阅读说明：本技术 一种细胞裂解液及其应用 (Cell lysis solution and application thereof ) 是由 郑丹丹 黄文静 叶海峰 于 2020-12-16 设计创作，主要内容包括：本发明公开了一种细胞裂解液及其应用。该细胞裂解液由A组分和B组分组成；其中,A组分由3～600mmol/L碱溶液和0.1mmol/L～7mmol/L SDS组成；B组分由3～600mmol/L Tris、8～1100mmol/L氯化盐和0.5～110mmol/L EDTA组成,pH为7～10；使用时A组分与B组分的体积比为(3～50)：1。该细胞裂解液不包含氯仿等有毒试剂,由常见的试剂组成,成本低,并且操作步骤简单,可快速将细胞中的核酸释放出来,大大节省核酸提取时间；并且可从微量的细胞(细胞个数小于等于2x10～4个)中提取出足够量的核酸用于PCR鉴定。(The invention discloses a cell lysis solution and application thereof. The cell lysate consists of a component A and a component B; wherein the component A consists of 3-600 mmol/L alkali solution and 0.1-7 mmol/L SDS; the component B consists of 3-600 mmol/L Tris, 8-1100 mmol/L chlorate and 0.5-110 mmol/L EDTA, and the pH value is 7-10; when the adhesive is used, the volume ratio of the component A to the component B is (3-50): 1. the cell lysate does not contain toxic reagents such as chloroform and the like, is composed of common reagents, has low cost and simple operation steps, can quickly release nucleic acid in cells, and greatly saves the time for extracting the nucleic acid; and can be used for treating a very small number of cells (the number of cells is 2X10 or less) 4 And) extracting a sufficient amount of nucleic acid for PCR identification.)

1. A cell lysate comprising: the cell lysate consists of a component A and a component B;

wherein the component A consists of 3-600 mmol/L alkali solution and 0.1-7 mmol/L SDS;

the component B consists of 3-600 mmol/L Tris, 8-1100 mmol/L chlorate and 0.5-110 mmol/L EDTA, and the pH value is 7-10.

2. A cell lysate according to claim 1, wherein:

the component A consists of 5-200 mmol/L alkali solution and 0.2-3.5 mmol/L SDS;

the component B consists of 5-500 mmol/L Tris, 10-1000 mmol/L chlorate and 1-100 mmol/L EDTA, and the pH value is 7-9.

3. Cell lysate according to claim 1 or 2, characterized in that:

when the adhesive is used, the volume ratio of the component A to the component B is (3-50): 1.

4. cell lysate according to claim 3, characterized in that:

when the adhesive is used, the volume ratio of the component A to the component B is (4-50): 1.

5. use of a cell lysate according to any one of claims 1 to 4 for the extraction of nucleic acids.

6. A method for extracting nucleic acid, comprising the steps of: 1) mixing a sample to be extracted with the component A of the cell lysate of any one of claims 1 to 4 to obtain a mixed solution A; 2) incubating the mixed solution A at 80-100 ℃ for 5-20 min; 3) mixing the incubated mixture A with the component B of the cell lysate according to any one of claims 1 to 4.

7. A kit, characterized in that: comprising a cell lysate according to any one of claims 1 to 4.

8. The kit of claim 7, wherein: the kit also comprises 2 xTaq Mix and sterile water.

9. The kit of claim 8, wherein: the 2 xTaq Mix comprises TaqDNA polymerase and MgCl₂、dNTPs。

10. Use of a cell lysate according to any one of claims 1 to 4 or a kit according to any one of claims 7 to 9 in a PCR assay.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cell lysate and application thereof.

Background

Gene editing technology is a technology for precisely manipulating DNA sequences in cells to change cell fates and biological characteristics, and provides an important tool for improving human understanding of genetics and treatment of genetic diseases. Particularly, the invention of the CRISPR-Cas9 gene editing technology brings revolutionary changes to the development of the gene editing technology. The CRISPR-Cas9 technology enables gene editing operation to be simple and quick, but still wastes time and labor in screening gene editing mutants, particularly cell gene editing mutant links. In order to obtain a homozygous gene editing cell strain, the gene editing cell is subjected to monoclonal screening, a single cell is inoculated into a 96-well microplate and cultured, the single cell is gradually amplified through a 48-well plate, a 24-well plate, a 12-well plate and a 6-well plate, and then a part of the cells is taken for nucleic acid extraction and genotype identification.

