Method for rapidly extracting yeast genome DNA and application

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a method for rapidly extracting yeast genome DNA and application. The method for rapidly extracting the yeast genome DNA mainly comprises the following steps: cell resuspension, alkaline solution treatment, microwave heating and centrifugal precipitation. According to the method, physical grinding means such as glass beads and a grinding pestle are not needed, and organic solvents with high toxicity such as phenol and chloroform are not needed for extraction. The method is simple in operation steps, high in safety and suitable for large-scale preparation of the yeast genome DNA, and the whole operation process can be completed only in 10-15 minutes. The yeast DNA solution prepared by the invention can be used as a template for PCR (Polymerase Chain Reaction) amplification, and all genes with the length of more than 5000bp can be efficiently amplified. The method is suitable for genetic engineering operation of various yeast cells such as pichia pastoris, saccharomyces cerevisiae and kluyveromyces lactis, and the efficiency of genetic modification of the yeast cells is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for rapidly extracting yeast genomic DNA and its application. Background Art

[0002] Yeast is widely used in the field of genetic engineering technology, and its core uses include efficient expression of exogenous proteins, fine regulation of metabolic pathways, and innovative practices in synthetic biology. Among them, Pichia pastoris, Saccharomyces cerevisiae, and Kluyveromyces lactis are the most commonly used yeast strains, and they have received a lot of attention and research in academic research and industrial production. For example, technicians can use genetic engineering methods to transform yeast cells to produce chemicals such as biofuels and organic acids. In addition, yeast can also be used to produce hepatitis B vaccines, bivalent HPV vaccines, etc., which are used in human immunity and animal health protection. In addition, yeast is often used in gene editing research because of its small genome, clear genetic background, and flexible operation. Through technologies such as CRISPR-Cas9, yeast has become an important carrier for exploring gene functions and disease mechanisms.

[0003] Studies have shown that the thickness of yeast cell walls is between 25 and 70 nm, usually accounting for 18% to 25% of the cell dry weight. In addition, yeast cell walls are complex in composition and arranged in a "sandwich" structure, with the outermost layer being mannan and the inner layer being glucan. There is a layer of protein molecules sandwiched between the inner and outer layers, most of which exist in various forms of enzyme proteins, such as glucanase, sucrase, alkaline phosphatase, etc. It is precisely because of the tough cell wall of yeast that the subsequent extraction of yeast genomic DNA and PCR detection have been greatly hindered, thus greatly affecting the identification and screening of yeast cell transformation.

[0004] At present, the commonly used methods for obtaining yeast genomic DNA mainly include direct method, boiling method, enzyme method, glass bead grinding method, ultrasonic method, etc., but these methods all have certain defects. Either the stability is poor and the effective template concentration is low, or the operation steps are cumbersome and the amount of template extracted at one time is small. For example, the template release process of direct genomic DNA mainly relies on the high temperature of PCR pre-denaturation and denaturation stage to heat treat the cell wall, but in fact the template release amount is very small and the randomness is relatively large, so the PCR results have no obvious rules, and the stability and repeatability are poor; although the boiling method has low requirements for equipment and reagents, it is not enough to damage the cells, the nucleic acid release is incomplete, and it is far from reaching the maximum effective template concentration. The stability is poor, and sometimes the PCR results of the same two tubes of bacterial solution are inconsistent; the enzyme method uses snail enzyme, lysozyme, etc. to treat the cell wall, and then adopts the classic nucleic acid purification method to extract the genomic DNA. Although this method has high template purity, it requires the use of highly toxic organic solvents such as phenol and chloroform for extraction. Moreover, only a small amount of bacteria can be screened and identified at a time, which cannot meet the needs of simple, safe and efficient yeast genomic DNA preparation; the glass bead grinding method adds an appropriate amount of acid-washed glass beads to destroy the cell wall during violent shaking, and then extracts it with a corresponding organic solvent to obtain yeast genomic DNA, but for the same reason as the enzymatic method, the operation process is time-consuming and requires the use of organic solvents. Only a small amount of template preparation can be performed at a time, which cannot meet actual needs; the ultrasonic method uses the mechanical shear force and hydrodynamic cavitation effect caused by oscillation to better achieve the cell wall breaking effect, but has high requirements on equipment, and the free radicals generated by ultrasonic treatment are harmful to biological molecules such as nucleic acids.

