction, the lysis solution comprising a chaotropic salt with a final concentration of 1 to 5 mol / L, 0.5wt% to 10wt% of a non-ionic surfactant, 0.1wt% to 10wt% of an anionic surfactant, a buffer with a final concentration of 10 to 100 mmol / L, and an ion chelator with a final concentration of 0.1 to 20 mmol / L.

[0007] In some embodiments, the chaotropic salt is one or more of sodium chloride, sodium periodate, sodium iodide, and sodium bromate.

[0008] For example, it can be a combination of sodium chloride, sodium periodate, sodium iodide and sodium bromate; it can also be a combination of sodium chloride, sodium periodate and sodium iodide, a combination of sodium chloride and sodium periodate, a combination of sodium iodide and sodium bromate, or a combination of sodium periodate, sodium iodide and sodium bromate, etc.

[0009] In some of the embodiments, the nonionic surfactant is one or more of PEG 2000 (polyethylene glycol 200), NP-40 (nonylphenol polyoxyethylene ether), Triton X-305 (Triton X-305), Tween-20 (Tween-20), Tween -80 (Tween-80), Brij-58 (hexadecyl polyoxyethylene ether), and AEO-9 (fatty alcohol polyoxyethylene ether (N=9)).

[0010] For example, it can be a combination of PEG 2000 and NP-40, a combination of Triton X-305 and Tween-20, a combination of Brij-58 and AEO-9, a combination of PEG 2000, NP-40, Triton X-305, Tween-20, Tween-80, Brij-58 and AEO-9, a combination of PEG 2000, NP-40 and Triton X-305, a combination of Tween-20, Tween-80, Brij-58 and AEO-9, or a combination of NP-40, Triton X-305, Tween-20, etc.

[0011] In some embodiments, the anionic surfactant is one or more of sodium dodecyl sulfate (SDS), sodium dodecyl sarcosine (SLS), triethanolamine dodecyl sulfate (TLS), sodium dodecyl sulfonate (SDS), lithium dodecyl sulfate (LDS), and sodium dodecylbenzene sulfonate (SDBS).

[0012] For example, it may be a combination of sodium dodecyl sulfate and sodium dodecyl sarcosinate, a combination of triethanolamine dialkyl sulfate and sodium dodecyl sulfonate, a combination of lithium dodecyl sulfate and sodium dodecylbenzene sulfonate, a combination of sodium dodecyl sulfate, sodium dodecyl sarcosinate, triethanolamine dialkyl sulfate, sodium dodecyl sulfonate, lithium dodecyl sulfate and sodium dodecylbenzene sulfonate, a combination of sodium dodecyl sulfate, sodium dodecyl sarcosinate and triethanolamine dialkyl sulfate, or a combination of sodium dodecyl sulfonate, lithium dodecyl sulfate and sodium dodecylbenzene sulfonate, etc.

[0013] In some embodiments, the buffer is one or more of Tris-HCl buffer, citric acid-sodium citrate buffer, and acetic acid-sodium acetate buffer.

[0014] For example, it may be a combination of Tris-HCl buffer, citric acid-sodium citrate buffer, a combination of citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, or a combination of Tris-HCl buffer and acetic acid-sodium acetate buffer.

[0015] In some embodiments, the ion chelator is one or more of EDTA (ethylenediaminetetraacetic acid), EDTA·Na2 (disodium ethylenediaminetetraacetic acid), EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), and 8-HQ (8-hydroxyquinoline).

[0016] For example, it may be a combination of EDTA, EDTA·Na2, EGTA, and 8-HQ, a combination of EDTA and EDTA·Na2, a combination of EGTA and 8-HQ, or the like.

[0017] In a first aspect, the present invention also provides a kit for efficiently extracting yeast genomic DNA, comprising the lysis solution.

[0018] In some embodiments, the kit further comprises a digestion solution, RNase A, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution; wherein the digestion solution comprises guanidine thiocyanate with a final concentration of 1 to 3 mol / L, 0.5wt% to 10wt% of a nonionic surfactant, 0.1wt% to 10wt% of an anionic surfactant, a buffer with a final concentration of 10 to 100 mmol / L, an ion chelator with a final concentration of 0.1 to 20 mmol / L and 10wt% to 50wt% of alcohol.

[0019] The washing solution 1 comprises a chaotropic salt with a final concentration of 1-5 mol / L, 0.5wt%-10wt% of a nonionic surfactant, 0.1wt%-10wt% of an anionic surfactant, a buffer with a final concentration of 10-100 mmol / L, an ion chelating agent with a final concentration of 0.1-20 mmol / L, and 30-50wt% of alcohol.

[0020] The washing solution 2 includes a buffer solution with a final concentration of 5-15 mmol / L and 60 wt %-80 wt % of alcohol.

[0021] The eluent comprises a buffer solution with a final concentration of 5 to 15 mmol / L and an ion chelator with a final concentration of 0.05 to 0.2 mmol / L.