The extraction of cell nucleic acid is mostly performed by a centrifugal column method and a magnetic bead method, phenol chloroform extraction is used less because of toxicity of reagents, and the methods can not extract enough DNA from trace cells for PCR genotype identification, the cells need to be amplified to more than 6 holes for extraction, the time for waiting for cell amplification is long, and the steps are complicated. Patent document CN108841729A discloses a cell lysate for fungi and bacteria, which consists of NaOH, Sodium Dodecyl Sulfate (SDS) and distilled water, wherein the concentration of NaOH is 4mmol/L, and the weight percentage content of SDS is 2%; patent document CN107574166A discloses a cell lysate for extracting soybean seed genomic DNA, wherein the cell lysate is a pure water solution containing 0.015 to 0.025g/mL SDS, 0.04 to 0.06mol/L Tris-HCl, 0.04 to 0.06mol/L EDTA, and 0.14 to 0.16mol/L NaCl; the pH value of the cell lysate is 8; however, none of the above cell lysates can extract sufficient amounts of DNA from a trace amount of cells for genotyping by PCR.

Disclosure of Invention

In order to overcome the defect that the existing cell lysate cannot extract enough DNA from a trace amount of cells for PCR genotype identification, the invention aims at providing a cell lysate in a first aspect.

The second aspect of the present invention is to provide the use of the cell lysate for extracting nucleic acid.

The third aspect of the present invention is directed to a method for extracting nucleic acid.

In a fourth aspect, the present invention provides a kit comprising the cell lysate.

The fifth aspect of the present invention is directed to use of the cell lysate of the first aspect or the kit of the fourth aspect in PCR assays.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a cell lysate comprising a fraction a and a fraction B;

wherein the component A consists of 3-600 mmol/L alkali solution and 0.1-7 mmol/L SDS (sodium dodecyl sulfate);

the component B consists of 3-600 mmol/L Tris, 8-1100 mmol/L chlorate and 0.5-110 mmol/L EDTA, and the pH value is 7-10.

Preferably, the cell lysate consists of a component A and a component B;

wherein the component A consists of 5-200 mmol/L alkali solution and 0.2-3.5 mmol/L SDS (sodium dodecyl sulfate);

the component B consists of 5-500 mmol/L Tris, 10-1000 mmol/L chlorate and 1-100 mmol/L EDTA, and the pH value is 7-9.

Further preferably, a cell lysate is composed of a component A and a component B;

wherein, the component A consists of 100mmol/L alkali solution and 2.5 mmol/L-3.5 mmol/L SDS (sodium dodecyl sulfate);

the component B consists of 200mmol/L Tris, 150mmol/L chloride salt and 20mmol/L EDTA, and has the pH value of 8.

Preferably, the base is NaOH.

Preferably, the chloride salt is NaCl or KCl.

Preferably, the volume ratio of the component A to the component B in use is (3-50): 1; further preferably, the volume ratio of the component A to the component B is (4-50): 1.

in a second aspect of the invention, there is provided the use of the cell lysate as described above for the extraction of nucleic acids.

In a third aspect of the present invention, there is provided a method for extracting nucleic acid, comprising the steps of: 1) mixing a sample to be extracted with the component A of the cell lysate to obtain a mixed solution A; 2) incubating the mixed solution A at 80-100 ℃ for 5-20 min; 3) and mixing the incubated mixed solution A with the component B of the cell lysate.

In a fourth aspect of the invention, there is provided a kit comprising a cell lysate of the first aspect.

A kit comprising the cell lysate.

The kit further comprises 2 xTaq Mix, sterile water (ddH)₂O)。

The 2 xTaq Mix comprises TaqDNA polymerase and MgCl₂、dNTPs。

In a fifth aspect of the invention there is provided the use of a cell lysate of the first aspect or a kit of the fourth aspect in a PCR assay.

Preferably, the PCR is identified as monoclonal cell PCR identification; the monoclonal cell is a cell mass obtained by proliferation of 1 cell.

The invention has the beneficial effects that:

the cell lysate provided by the invention does not contain toxic reagents such as chloroform and the like, is composed of common reagents, has low cost and simple operation steps, can quickly release nucleic acid in cells, and greatly saves the time for extracting the nucleic acid; and can be used for treating a very small number of cells (the number of cells is 2X10 or less)⁴And) extracting a sufficient amount of nucleic acid for PCR identification.

Drawings

FIG. 1 is a photograph of agarose gel electrophoresis after PCR reaction after treatment of different modes of addition of the A component and the B component in example 1.

FIG. 2 is an agarose gel electrophoresis image of the lysates obtained in examples 4-7 after PCR reaction.