[0005] In order to solve the above problems encountered in extracting yeast genomic DNA, it is necessary to invent a simple and rapid method for preparing yeast genomic DNA to meet the needs of yeast genetic engineering operations using PCR technology as the main means. Summary of the invention

[0006] The purpose of the present invention is to provide a method for rapidly preparing yeast genomic DNA in view of the deficiencies in the prior art, thereby solving the key problems of the prior art in extracting yeast gene DNA, such as complicated operation steps, use of toxic reagents, high cost, poor stability, and unsuitability for large-scale preparation.

[0007] In order to achieve the above object, the present invention provides a method for extracting yeast genomic DNA, the method comprising the following steps:

[0008] (1) Dispense solution I into a 96-well plate (A), directly pick a single yeast colony with a growth period of 2 to 3 days and add it to solution I, and mix well; the solution I is 10 to 20 mmol / L PBS, with a pH of 7.0 to 7.5;

[0009] (2) Dispense solution II into a 96-well plate (B), pipette the suspended bacteria in the 96-well plate (A) into the 96-well plate (B), and after sufficient suspension, cover the 96-well plate (B) with a sealing film and place it at 25 to 37°C to allow the components in solution II to fully act on the yeast cell wall;

[0010] The composition of the solution II is: 50-60 mmol / L NaOH, 30-50 mmol / L Tris-HCl, 5-10 mmol / L EDTA, 0.5-1‰ TritonX-100, and the pH of the solution II is 9.5-10.5;

[0011] (3) Place the 96-well plate (B) in a microwave oven and heat at 400-800 W for 1-2 min to heat Solution II containing the suspended bacteria;

[0012] (4) After taking the 96-well plate (B) out of the microwave oven, it is shaken vigorously several times to fully release the genomic DNA, and then placed in a centrifuge to precipitate impurities. The resulting supernatant is the yeast genomic DNA solution.

[0013] According to a preferred embodiment of the present invention, the solution I is 10mmol / L PBS pH7.2; the composition of the solution II is: 55mmol / L NaOH, 40mmol / L Tris-HCl pH7.0, 5mmol / L EDTA, 0.5‰ TritonX-100.

[0014] According to a preferred embodiment of the present invention, in step (1), the volume of solution I dispensed into the 96-well plate (A) is 10-15 μL. In step (2), the volume of solution II dispensed into the 96-well plate (B) is 90-95 μL, and the volume of suspended bacteria in the 96-well plate (A) aspirated is 5-10 μL.

[0015] According to a preferred embodiment of the present invention, in step (2), the standing time is 3 to 5 minutes.

[0016] According to a preferred embodiment of the present invention, in step (3), the power of the microwave oven heating is 500-700 W. Specifically, the medium heat, medium-high heat or high heat of a commercially available conventional microwave oven can be selected.

[0017] According to a preferred embodiment of the present invention, in step (4), after taking the 96-well plate (B) out of the microwave oven, the 96-well plate (B) is flicked to vibrate violently 5 to 10 times. For ease of operation, the flicking can be performed directly with fingers.

[0018] According to a preferred embodiment of the present invention, in step (4), the centrifugal conditions include: centrifugation at 10000-12000 rpm for 30s-60s.

[0019] According to a preferred embodiment of the present invention, the 96-well plate is a planar PCR plate. The present invention has no particular limitation on the pore volume of the 96-well plate, and a planar PCR plate with a volume of 0.2 mL is preferred; the sealing film is a transparent PCR sealing film.

[0020] The method of the present invention is applicable to the extraction of DNA from various yeasts, including but not limited to Pichia pastoris, Saccharomyces cerevisiae or Kluyveromyces lactis.

