KR100563621B1 - Kit for extracting genomic dna from human blood and method for extracting genomic dna using the same - Google Patents

Abstract

The present invention relates to a kit consisting of reagents capable of extracting genomic DNA from human blood and a method for extracting genomic DNA from human blood using the same, wherein tris-HCl buffer of 20 mM, pH 7.5 in uncoagulated human blood Adding a solution kit for erythrocyte lysis comprising a solution to remove erythrocytes, plasma, platelet components, and the like and to obtain leukocytes only; A second step of eluting the leukocyte nuclei by adding a leukocyte lysis solution kit consisting of 10 mM, pH 8.0 Tris-HCl buffer solution, 1.0 mM EDTA and 1.0% SDS to the obtained leukocytes; A third step of separating genomic DNA and nuclear protein from the nuclei of the white blood cells by adding a solution kit for protein precipitation consisting of a sodium acetate solution of 3M and pH 5.2 to the nuclei of eluted white blood cells; And a fourth step of recovering the genomic DNA by adding a TE buffer solution kit for genomic DNA recovery consisting of 10 mM, pH 8.0 Tris-HCl buffer solution and 1.0 mM EDTA, to recover the genomic DNA. Provide extraction methods.

Blood, genomic DNA, genomic DNA extraction kit

Description

Kit for extracting genome DNA from human blood and method for extracting genome DNA using same {KIT FOR EXTRACTING GENOMIC DNA FROM HUMAN BLOOD AND METHOD FOR EXTRACTING GENOMIC DNA USING THE SAME}             

Figure 1a is a photograph showing a solution kit for erythrocyte lysis according to the present invention.

Figure 1b is a photograph showing a solution kit for leukocyte lysis according to the present invention.

Figure 1c is a photograph showing a solution kit for protein precipitation according to the present invention.

Figure 1d is a photograph showing a TE buffer solution kit for genomic DNA recovery according to the present invention.

Figure 2 is a photograph showing the results of Experimental Example 1 and Comparative Example 1 according to the present invention.

Figure 3 is a photograph showing the results of Experimental Example 2 according to the present invention.

Figure 4 is a photograph showing the results of Experimental Example 3 according to the present invention.

Figure 5a is a photograph showing the results of nucleotide sequence analysis of CYP450 3A4 in Experimental Example 4 according to the present invention.

Figure 5b is a photograph showing the results of the nucleotide sequence analysis of the photoliase in Experimental Example 4 according to the present invention.

Figure 5c is a photograph showing the results of nucleotide sequence analysis of RANKL in Experimental Example 4 according to the present invention.

Figure 5d is a photograph showing the nucleotide sequence analysis of the p53 gene in Experimental Example 4 according to the present invention.

Figure 5e is a photograph showing the results of the nucleotide sequence analysis of the pre-genelin-1 in Experimental Example 4 according to the present invention.

Figure 5f is a photograph showing the results of nucleotide sequence analysis of BRCA2 in Experimental Example 4 according to the present invention.

Figure 5g is a photograph showing the results of the nucleotide sequence analysis of aldehyde dehydrogenase 2 in Experimental Example 4 according to the present invention.

The present invention relates to a kit comprising reagents capable of extracting genomic DNA from human blood and a method of extracting genomic DNA from human blood using the same.

Recently, after the nucleotide sequence of human genomic DNA is known, molecular biological testing at the level of genomic DNA has been actively conducted for the purpose of curing or preventing human diseases. That is to say, for example, disease testing at the genetic level, testing of effects on newly developed drugs, tests to know in advance whether a blood donor is infected with a virus or bacteria, as well as forensics for identification of relatives or offenders. In various fields, the analysis of genomic DNA extracted from human cells is commonly performed today.

In order to ensure accuracy in such a gene-level test, it is important to extract a large amount of high-purity genomic DNA from human cells and repeat the experiment, thereby obtaining reproducible results. The conventional method of extracting genomic DNA from human cells, which has been developed so far to obtain high purity DNA, uses harmful organic solvents such as phenol and isoamyl alcohol, and various reagents such as proteolytic enzymes. Several steps are included, such as required washing steps, proteolytic steps, and the like. This method not only takes a lot of time by going through several steps, but also limits the processing of large amounts of samples simultaneously (Sambrook, J., EFFritsch, and T. Manatis, 1989. Molecular Cloning: A Laboratory Manual. CHS Laboratory Press, Cold Spring Harbor, NY.