FIG. 3 is an agarose gel electrophoresis image of the cell lysate of example 3, the cell lysate of comparative example 1, and the cell lysate of comparative example 1 after 10-fold dilution after PCR reaction: wherein A is an agarose gel electrophoresis image after PCR reaction after treatment of the cell lysate of comparative example 1; b is an agarose gel electrophoresis picture of the cell lysate of the comparative example 1 diluted by 10 times after the PCR reaction; c is the agarose gel electrophoresis image after PCR reaction after treatment of the cell lysate of example 3.

FIG. 4 is a graph showing the success rate of PCR after treatment of the cell lysate of example 3, the cell lysate of comparative example 1, and the cell lysate of comparative example 1 diluted 10 times.

FIG. 5 is an agarose gel electrophoresis image after PCR reaction after treatment of cell lysate of example 3, comparative example 2, and comparative example 3.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Example 1 cell lysate

A cell lysate is composed of a component A and a component B, wherein the component A and the component B are prepared from the following raw materials:

the component A comprises: 5mmol/L NaOH, 0.2mmol/L sodium dodecyl sulfate;

and B component: 5mmol/L Tris, 10mmol/L KCl and 1mmol/L EDTA, pH 7.0;

when in use, the volume ratio of the component A to the component B is 3: 1.

Example 2 cell lysis solution

A cell lysate is composed of a component A and a component B, wherein the component A and the component B are prepared from the following raw materials:

the component A comprises: 100mmol/L NaOH, 2.5mmol/L sodium dodecyl sulfate;

and B component: 200mmol/L Trisl, 150mmol/L KCl, 20mmol/L EDTA, pH8.0;

when in use, the volume ratio of the component A to the component B is 3: 1.

Example 3 cell lysis solution

A cell lysate is composed of a component A and a component B, wherein the component A and the component B are prepared from the following raw materials:

the component A comprises: 200mmol/L NaOH, 3.5mmol/L sodium dodecyl sulfate;

and B component: 500mmol/L Tris, 1000mmol/L KCl, 100mmol/L EDTA, pH9.0;

when in use, the volume ratio of the component A to the component B is 3: 1.

Example 4A cell lysate

The cell lysate of this example is the same as that of example 2, except that: when in use, the volume ratio of the component A to the component B is 1: 1.

Example 5 cell lysate

The cell lysate of this example is the same as that of example 2, except that: when in use, the volume ratio of the component A to the component B is 4: 1.

Example 6 cell lysate

The cell lysate of this example is the same as that of example 2, except that: when in use, the volume ratio of the component A to the component B is 20: 1.

Example 7A cell lysate

The cell lysate of this example is the same as that of example 2, except that: when in use, the volume ratio of the component A to the component B is 50: 1.

Comparative example 1 cell lysate from ultra light velocity mix (available from polymere technologies, inc. of beijing) of M5.

Comparative example 2 patent document CN 108841729A.

Comparative example 3 patent document CN107574166A provides a cell lysate of example 1.

Effect example 1 Effect of addition of A-and B-Components on DNA extraction

(1) Fractional treatment of samples with component A and component B

1) 6 wells of the 96-well cell culture plate were inoculated with the same amount of 293T cells per well, followed by the next day and the like with adherent confluency (the number of cells was 1X 10)⁴～2x10⁴) Taking out from the incubator;

2) sucking the culture medium in the culture plate by using a pipette, adding 80 mu LA component into each hole, and then blowing and beating by using the pipette;

3) transferring the liquid into a 96-hole PCR plate, putting the plate into a PCR instrument, and incubating for 5 minutes at 95 ℃;

4) and (3) taking the 96-hole PCR plate out of the PCR instrument, adding 20 mu L B components into each hole, and blowing and uniformly mixing by using a pipette to obtain a cleavage product.

(2) The A component and the B component are added to the sample at the same time

1) 6 wells of the 96-well cell culture plate were inoculated with the same amount of 293T cells per well, followed by the next day and the like with adherent confluency (the number of cells was 1X 10)⁴～2x10⁴) Taking out from the incubator;

2) sucking the culture medium in the culture plate by using a pipette, adding 80 mu L A components and 20 mu L B components into each hole, and then blowing and beating by using the pipette;

3) transferring the liquid into a 96-hole PCR plate, putting the plate into a PCR instrument, and incubating for 5 minutes at 95 ℃;

4) the 96-well PCR plate was removed from the PCR instrument to obtain a lysate.