[0021] The method of the present invention can be used in yeast genetic engineering, and the yeast genomic DNA obtained by the method of the present invention can be used as a template for a PCR amplification system.

[0022] The main feature of the present invention is: a method of coupling yeast cells with alkaline buffer solution and microwave treatment, which has significant advantages. First, the bacteria are suspended in solution II. The alkaline environment and components of solution II help to destroy the yeast cell wall and cell membrane, and stabilize the genomic DNA. Subsequently, the treated yeast liquid is placed in a microwave environment. The microwave penetrates the material at a frequency of 2450MHz per second, causing polar molecules such as water, polysaccharides, and proteins in the yeast cells to oscillate at the same frequency. The large amount of friction heat generated by the collision between molecules causes the temperature of the intracellular water to rise and vaporize, thereby achieving cell expansion and rupture. Unlike the boiling method through external heating, the microwave method can heat the yeast cells uniformly inside and outside at the same time, and the heating rate is faster. Impurities such as proteins released by yeast lysis are rapidly denatured under the action of alkali, reducing interference with subsequent genetic operations.

[0023] Compared with the traditional yeast genomic DNA preparation method, the method for rapidly preparing yeast genomic DNA provided by the present invention improves and innovates the solution components and extraction method. Its advantages include:

[0024] ① The ratio of NaOH and 1M Tris-HCl pH7.0 used in solution II has been optimized to maintain the final solution pH between 9.5 and 10.5. Such a pH environment can not only effectively destroy the cell wall of yeast cells and promote their lysis, but also denature and precipitate most impurities. In addition, this pH environment will not affect the phosphodiester bonds of DNA and avoid DNA degradation. However, if the adjustment ratio of the two components of NaOH and 1M Tris-HCl pH7.0 is improper, so that the pH value of the solution is lower than 9.5, the damage to the cell wall of yeast cells by the solution will decrease rapidly; on the contrary, if the pH value is higher than 10.5, its damage to the phosphodiester bonds of DNA will begin to increase, resulting in the breakage of genomic DNA molecules, thereby affecting the effective DNA concentration.

[0025] ② Take 1 μL of the prepared yeast DNA solution and use it in a 10 μL PCR system. Due to the appropriate ratio of NaOH and 1M Tris-HCl pH 7.0 in solution II, the solution will not affect the pH environment of the PCR system after dilution.

[0026] ③EDTA used in the extract is a metal ion chelating agent that can chelate divalent metal ions (such as Mg2+, Ca 2 +, etc.), inhibiting the activity of nucleases that depend on these ions, thereby preventing the degradation of genomic DNA.

[0027] ④In addition, TritonX-100 used in the extract is a non-ionic surfactant that is used to destroy the lipid bilayer structure of the cell membrane, promote cell lysis, and help release intracellular DNA components.

[0028] ⑤ The preparation method is carried out by coupling alkali treatment with microwaves. The superposition of these two operation steps can effectively release genomic DNA and improve the yield of genomic DNA. At the same time, the alkaline buffer can denature the protein released by cleavage during microwave treatment, thereby obtaining good DNA purity. However, the effect of using alkali treatment and microwave treatment alone on extracting genomic DNA is not ideal.

[0029] ⑥ This preparation method is highly efficient and can significantly shorten the working time for screening and identifying a large number of yeast colonies. The operation process can be completed in only 10 to 15 minutes.

[0030] ⑦ In addition, the equipment requirements are low and the safety is high. Only a common microwave oven in the laboratory is required, and there is no need to use highly toxic reagents such as phenol and chloroform.

[0031] ⑧The present invention has a wide range of applications and is suitable for various genetic engineering operations of various yeast cells such as Pichia pastoris, Saccharomyces cerevisiae, and Kluyveromyces lactis. For example, when using CRISPR-Cas9 or other CRISPR technologies for gene knockout or knock-in, a large number of single colony DNA templates can be quickly prepared by the method of the present invention, identification primers can be designed, and the amplification bands can be used to determine whether the yeast cells are successfully edited, thereby efficiently identifying positive colonies; when using the homologous recombination method in traditional gene editing technology to transform yeast cells, the method of the present invention can also achieve rapid screening of high-throughput colonies; when performing heterologous expression of yeast cells, the present invention can quickly extract genomic DNA and eliminate false positive colonies on the screening plate by the PCR method.