Recently, various methods have been devised to extract genomic DNA more quickly and easily, but these methods are also similar to the conventional methods using harmful organic solvents such as phenol after protease treatment (Parzer, S., and C. Mannhalter. 1991. A rapid method for the isolation of genomic DNA from citrated whole blood.Biochem. J. 273 (Pt 1): 229-231 .; Albarino, CG and V. Romanowski. 1994. Phenol extraction revisited: a rapid methods for the isolation and preservation of human genomic DNA from whole blood.Mol. Cell Probes 8: 423-427) or time-consuming methods by requiring multiple steps in the extraction of genetic DNA from human blood (Wang, L., K. Hirayasu, M. Ishizawa, and T. Kobayashi. 1994. Purification of genomic DNA from human whole blood by isopropanol-fractionation with concentrated NaI and SDS.Nuclecic Acids Res. 22: 1774-1775 .; Robbins, V , MPAguinaga, and MS Valenzuela. 1995. Efficiencet isolation of whole genomic DNA frome cell cultures and blood samples.BioTechniques 18: 414-418. In 2001, Sambrook, J. et al. Used harmful organic solvents such as phenol by omitting the process of proteolytic enzymes, which take up to 16 hours of genomic DNA extraction from human blood, to ameliorate these problems. We devised a method that shortened the time and reduced the time (Sambrook. J, and DWRussell. 2001. Molecular Cloning: A Laboratory Manual. CHS Laboratory Press. Cold Spring Harbor. NY) It is not suitable for the purpose of extracting various genomic DNA from. In addition, a genome DNA extraction kit (QIAGEN Inc. Valencia. USA) has been released that improves the above problems, but this kit also has a problem in that it is expensive to extract a large amount of genomic DNA from a large amount of human blood.

An object of the present invention is to provide a method for extracting a large amount of high-purity genomic DNA quickly and easily from a large amount of human blood.                         

Another object of the present invention is to provide a commercialized kit consisting of a reagent capable of quickly and simply extracting a large amount of high purity genomic DNA from a large amount of human blood.

In the present invention, as a result of repeated studies to achieve the above technical problem, by limiting the target of the sample for genomic DNA extraction only in human blood, it is used to extract genomic DNA from the existing human cells The present invention has been completed by focusing on the fact that various reagents can be simplified with only a few reagents, and even a small amount of reagents can be used to quickly and easily extract high-purity genomic DNA from a large amount of samples.

The present invention, (a) solution kit for erythrocyte lysis consisting of Tris-HCl buffer solution of pH 7.5 of 20mM; (b) a solution kit for leukocyte lysis consisting of Tris-HCl buffer solution at pH 8.0 of 10 mM, 1.0 mM EDTA and 0.1% sodium dodecyl sulfate (SDS); (c) a solution kit for protein precipitation consisting of sodium acetate at pH 5.2 of 3M; And (d) TE (Tris-HCl.EDTA) buffer solution kit for genome DNA recovery consisting of Tris-HCl buffer solution at pH 8.0 of 10 mM and 1.0 mM EDTA. will be.

In addition, the present invention, the first step of obtaining only white blood cells by removing red blood cells, plasma, platelet components and the like in human blood; Eluting the nuclei of the obtained leukocytes; A third step of separating genomic DNA and nuclear protein from the nuclei of eluted white blood cells; And a fourth step of recovering the separated genomic DNA. The present invention relates to a method of extracting genomic DNA from human blood. More specifically, by adding Tris-HCl buffer solution of 20 mM, pH 7.5 to uncoagulated human blood, A first step of removing red blood cells, plasma, platelet components and the like and obtaining only white blood cells; A second step of eluting the leukocyte nuclei by adding 10 mM, Tris-HCl buffer solution of pH 8.0, 1.0 mM EDTA and 1.0% SDS to the obtained leukocytes; A third step of separating genomic DNA and nuclear protein from the nuclei of the white blood cells by adding a sodium acetate solution of 3M, pH 5.2 to the nuclei of eluted white blood cells; And a fourth step of recovering the genomic DNA by adding 10 mM, Tris-HCl buffer solution at pH 8.0, and 1.0 mM EDTA to the separated genomic DNA; and a method for extracting genomic DNA from human blood.