[0022] The nonionic surfactant is one or more of PEG 2000, NP-40, Triton X-305, Tween-20, Tween -80, Brij-58, and AEO-9.

[0023] The anionic surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sarcosinate, triethanolamine dodecyl sulfate, sodium dodecyl sulfonate, lithium dodecyl sulfate, and sodium dodecylbenzene sulfonate.

[0024] The buffer is one or more of Tris-HCl buffer, citric acid-sodium citrate buffer, and acetic acid-sodium acetate buffer.

[0025] The ion chelating agent is one or more of EDTA (ethylenediaminetetraacetic acid), EDTA·Na2 (disodium ethylenediaminetetraacetic acid), EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), and 8-HQ (8-hydroxyquinoline).

[0026] The alcohol is one or more of ethanol and isopropanol.

[0027] In some embodiments, the concentration of the proteinase K solution is 20 mg / mL; the concentration of the RNase A is 10 mg / mL.

[0028] In a third aspect, the present invention also provides the use of the kit for efficiently extracting yeast genomic DNA in extracting yeast genomic nucleic acid.

[0029] Specifically, the yeast is conventional yeast such as Pichia pastoris, Saccharomyces cerevisiae, Candida tropicalis, Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, etc.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a kit for efficiently extracting yeast genomic DNA. By using a lysate prepared by a chaotropic salt, a nonionic surfactant, an anionic surfactant, a buffer, alcohols and an ion chelating agent, and cooperating with a digestion solution, RNaseA, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution, the yeast genome extracted has not only a high yield but also a high purity, can be directly used for downstream molecular biology applications, provides good conditions for subsequent PCR amplification, and helps to obtain accurate and reliable experimental results. At the same time, the cost and stability problems caused by the use of biological enzymes in the prior art are avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is the electrophoresis diagram of yeast genome extraction results using different kits. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0035] In the present invention, the test methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0036] Example 1 An efficient yeast genomic DNA extraction kit includes a lysis solution, a digestion solution, RNase A, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution. The specific formula of each component of the kit is shown in Table 1 below: Table 1 Kit formula

[0037] In this example, the concentration of the proteinase K solution is 20 mg / mL; the concentration of the RNase A is 10 mg / mL.

[0038] Example 2 An efficient yeast genomic DNA extraction kit includes a lysis solution, a digestion solution, RNase A, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution. The specific formula of each component of the kit is shown in Table 2 below: Table 2 Kit formula

[0039] In this example, the concentration of the proteinase K solution is 20 mg / mL; the concentration of the RNase A is 10 mg / mL.

[0040] Example 3 An efficient yeast genomic DNA extraction kit includes a lysis solution, a digestion solution, RNase A, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution. The specific formula of each component of the kit is shown in Table 3 below: Table 3 Kit formula

[0041] In this example, the concentration of the proteinase K solution is 20 mg / mL; the concentration of the RNase A is 10 mg / mL.

[0042] Example 4 An efficient yeast genomic DNA extraction kit includes a lysis solution, a digestion solution, RNase A, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution. The specific formula of each component of the kit is shown in Table 4 below: Table 4 Kit formula

[0043] In this embodiment, the concentration of the proteinase K solution is 20 mg / mL; the concentration of the RNase A is 10 mg / mL.

[0044] Example 5 An efficient yeast genomic DNA extraction kit includes a lysis solution, a digestion solution, RNase A, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution. The specific formula of each component of the kit is shown in Table 5 below: Table 5 Kit formula

[0045] In this embodiment, the concentration of the proteinase K solution is 20 mg / mL; the concentration of the RNase A is 10 mg / mL.

[0046] Example 6 The yeast genomic DNA was extracted using the kit of Example 1, and the extraction method included the following steps: 1. Cultivate yeast (Pichia pastoris): Take 1-1.5 mL of culture medium (<5×10 yeast cells) in a 2 mL centrifuge tube, centrifuge at 5,000 x g for 5 minutes to collect yeast cells, and carefully aspirate and discard the culture medium; 2. Add 300 µL of lysis buffer to the sample in step 1, vortex to resuspend the bacteria, and incubate at 37°C for 30 minutes (if RNA needs to be removed, after heating, wait for the liquid to cool to room temperature and add 5 µL of RNase A to the sample); 3. Add 300 µL digestion solution and 10 µL proteinase K solution to the sample, vortex to mix, and incubate at 70°C for 10 minutes; 4. Centrifuge at 10,000 x g for 3 minutes to remove undigested impurities and transfer the supernatant to a new 2 mL centrifuge tube; 5. Add 250 µL of anhydrous ethanol to the supernatant and vortex to mix; 6. Install the adsorption column in the collection tube, transfer the mixed solution to the adsorption column, and centrifuge at 10,000 x g for 1 minute; 7. Discard the filtrate, put the adsorption column back into the collection tube, add 500 μL of washing solution 1 to the adsorption column, and centrifuge at 10,000 x g for 1 minute; 8. Discard the filtrate, put the adsorption column back into the collection tube, add 600 μL of washing solution 2 to the adsorption column, and centrifuge at 10,000 x g for 1 minute; 9. Discard the filtrate, put the adsorption column back into the collection tube, and centrifuge at 13,000xg for 2 minutes to dry the adsorption column; 10. Transfer the column to a new 1.5 mL centrifuge tube, add 30-50 μL of eluent preheated to 65°C to the center of the adsorption column membrane, place at 65°C for 3 minutes, and centrifuge at 12,000 rpm for 1 minute. The resulting eluate is the yeast genome.