[0032] In summary, the present invention provides a method for rapidly preparing yeast genomic DNA, the main steps of which include: cell resuspension, alkaline solution treatment, microwave heating and centrifugal precipitation. The method of the present invention does not require the use of physical grinding means such as glass beads and grinding pestles, which increases the time for processing samples, and does not require phenol, chloroform and other highly toxic organic solvents for extraction. The method has simple operating steps, low equipment requirements, high safety, and is suitable for large-scale preparation of yeast genomic DNA. The entire operating process only takes 10 to 15 minutes to complete, and has the advantages of high template concentration and strong stability. The yeast DNA solution prepared by the present invention can be used as a template for PCR amplification, and genes with a length greater than 5000bp can be efficiently amplified. The method of the present invention is suitable for genetic engineering operations of various yeast cells such as Pichia pastoris, Saccharomyces cerevisiae, and Kluyveromyces lactis, and significantly improves the efficiency of genetic modification of yeast cells.

[0033] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0035] Figure 1 Shown are bar graphs showing the stability of the HIS4 gene of the Pichia pastoris X-33 genome amplified by direct method (A), alkali treatment (B), microwave method (C), and the present invention (D).

[0036] Figure 2 Shown are bar graphs of the genomic DNA concentration and A260 / A280 average values ​​of the supernatants prepared by alkali treatment (B), microwave method (C), and the present invention (D).

[0037] Figure 3Shown are line graphs of genomic DNA concentration in supernatants prepared by alkali treatment (B), microwave method (C), and the present invention (D).

[0038] Figure 4 Shown are the A260 / A280 line graphs of the supernatant genomic DNA prepared by alkali treatment (B), microwave method (C), and the present invention (D).

[0039] Figure 5 Shown are 0.8% agarose gel electrophoresis images of the TDH3 gene (A), HIS4 gene (B), and ESP1 gene (C) of the Pichia pastoris X-33 genome amplified by the method of the present invention, wherein M: Takara DNA Marker 5000bp, and the target band sizes are 1509bp, 2814bp, and 5025bp, respectively.

[0040] Figure 6 Shown are 0.8% agarose gel electrophoresis images of the HIS4 gene (A) of the Saccharomyces cerevisiae S288C genome and the LAC4 gene (B) of the Kluyveromyces lactis GG799 genome amplified by the method of the present invention, wherein M: Takara DNA Marker 5000bp, and the target band sizes are 2829bp and 3078bp, respectively. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0042] If no specific conditions are specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments without manufacturer's information are all conventional products available from commercial channels.

[0043] Preparation Examples Reagents used in the preparation of the examples

[0044] Solution I: 10 mmol / L PBS pH 7.2;

[0045] Accurately weigh 2.87 g Na2HPO4·12H2O, 0.31 g NaH2PO4·2H2O and 9 g NaCl into a container, make the solution volume up to 1 L, and stir evenly to complete the preparation of Solution I.

[0046] Solution II: 55mmol / LNaOH, 40mmol / L Tris-HCl pH7.0, 5mmol / L EDTA, 0.5‰TritonX-100.

[0047] Accurately weigh 12.11 g of Tris (tris(hydroxymethyl)aminomethane)) into a container, and add about 90 mL of purified water to the container, stir at room temperature to fully dissolve it, adjust the pH value to 7.0 with concentrated hydrochloric acid, make the solution dilute to 100 mL, and stir again to mix well, thus completing the preparation of 1M Tris-HCl pH7.0 solution.

[0048] Accurately weigh 2.2 g NaOH, 1.46 g EDTA, 40 mL 1 M Tris-HCl pH 7.0, and 500 μL Triton X-100 into a container, adjust the volume of the solution to 1 L, and stir evenly to complete the preparation of Solution II.