The reagent kit for genomic DNA extraction and the genomic DNA extraction method of the present invention described above are focused on the characteristics of blood. Blood is composed of plasma and cellular components, and blood coagulants, such as heparin, are injected into the test tube, and the blood is separated into transparent pale yellow liquid (plasma) and cellular components (red blood cells, platelets, white blood cells, etc.). . After removing the plasma components by tilting the test tube, when the saline solution with a lower osmotic pressure than the blood is introduced into the cell components, water is sucked into the blood cells to cause a hemolysis phenomenon. In the present invention, the anticoagulant is first treated using the characteristics of the blood. Plasma components are removed from the blood, and a low concentration buffer solution is added to the blood to remove red blood cells, platelets, and a small amount of plasma proteins that do not contain nuclei in advance. In this case, the plasma component may be removed together with a low concentration buffer solution to remove red blood cells, platelets, and the like. Subsequently, the remaining white blood cells are contacted with a surfactant such as SDS at a low pH so that the nucleus containing the genomic DNA in the white blood cells is separated and eluted from the cell membrane, cytoplasm, and the like forming the white blood cells. When an acetate such as sodium acetate is added thereto, the genomic DNA and the nuclear protein are separated from the eluted nucleus, and the nuclear protein is present in the precipitated state by centrifugation. Thus, the genomic DNA is obtained by taking only the supernatant. can do. The genomic DNA is precipitated by isopropyl alcohol, and then purified by ethanol washing to form high-purity genomic DNA containing no contaminants, and the polymerase chain reaction (PCR) and restriction enzymes are applied to the genomic DNA. Molecular biological tests such as treatment, sequencing and the like can be preferably performed.

Hereinafter, a kit for extracting genomic DNA from human blood and a method of extracting genomic DNA using the same according to the present invention will be described in more detail with reference to the following examples and accompanying drawings. The following examples and the accompanying drawings are not intended to limit the invention, the invention may be appropriately modified and modified based on the matter described in the claims.

Example

Step 1: 300 μl of blood treated with heparin and 1.2 mL of Tris-HCl buffer solution at pH 7.5 of 20 mM were placed in a 1.5 mL microcentrifuge tube, vortex mixed, and allowed to stand at room temperature for 10 minutes. Subsequently, this was centrifuged at 16,000 xg for 30 seconds in a microcentrifuge, and the supernatant containing hemolyzed red blood cells, platelets, serum, and the like was tilted to remove the tube. The white blood cells remaining at the bottom of the tube were added to Tris-HCl buffer solution at pH 7.5 of 20 mM again and washed three times. The 20 mM pH 7.5 Tris-HCl buffer solution was kitted as a solution for erythrocyte dissolution (see FIG. 1A, solution kit for red blood cell lysis) and stored at room temperature.

Step 2: After releasing the white blood cells remaining in the bottom of the tube after washing in the first step with a vortex mixer, a mixed solution of Tris-HCl buffer solution, 1.0 mM EDTA, and 0.1% SDS 600, pH 8.0 of 10 mM After addition of [mu] l, the nuclei of white blood cells were eluted by stirring in an orbital shaker for 30 minutes. The mixed solution of Tris-HCl buffer solution, 1.0 mM EDTA, and 0.1% SDS at pH 8.0 of 10 mM was kitted as a solution for leukocyte dissolution (see FIG. 1B, solution kit II for leukocyte lysis) and stored at room temperature.

Step 3: After adding 200 μl of 3M sodium acetate having a pH of 5.2 to the tube from which the nuclei of the leukocyte cells were eluted, vortex-mixed and centrifuged at 16,000 × g for 3 minutes in a 4 ° C. microcentrifuge, the nuclear protein By precipitating a large proportion of components, including the genomic DNA and nuclear protein of the leukocyte nucleus was separated from each other. The 3M sodium acetate solution at pH 5.2 was kitted as a solution for protein precipitation (see FIG. 1C, solution kit III for protein precipitation) and stored at room temperature.

