JPH0824583B2 - DNA extraction method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Field of Industrial Application> The present invention relates to Southern hybridization (Southern
hybridization) and the like for an efficient DNA extraction method for extracting white blood cell DNA.

<Conventional Technology> In recent years, with the remarkable development of gene manipulation technology, it is becoming possible to analyze various diseases at the gene level. For example, the genomic DNA is extracted from leukocytes of blood collected from peripheral blood vessels by a complicated method and then analyzed using a technique such as Southern hybridization to diagnose a genetic disease or the like.

In the Southern hybridization method, DNA is extracted from cells (white blood cells and lymphocytes), the DNA is cut with a restriction enzyme, the cut DNA is electrophoresed, and then transferred to a membrane filter (called blotting), Then, wash the membrane filter lightly and then dry it in an oven, preferably perform an operation to reduce nonspecific adsorption of the filter, and then add denatured probe DNA labeled with ³² P to perform hybridization. Radiographic defects in proviral DNA and genes,
It detects polymorphisms such as mutations, and conventionally, DNA has generally been extracted from leukocytes separated by the Ficoll method.

However, the Ficoll method described above is similar to that of "equal amount of phosphate-buffered saline (PB
S, PH7.4) and mixed well, gently injecting this into the Spitz tube containing the separating agent, overlaying the diluted blood sample on the separating agent, centrifuging at 400 g for 30 minutes, and then removing white blood cells. The upper layer was removed so as not to disturb the intermediate layer containing it, and the intermediate layer containing a little upper and lower layers was collected, collected in another Spitz tube, mixed with PBS and washed, and this time 160 g 10
Centrifuge for minutes, remove the supernatant, add PBS and mix.
After repeating the operation of centrifuging at 00g for 10 minutes twice, the supernatant is removed and suspended in a required solvent, and finally the number is counted with a microscope to adjust the number of white blood cells to the required number. The operation is very complicated, and the skill of the operator is required.

<Problems to be Solved by the Invention> The present invention has been made in view of the above circumstances, and an object thereof is to provide a DNA extraction method that is easy to operate, does not require skill, and is efficient. .

<Means for Solving the Problems> In order to solve the above problems, the present invention (a) adsorbs and separates leukocytes from blood using a filter having a container filled with a fibrous substance having an average diameter of 10 μm or less. To do. (I)
The white blood cells adsorbed on the filter are washed with a washing solution,
Remove proteins such as hemoglobin. (C) The white blood cells are frozen together with the filter at a low temperature of -20 ° C or lower, and then thawed at room temperature. The DNA extraction method including each step of is adopted.

<Operation> According to the present invention, when blood is passed through the filter, most leukocytes in the blood are adsorbed by the fibrous substance and separated from other components of the blood. For example, TE (10 mM tris [hydroxymethyl] aminoethane 1 mM EDTA, PH7.
Washing through 6) removes proteins such as hemoglobin. The white blood cells separated and washed in this manner are frozen together with the filter at, for example, −80 ° C., and then left at room temperature to thaw the white blood cells. Then, TE-mix (TE, 10 mM Na
cl, 1.5 mM Mgcl ₂ , PH7.5), leukocytes adsorbed on the fibrous material of the filter are easily recovered from the fibrous material.

The operation of extracting genomic DNA from the collected white blood cells is
It can be performed by a known method.

<Example> Next, an example of the present invention will be described.

In the DNA extraction method of the present invention, (a) leukocytes are adsorbed and separated from blood using a filter in which a container is filled with a fibrous substance having an average diameter of 10 μm or less. (A) The white blood cells adsorbed on the filter are washed with a washing solution to remove proteins such as hemoglobin. (C) minus 20 of the white blood cells
Freeze the whole filter at a low temperature below ℃ and thaw it at room temperature. After each step of thawing white blood cells,
Remove the fibrous material from the filter and put it into
-Add a buffer such as mix to collect leukocytes in an appropriate test tube such as a Spitz tube, and add 10% to this.
A surfactant such as sodium dodecyl sulfate (SDS) and a proteinase (Proteinase K) were added, and the mixture was incubated at 65 ° C for 15 hours, and then heat-treated, followed by RNAase (RNa
seA) and incubate at 37 ° C for 1 hour. Then, this is treated with a phenol reagent and the DNA is precipitated with ethanol for purification. The addition of proteolytic enzyme is for degrading the protein of cells containing DNA and the protein for binding DNA (Histone etc.), and the addition of RNaseA is due to the affinity of RNA and DNA.
This is because when the RNA is mixed with the DNA because it is larger than that of the DNA and the DNA, it may show an inhibitory effect on the probe DNA when the hybridization is performed.

As the fibrous substance, polyester or polyamide,
Cotton and polyacrylonitrile can be used, and polyester is particularly preferable because of high leukocyte recovery rate. The container filled with the fibrous substance has an opening through which blood can flow in and out, and examples thereof include a syringe and a case of a commercially available leukocyte removal filter. The washing solution for washing the leukocytes adsorbed on the filter is for removing proteins such as hemoglobin attached to the fibrous substance, and TE is generally used.