[0049] Example 1 Comparison of results of different preparation methods

[0050] 1. Pick 24 single colonies of Pichia pastoris X-33 with a growth cycle of 2 days and prepare yeast genomic DNA according to the following 4 methods.

[0051] Direct method (A): The picked single colony was suspended in a PCR tube containing 100 μL ultrapure water, and the untreated suspension was used as a PCR amplification template.

[0052] Alkali treatment (B): The picked single colony was suspended in a PCR tube containing 100 μL of solution II, placed at room temperature 25°C for 5 minutes, and then centrifuged at 12,000 rpm for 1 minute. The supernatant was used as a PCR amplification template.

[0053] Microwave method (C): Suspend the picked single colony in a PCR tube containing 100 μL TE buffer, place it in a microwave oven, and heat it on high for 60 seconds. Cover it with a sealing film, flick the bottom of the PCR tube repeatedly with your fingers 8 to 10 times, place it in a centrifuge, centrifuge at 12,000 rpm for 1 minute, and use the supernatant obtained as a PCR amplification template.

[0054] The present invention (D): the single colony picked is suspended in a PCR tube containing 100 μL of solution II, and placed at room temperature of 25° C. for 5 minutes, and then the PCR tube is placed in a microwave oven at high temperature for 60 seconds. Then, the PCR tube is taken out of the microwave oven, covered with a sealing film, and repeatedly flicked with fingers for 8 to 10 times, and placed in a centrifuge at 12,000 rpm for 1 minute, and the supernatant obtained is used as a PCR amplification template.

[0055] 2. Take 1 μL of the above PCR template and prepare 10 μL PCR system as shown in Table 1.

[0056] Table 1 Reaction system

[0057]

[0058] 3. The validation primers used are:

[0059] HIS4-F(5'-CCCAATCACTTGAGTACGAAC-3', SEQ ID NO: 1)

[0060] HIS4-R (5'-CAGAGTGAGACTTGATCTCG-3', SEQ ID NO: 2).

[0061] 4. The reaction conditions used are shown in Table 2.

[0062] Table 2 Reaction conditions

[0063]

[0064] 5. After the PCR reaction is completed, 10 μL of PCR product is taken for 0.8% agarose gel electrophoresis detection, and the number of amplified positive bands is counted.

[0065] 6. The above four methods were repeated for three batches, and the statistical results are shown in Table 3. Figure 1 .

[0066] Table 3 Positive rate of HIS4 gene amplified by different methods

[0067]

[0068] Summary: The test results of three batches of the above four methods show that the direct method has the lowest positive rate of amplified bands, and the worst stability and repeatability; although the solution II alkali treatment alone can increase the positive rate of amplified bands, and the stability and repeatability are also slightly improved, it still cannot meet the actual needs; compared with the first two methods, the microwave method can further improve the positive rate of amplified bands, and the stability and repeatability are also significantly improved, but it is still not the optimal method; the verification results show that in terms of the positive rate, stability and repeatability of the amplified bands, the method of the present invention is the best of the four methods, and the positive rate is 100% in three repeated experiments.

[0069] 7. 2 μL samples were taken from the alkaline treatment (B), microwave method (C), and the system of the present invention (D) respectively, and the genomic DNA template concentration and genomic DNA template purity A260 / A280 were tested using NanoDrop 1000 micro-nucleic acid protein analyzer (Thermo). After the results of the above three batches were statistically analyzed, the average values ​​were obtained and are shown in Table 4. Figure 2 .

[0070] Table 4 Average data parameters of genomic DNA detected by different methods

[0071]

[0072] Summary: The average values ​​of the three batches of test results of the above three methods show that the effect of using alkali to treat yeast cells alone on increasing the concentration of genomic DNA in the supernatant is not obvious, and the A260 / A280 ratio is too large, which indicates that there are more nucleic acid impurities in the solution and the purity is poor; the microwave method alone can significantly increase the concentration of genomic DNA in the supernatant compared with alkali treatment, but the A260 / A280 ratio is lower, indicating that there are more protein impurities in the solution and the purity is poor; the present invention can effectively lyse yeast cells by coupling solution II and microwave, thereby increasing the concentration of genomic DNA in the supernatant with good purity.