Step 4: In the third step, 800 µl of the supernatant present in the tube after centrifugation was transferred to another 1.5 ml microcentrifuge tube, and 600 µl of isopropyl alcohol was added thereto, followed by mixing well to obtain genomic DNA. Precipitated. Centrifuge the tube for 1 min at 16,000 × g in a micro-centrifuge, then carefully decant the supernatant and use sorbent paper to completely remove the residual fluid around the pellet without loss of the genomic DNA pellets present at the bottom of the tube. Removed. 300 μl of 70% ethanol was added to the genomic DNA pellet, and the tube was shaken up and down several times to wash the genomic DNA pellet. After centrifugation for 1 minute at 16,000 x g again, the supernatant was discarded and the remaining liquid around the genomic DNA pellet at the bottom of the tube was removed by absorbing with absorbent paper and dried at room temperature for 10 minutes.

The genomic DNA pellet was dissolved by adding 100 μl of a 10 mM pH 8.0 Tris-HCl buffer solution and 1.0 mM EDTA mixture to the genomic DNA pellet at the bottom of the tube. The lysed genomic DNA was stored at -20 ~ -80 ℃ for long term storage. The 10 mM pH 8.0 Tris-HCl buffer solution and 1.0 mM EDTA mixed solution were kitted as TE buffer solution for genomic DNA recovery (see FIG. 1D, TE buffer solution kit IV for genomic DNA recovery) and stored at room temperature. It took a total of about 1 hour to perform the above four steps.

According to the present invention, the following experiments were performed on genomic DNA obtained by a method of extracting genomic DNA from human blood to determine its purity.

Experimental Example 1 Electrophoresis

A 0.7% agarose gel and 1 × TBE buffer solution were prepared and the electrophoresis of the genomic DNA obtained by the method of the present invention under electrophoresis conditions of 10 V / cm. After staining with 0.5 µg / ml ethidium bromide, the result of electrophoresis was shown in a polaroid photograph under a 300 nm ultraviolet light emitter.

Comparative Example 1 Electrophoresis

Under the same conditions as in Experimental Example 1, electrophoresis was performed on the genomic DNA obtained by the Sambrook method and the commercial DNA extraction kit using the commercial gene extraction kit. Shown in

In Fig. 2, lane M shows the size marker using the NEB 2-log DNA ladder as the standard DNA marker, and lane 1 and lane 2 show the results of electrophoresis of genomic DNA obtained by the method of the present invention. In addition, lanes 3 and 4 show electrophoresis results of genomic DNA obtained by the Sambrook method, and lanes 5 and 6 show electrophoresis results of genomic DNA obtained using a commercially available gene extraction kit.

As shown in Fig. 2, the electrophoresis results of the genomic DNA obtained by the method of the present invention are the electrophoresis results of the genomic DNA obtained by the conventional Sambrook method or the genomic DNA obtained by using a commercial gene extraction kit. There was little difference from the electrophoresis results and only a single clean light emission was reproduced. Therefore, it can be confirmed that even by the method of the present invention, highly contaminated genomic DNAs such as proteins and RNAs can be obtained.

Experimental Example 2 Restriction Enzyme Cleavage

Restriction enzymes such as HindIII, MspI, PvuII, RsaI, NotI and SacI were prepared, and the genomic DNA obtained by the method of the present invention was treated with the restriction enzymes, followed by electrophoresis under the same conditions as in Experimental Example 1. The results are shown in FIG.

In Fig. 3, lane M represents a size marker using the NEB 2-log DNA ladder as a standard DNA marker, and lane 1, lane 2, lane 3, lane 4, lane 5 and lane 6 in turn, HindIII, MspI, PvuII. , RsaI, NotI and SacI were treated with genomic DNA obtained by the method of the present invention, followed by electrophoresis.

As shown in Fig. 3, the result of electrophoresis after treatment of the genomic DNA obtained by the method of the present invention with a restriction enzyme showed that the genomic DNA was in the correct position by the restriction enzymes as compared with the standard DNA marker. It can be confirmed that the cut. Therefore, according to the method of the present invention, it can be seen that high-purity genomic DNA can be obtained in which contamination by proteins, RNA, or the like and mutations due to specific factors do not occur.