The freezing temperature of white blood cells is −2 ° C. or lower, preferably −20 ° C. or lower, particularly around −80 ° C. This is because cells are not damaged if they are frozen rapidly at around -80 ° C. It should be noted that the purpose of thawing at room temperature is to facilitate leukocyte detachment from the filter, and the buffer solution after thawing is used that keeps the pH constant without harming cells, and TE-mix is generally used. To be done. The reason that EDTA is added as a constituent element of TE is that it absorbs a divalent cation with EDTA and thereby inhibits the reaction of a DNA degrading enzyme (DNAase) that easily binds to the divalent cation. .

The surfactant is for lysing leukocytes, and when this is added, leukocytes are rapidly lysed and internal DNA and the like are released to make the solution viscous. The surfactant should be non-ionic and biochemically used.
Besides, NP-40, Tween, etc. can be used. A mixture of a buffer solution and a surfactant may be used to recover leukocytes, and a surfactant may be mixed with a protease to lyse cells.

For the operation after recovery of the white blood cells after thawing, for example, the method introduced in the left column of page 393, line 4 to 37 of the magazine “Clinical Examination” (vol.32 no.4 April 1988), etc. An appropriate method can be selected.

[Example 1] 10 ml of heparin blood (blood 9.5 ml plus heparin 0.5 ml) was taken in a test tube, and physiological saline 10 ml was added to the test tube. 3.5
In a filter filled with 80 mg of polyester fiber of μm,
It was poured while pulling with a peristaltic pump (dial scale 3) to adsorb leukocytes on the polyester fiber. Next, the blood in the syringe was discharged, and proteins such as hemoglobin attached to the polyester fiber were washed and removed with 10 ml of TE (which can be washed with any amount of 4 to 10 ml). Then, the white blood cells were frozen together with the syringe at −80 ° C. and then left at room temperature for thawing. Then, the polyester fiber was taken out from the syringe, and 4 ml of TE-mix was added thereto to wash and collect leukocytes. Next, 200 μl of 10% SDS and 50 μl of proteolytic enzyme (5 mg / ml) were added to the collected leukocytes and incubated at 65 ° C. for 15 hours. Then, 3 ml (10 mg / ml) of heat-treated RNA degrading enzyme was added thereto, and the mixture was incubated at 37 ° C. for 1 hour, treated with a phenol reagent, and leukocytes were precipitated with ethanol to purify DNA.

Table 1 shows the results of measuring the amount of white blood cells in the blood before and after passing the blood through the filter. It can be seen that about 93% of white blood cells were adsorbed and separated.

Further, FIG. 1 shows the results of measuring the absorbance by dissolving DNA in distilled water after ethanol precipitation. DNA recovery using Ficoll Hyperc (Pharmacia)
The pattern is almost the same as that obtained when the method was performed), indicating that the DNA has been recovered. Extinction coefficient is 10D = 50mg / ml
Since the DNA appears at a wavelength of 260 nm, the amount of DNA recovered using this method is 33.5 mg / ml, which is higher than that using the Ficoll method (more than 5.6 mg / ml).

[Example 2] DNA recovered by this method and Ficol method was treated with restriction enzyme Bg.
When digested with 11 and electrophoresed, a pattern as shown in FIG. 2 was obtained.

From each pattern, it is estimated that the method invented this time can be used as a material for genomic Southern.

The filter used in this method is the same as that used in the first embodiment.

[Example 3] In order to confirm that leukocytes recovered by this method can be used for Southern hybridization, DNA extracted by this method and Ficol method was digested with restriction enzyme BglI to stimulate thyroid gland. Hybridization was attempted using a gene encoding the β chain of the hormone as a probe (about 500 bp). The resulting autoradiogram is the third
Shown in the figure.

In both this method and Ficol method, one band was observed at around 4.4 kb. From this, the DNA extracted using this method is also Fic.
It can be seen that it can be used for actual analysis as well as the DNA extracted using the ol method.

The filter used in this method is the same as that used in the first embodiment.

<Effects of the Invention> According to the present invention, the following effects can be achieved.

(1) Since leukocytes are adsorbed and separated on fibrous substances and washed and collected, the operation is simple and the purity of leukocytes is high.

(2) Since white blood cells are frozen and thawed at room temperature,
Since the fibrous substance has almost no ability to be adsorbed, almost all the amount of adsorbed leukocytes can be easily and efficiently recovered.

(3) Since highly pure white blood cells can be easily and efficiently recovered, DNA can be extracted easily and efficiently.

[Brief description of drawings]

FIG. 1 shows that the DNA of the present invention was recovered by the Ficoll method.
FIG. 4 is a graph showing the absorbance of, wherein − ◯ − is the case where the present method is used, and −Δ− is the case where the Ficoll method is used. Also,
Fig. 2 is an electropherogram showing the pattern of DNA digested with BglI, and Fig. 3 is an autoradiogram of the same DNA used in Fig. 2 when hybridized with ³² P TSHβ probe. FIG.

Claims (3)

[Claims] 1. A) Leukocytes are adsorbed and separated from blood by using a filter having a container filled with a fibrous substance having an average diameter of 10 μm or less. (A) Leukocytes adsorbed on the filter are washed with a washing solution to remove proteins such as hemoglobin. (C) The white blood cells are frozen together with the filter at a low temperature of -20 ° C or lower, and then thawed at room temperature. Each step of
DNA extraction method. 2. The fibrous material is polyester, polyamide,
The method for extracting DNA according to claim 1, which comprises cotton or polyacrylonitrile. 3. The method for extracting DNA according to claim 2, wherein the washing solution is TE.