[0073] 8. The second batch of experimental data is selected as a typical result of verification of different experimental methods for supplementary explanation. The statistical results are shown in Table 5. Figure 3 , Figure 4 .

[0074] Table 5 Statistical results of the second batch of data for different experimental methods

[0075]

[0076]

[0077] Example 2 Preparation of Yeast Genomic DNA Solution

[0078] 1. Pipette 15 μL of solution I into three rows of 8 PCR tubes (A), then pick 24 yeast colonies with a growth cycle of 2 days, good growth and uniform size, place them in the prepared PCR tubes (A) respectively, and mix them thoroughly.

[0079] 2. Pipette 90 μL of solution II into another three rows of 8-row PCR tubes (B), then pipette 10 μL from PCR tube (A) (if this method is used for positive yeast colony screening, the remaining 5 μL of bacterial solution in PCR tube "A" can be used for positive colony amplification) and add it to PCR tube (B), mix thoroughly, cover with sealing film, and let stand at room temperature 25°C for 5 minutes.

[0080] 3. After standing at room temperature, place the PCR tube (B) in a microwave oven and set on high heat for 70 seconds.

[0081] 4. Take out the PCR tube (B) from the microwave, flick it repeatedly with your fingers 8 to 10 times, place it in a centrifuge, centrifuge at 12000rpm, centrifuge for 1 minute, and the supernatant obtained is the yeast genomic DNA solution.

[0082] The yeast used in this embodiment is Pichia pastoris X-33 strain. Other types of Pichia pastoris, Kluyveromyces and Saccharomyces cerevisiae are all suitable for this method.

[0083] Example 3 Amplification of TDH3, HIS4, and ESP1 genes in the genome of Pichia pastoris X-33

[0084] 1. Take 1 μL of supernatant as PCR template and prepare 10 μL PCR system as shown in Table 1.

[0085] 2. The amplification primers used are shown in Table 6. Each pair of primers amplified 8 X-33 genomic DNAs.

[0086] Table 6 Primers used

[0087]

[0088] 3. The reaction conditions used are shown in Table 2.

[0089] 4. After the PCR reaction is completed, take 10 μL of PCR product for 0.8% agarose gel electrophoresis detection. The test results are shown in Figure 5 .

[0090] The TDH3 gene in the Pichia pastoris X-33 gene amplified in this example is located at 808.503 to 810.011 of chromosome 2 (sequence number: CP014716), encoding glyceraldehyde-3-phosphate dehydrogenase, which is a key enzyme in the glycolysis pathway, participates in energy metabolism and biosynthesis, and is crucial to cell growth and fermentation processes; the HIS4 gene is located at 1.701.721 to 1.704.574 of chromosome 1 (sequence number: CP014715), encoding an enzyme for histidine synthesis, and is an important gene in the histidine biosynthesis pathway. The lack of this gene will lead to a histidine synthesis disorder, which has an important impact on the nutritional requirements of the culture medium; the ESP1 gene is located at 2.400.022 to 2.405.046 of chromosome 1, encoding an exogenous esterase, which participates in the secretion and metabolism of proteins and is of great significance for optimizing the expression system and improving the secretion efficiency of recombinant proteins. The sizes of the three target bands were 1509 bp, 2814 bp, and 5025 bp, respectively.

[0091] Depend on Figure 5 It can be seen that, using the method of the present invention, each group of experiments clearly amplified the target band, and the band brightness was very high, indicating that the amplification amount was large.

[0092] Example 5 Amplification of the HIS4 gene in the genome of Saccharomyces cerevisiae S288C and the LAC4 gene in the genome of Kluyveromyces lactis GG799

[0093] 1. Pick 8 single colonies of Saccharomyces cerevisiae S288C with a growth cycle of 2 days and 8 single colonies of Kluyveromyces lactis GG799 with a growth cycle of 2 days respectively, and prepare yeast genomic DNA solutions according to the method described in Example 2.