[0047] Comparative Example 1 Yeast genomic DNA extraction kit (spin column method) commercially available from T Company.

[0048] Comparative Example 2 Yeast DNA extraction kit (proteinase K method) commercially available from Company M.

[0049] Test Example 1 The yeast genomes extracted by the kits of Example 1, Control Example 1 and Control Example 2 were respectively subjected to UV spectrophotometer detection, Qubit detection and agarose gel electrophoresis detection. The kit of Example 1 extracted the yeast genome according to the extraction method of Example 2, and Control Examples 1 and 2 respectively performed yeast genome extraction operations according to their instructions.

[0050] Table 6 UV spectrophotometer and Qubit test results

[0051] like Figure 1 As shown, the yeast genome concentration extracted by the control example is low, while the band of Example 1 is clear and bright; further, it can be seen from Table 6 that the A260 / A280 ratio of Example 1 is better than that of Control Example 1, and there is no significant difference with that of Control Example 2, while the nucleic acid concentration is much higher than that of Control Example 1 and Control Example 2, indicating that the kit of Example 1 can extract yeast genomes with higher yield and better quality than commercially available kits.

[0052] In summary, the kit of the present invention does not require the use of toxic reagents such as phenol and chloroform, and can not only effectively extract the yeast genome, but also is easy to operate, and the extracted genome has high content and good quality, which is conducive to stable results and can be directly used in experiments such as PCR.

[0053] The present invention uses Pichia pastoris as a test sample. It should be noted that the kit of the present invention has wide practicability and is not limited to extracting the genome of Pichia pastoris, but can also be applied to the genome extraction of conventional yeasts such as Saccharomyces cerevisiae, Candida tropicalis, Kluyveromyces lactis, Hansenula polymorpha, and Schizosaccharomyces pombe. Moreover, under the protection method of the present invention, as in Examples 2-5, the chaotropic salt, nonionic surfactant, anionic surfactant, and buffer of the lysate are replaced, and the prepared kit can still achieve the extraction of the yeast genome, and the extracted nucleic acid concentration is high, showing a good extraction efficiency.

[0054] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A lysis solution for yeast genome extraction, characterized in that: The lysate comprises a chaotropic salt with a final concentration of 1-5 mol / L, a nonionic surfactant of 0.5wt%-10wt%, an anionic surfactant of 0.1wt%-10wt%, a buffer with a final concentration of 10-100 mmol / L, and an ion chelating agent with a final concentration of 0.1-20 mmol / L.

2. The lysate according to claim 1, characterized in that The chaotropic salt is one or more of sodium chloride, sodium periodate, sodium iodide and sodium bromate.

3. The lysate according to claim 1, characterized in that The nonionic surfactant is one or more of PEG 2000, NP-40, Triton X-305, Tween-20, Tween-80, Brij-58, and AEO-9.

4. The lysate according to claim 1, characterized in that The anionic surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sarcosinate, triethanolamine dodecyl sulfate, sodium dodecyl sulfonate, lithium dodecyl sulfate, and sodium dodecylbenzene sulfonate.

5. The lysate according to claim 1, characterized in that The buffer is one or more of Tris-HCl buffer, citric acid-sodium citrate buffer, and acetic acid-sodium acetate buffer.

6. The lysate according to claim 1, characterized in that The ion chelating agent is one or more of EDTA, EDTA·Na2, EGTA, and 8-HQ.

7. A kit for efficiently extracting yeast genomic DNA, characterized in that: The method comprises the lysate according to any one of claims 1 to 6.

8. The kit for efficiently extracting yeast genomic DNA according to claim 7, characterized in that: The kit also includes a digestion solution, RNase A, a proteinase K solution, a washing solution 1, a washing solution 2 and an elution solution; wherein the digestion solution includes guanidine thiocyanate with a final concentration of 1-3 mol / L, 0.5wt%~10wt% non-ionic surfactant, 0.1wt%~10wt% anionic surfactant, a buffer with a final concentration of 10~100 mmol / L, an ion chelator with a final concentration of 0.1~20 mmol / L and 10wt%~50wt% alcohol.

9. The kit for efficiently extracting yeast genomic DNA according to claim 7, characterized in that: The concentration of the proteinase K solution is 20 mg / mL; the concentration of the RNase A is 10 mg / mL.

10. Use of the kit for efficiently extracting yeast genomic DNA according to any one of claims 7 to 9 in extracting yeast genomic nucleic acid.

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