(3) PCR identification of cleavage Effect

3uL of the cleavage products were taken as templates, respectively, and subjected to PCR reaction (the PCR reaction system is shown in Table 1: primer F: CTTATTTTGATTTTACAAAGACAGTTAAG (SEQ ID NO. 1); primer R: CACAGTTCCTTTTTCTTTTGAATATAAC (SEQ ID NO. 2); and the reaction procedure is shown in Table 2), and then 10uLPCR products were taken and subjected to agarose gel electrophoresis, the results are shown in FIG. 1: the left side of the Mmarker is a result obtained after the samples are processed by the component A and the component B step by step, six strips exist, and the PCR success rate is 100 percent; the right side of the Mmarker is a result obtained after the component A and the component B are simultaneously added to the sample, four strips exist, the PCR success rate is 66.7%, and the brightness of the strip on the left side of the Mmarker is stronger than that of the strip on the right side of the Mmarker; the results show that the samples treated by the component A and the component B step by step have better effects.

TABLE 1 PCR reaction System

Reagent	Volume of
		Cleavage products	3μL
2x Taq Mix	25μL
		Primer F (10. mu.M)	1μL
Primer R (10. mu.M)	1μL
		Sterile water	To 50μL

TABLE 2 PCR reaction procedure

Effect example 2 Effect of volume ratio of A-component to B-component on cracking Effect

The lysates of examples 4-7 were taken to treat 293T cells (see (1) of reference effect example 1), 3uL lysates were taken as templates for PCR reaction (PCR reaction system is shown in table 1, reaction procedure is shown in table 2), 10uL PCR products were taken for agarose gel electrophoresis, and the results are shown in fig. 2: from left to right, the 1 st to 4 th wells showed the results of the lysate treatment of example 4, the 5 th and 18 th wells showed Mraker, the 6 th to 9 th wells showed the results of the lysate treatment of example 7, the 10 th to 13 th wells showed the results of the lysate treatment of example 6, and the 6 th to 9 th wells showed the results of the lysate treatment of example 5, which revealed that the best effect was obtained in example 4 (the volume ratio of the component A to the component B was 2:1) and the best effect was obtained in example 5 (the volume ratio of the component A to the component B was 4: 1).

EXAMPLE 3 comparison of the Effect of the cell lysate of the present invention and that of a commercially available cell lysate

293T cells were treated with the cell lysate of example 3 (part (1) of reference Effect example 1, the specific procedure), the cell lysate of M5 ultrafast mix (cell lysate provided in comparative example 1) and the cell lysate of M5 ultrafast mix diluted 10 times (part (2) of reference Effect example 1), respectively, and then 3uL lysate was used as a template for PCR reaction (PCR reaction system is shown in Table 1, reaction procedure is shown in Table 2), 10uLPCR products were subjected to agarose gel electrophoresis, and the results are shown in FIGS. 3A-C, 4: c is the result of the cell lysate treatment of example 3, with 24 bands and a PCR success rate of 100%; a is the result of cell lysate treatment in M5 ultra-light velocity mix, 9 strips exist, and the PCR success rate is 37.5%; b is the result of 10-fold dilution post-treatment of cell lysate in M5 ultra-light speed mix, 4 strips exist, and the PCR success rate is 16.7%; the effect of the cell lysate provided by the invention is obviously better than that of the commercial product.

EXAMPLE 3 comparison of the Effect of the cell lysate of the present invention with that of the cell lysate of patent documents CN108841729A and CN107574166A

The cell lysate of example 3 (part (1) of reference effect example 1, specific step, part (2) of reference effect example 1), the cell lysate of comparative example 2 (part (2) of reference effect example 1, specific step, and comparative example 3 (part (2) of reference effect example 1) were taken to treat 293T cells, 3uL lysate was taken as a template, PCR reaction was performed (PCR reaction system is shown in table 1, reaction procedure is shown in table 2), 10uL PCR product was taken to perform agarose gel electrophoresis, and the results are shown in fig. 5: from left to right, the 1 st to 6 th holes are the results after the cell lysate treatment of the comparative example 2, the PCR reaction fails, the target band is not amplified, and a plurality of miscellaneous bands exist; mraker is used in the 7 th and 14 th holes; 8 th to 13 th holes are the results of the cell lysate treatment of the comparative example 3, the PCR reaction fails, a target strip is not amplified, and a plurality of miscellaneous bands exist; the 15 th to 20 th holes are the results of the cell lysate treatment of the embodiment 3, the PCR success rate is 100%, and the strip specificity is single; the effect of the cell lysate provided by the invention is better than that of comparative examples 2 and 3.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Guangzhou Source well Biotechnology Ltd

<120> cell lysate and application thereof

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> DNA

<213> Artificial sequence

<400> 1

cttattttga ttttacaaag acagttaag 29

<210> 2

<211> 28

<212> DNA

<213> Artificial sequence

<400> 2

cacagttcct ttttcttttg aatataac 28