[0094] 2. Take 1 μL of supernatant as PCR template and prepare 10 μL PCR system as shown in Table 1.

[0095] 3. The amplification primers used are shown in Table 7.

[0096] Table 7 Primers used

[0097]

[0098] 4. The reaction conditions used are shown in Table 2.

[0099] 5. After the PCR reaction is completed, take 10 μL of PCR product for 0.8% agarose gel electrophoresis detection. The detection results are shown in Figure 6 .

[0100] The HIS4 gene in the Saccharomyces cerevisiae S288C genome amplified in this example is located at 65.346-68.174 of chromosome 3 (sequence number: CP046083), encoding a key enzyme in the histidine biosynthesis pathway, participating in the biosynthesis of histidine, and is essential for the growth and metabolism of yeast; the LAC4 gene in the Kluyveromyces lactis GG799 genome amplified is located at 1.309.173-1.312.250 of chromosome 3 (sequence number: CP021240), encoding β-galactosidase, participating in the metabolism of lactose, hydrolyzing lactose into glucose and galactose, and is essential for the lactose utilization and fermentation process. The sizes of the two target bands are 2829bp and 3078bp respectively.

[0101] Depend on Figure 6 It can be seen that, using the method of the present invention, each group of experiments clearly amplified the target band, and it is shown that the method of the present invention is applicable to various types of yeast.

[0102] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for rapidly extracting yeast genomic DNA, characterized in that: The method comprises the following steps: (1) Dispense solution I into a 96-well plate (A), directly pick a single yeast colony with a growth period of 2 to 3 days and add it to solution I, and mix well; the solution I is 10 to 20 mmol / L PBS, with a pH of 7.0 to 7.5; (2) Dispense solution II into a 96-well plate (B), pipette the suspended bacteria in the 96-well plate (A) into the 96-well plate (B), and after sufficient suspension, cover the 96-well plate (B) with a sealing film and place it at 25 to 37°C to allow the components in solution II to fully act on the yeast cell wall; The composition of the solution II is: 50-60 mmol / L NaOH, 30-50 mmol / L Tris-HCl, 5-10 mmol / L EDTA, 0.5-1‰ TritonX-100, and the pH of the solution II is 9.5-10.5; (3) Place the 96-well plate (B) in a microwave oven and heat at 400-800 W for 1-2 min to heat solution II containing suspended bacteria; (4) After taking the 96-well plate (B) out of the microwave oven, it is shaken vigorously several times to fully release the genomic DNA, and then placed in a centrifuge to precipitate impurities. The resulting supernatant is the yeast genomic DNA solution.

2. The method according to claim 1, characterized in that The solution I is 10mmol / L PBS pH7.2; The composition of the solution II is: 55mmol / L NaOH, 40mmol / L Tris-HCl pH7.0, 5mmol / L EDTA, and 0.5‰ TritonX-100.

3. The method according to claim 1, characterized in that In step (1), the volume of solution I dispensed into a 96-well plate (A) is 10 to 15 μL.

4. The method according to claim 3, characterized in that In step (2), the volume of solution II dispensed into the 96-well plate (B) is 90 to 95 μL, and the volume of suspended bacteria in the 96-well plate (A) is 5 to 10 μL; the standing time is 3 to 5 minutes.

5. The method according to claim 1, characterized in that In step (3), the power of microwave heating is 500-700W.

6. The method according to claim 1, characterized in that In step (4), after taking the 96-well plate (B) out of the microwave oven, flick the 96-well plate (B) to make it vibrate violently 5 to 10 times.

7. The method according to claim 1, characterized in that In step (4), the centrifugal conditions include: centrifugation at 10000-12000 rpm for 30s-60s.

8. The method according to claim 1, characterized in that The 96-well plate is a flat PCR plate; the sealing film is a transparent PCR sealing film.

9. The method according to claim 1, characterized in that: The yeast is Pichia pastoris, Saccharomyces cerevisiae or Kluyveromyces lactis.

10. Use of the method according to any one of claims 1 to 9 in yeast genetic engineering.

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