Experimental Example 3 PCR-electrophoresis

The genomic DNA obtained by the method of the present invention was PCR for the CYP450 3A4, photolyase, RANKL, p53 gene, presenilin-1, BRCA2 and aldehyde dehydrogenase2 sites, respectively. A 1.5% agarose gel and a 1 × TBE buffer solution were prepared, and the product of the PCR result was subjected to electrophoresis under electrophoresis conditions of 10 V / cm, followed by staining with 0.5 µg / ml ethidium bromide. Indicated.

In Figure 4, lane M represents the size marker using the NEB 2-log DNA ladder as the standard DNA marker, lane 1, lane 2, lane 3, lane 4, lane 5, lane 6 and lane 7 in turn, of the present invention The result of electrophoresis after PCR on the CYP450 3A4, photolyase, RANKL, p53 gene, pregenillin-1, BRCA2 and aldehyde dehydrogenase2 sites in the genomic DNA obtained by the method,

As shown in FIG. 4, the result of electrophoresis after PCR of a portion corresponding to a specific gene in the genomic DNA obtained by the method of the present invention, shows a distinct luminescence at the exact position expected when compared with a standard DNA marker. From the genomic DNA, the parts corresponding to the specific genes are exactly present, and it can be confirmed that the PCR was amplified without contamination. Therefore, according to the method of the present invention, it can be seen that high-purity genomic DNA can be obtained without contamination by protein, RNA, or the like and without mutation.

Experimental Example 4 PCR-Base Sequence Analysis

The genomic DNA obtained by the method of the present invention was PCR for the CYP450 3A4, photolyase, RANKL, p53 gene, pregenillin-1, BRCA2 and aldehyde dehydrogenase2 sites, respectively. Each base sequence was analyzed for the PCR product, and the results are sequentially shown in FIGS. 5A to 5G.

As shown in Figures 5a to 5g, each of the genomic DNA obtained by the method of the present invention after PCR, the results of each sequence analysis of the PCR product, was analyzed without error with the sequence of each gene analyzed previously, Therefore, according to the method of the present invention, it can be seen that high-purity genomic DNA can be obtained without contamination by protein, RNA, or the like and without mutation.

According to the present invention, since genome DNA in the leukocyte nucleus is to be extracted from only white blood cells, which are blood cells among human cells, genomic DNA can be obtained economically and simply by simplifying the reagents used and by using a small amount of the reagents. have.

In addition, by kitting a reagent capable of extracting genomic DNA from human blood, the genome DNA extraction step can be simplified in four steps, and genomic DNA can be extracted from a large volume of samples more quickly. The resulting genomic DNA has a purity that is similar to that of using conventional methods or commercially available kits, and thus can be suitably used for genetic level inspection and the like.

Claims (3)

(a) a solution kit for erythrocyte lysis consisting of Tris-HCl buffer solution at pH 7.5 of 20 mM; (b) a solution kit for leukocyte lysis consisting of Tris-HCl buffer solution at pH 8.0 of 10 mM, 1.0 mM EDTA and 0.1% SDS; (c) a solution kit for protein precipitation consisting of sodium acetate at pH 5.2 of 3M; And (d) a TE buffer solution kit for genomic DNA recovery comprising Tris-HCl buffer solution at pH 8.0 of 10 mM and 1.0 mM EDTA; Reagent kit for genomic DNA extraction from human blood. delete Adding a 20 mM, pH 7.5 Tris-HCl buffer solution to uncoagulated human blood to remove erythrocytes, plasma, platelet components, and the like and obtain only white blood cells; A second step of eluting the leukocyte nuclei by adding 10 mM, Tris-HCl buffer solution of pH 8.0, 1.0 mM EDTA and 1.0% SDS to the obtained leukocytes; A third step of separating genomic DNA and nuclear protein from the nuclei of the white blood cells by adding a sodium acetate solution of 3M, pH 5.2 to the nuclei of eluted white blood cells; And A fourth step of recovering the genomic DNA by adding 10 mM, Tris-HCl buffer solution at pH 8.0 and 1.0 mM EDTA to the separated genomic DNA; A method of extracting genomic DNA from human blood.

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