KR20020028386A - PURITY SEPARATION METHOD OF mtDNA FROM ANIMALS OF A SMALL AMOUNT - Google Patents

Abstract

The present invention relates to a method for pure separation of mitochondrial D.A. (mtDNA) from a small amount of animal tissue. In particular, the present invention relates to a pure separation method of mtDNA which can extract purely mtDNA from a small amount of animal tissue or animal cells without using chemicals such as phenol when extracting mtDNA.

According to the present invention, a predetermined amount of sample tissue is placed in a centrifuge tube, a predetermined amount of the first buffer is added, and then the tissue is crushed and centrifuged to transfer the supernatant to another centrifuge tube, followed by centrifugation. A first step of removing all of the supernatant; A second step of mixing a predetermined amount of the second buffering agent, then adding a predetermined amount of the third buffering agent and shaking it a predetermined number of times and leaving it on ice; A third step of putting a predetermined amount of the third buffer and shaking it a predetermined number of times and leaving it on ice; Inserting a predetermined amount of the fourth buffer agent, shaking a predetermined number of times, leaving it on ice, and centrifuging to transfer a predetermined amount of supernatant to a new centrifuge tube; A fifth step of adding a predetermined amount of the fifth buffer and the sixth buffer and shaking and centrifuging to remove the supernatant; A sixth step of inserting a predetermined amount of the fifth buffer and shaking and centrifuging to remove the supernatant; A seventh step of inserting a predetermined amount of the seventh buffer and shaking and centrifuging to remove the supernatant; An eighth step of completely drying using a vacuum pump; A pure separation method of mtDNA from a small amount of animal tissue is provided, which includes a ninth step of adding a predetermined amount of the eighth buffer and shaking and centrifuging the supernatant to a clean centrifuge tube.

Therefore, when mtDNA is extracted by the conventional method, foreign substances such as RNA are extracted together, but when mtDNA is extracted according to the present invention, only pure mtDNA is extracted, and thus better results can be expected in the following process.

Description

Purification method of mitochondrial D.N.A from small amount of animal tissue {PURITY SEPARATION METHOD OF mtDNA FROM ANIMALS OF A SMALL AMOUNT}

The present invention relates to a pure separation method of mitochondrial D. N. (hereinafter referred to as mtDNA) from a small amount of animal tissue. In particular, the present invention relates to a pure separation method of mtDNA capable of purely extracting mtDNA from a small amount of tissues or cells without using chemicals such as phenol when extracting mtDNA.

In general, extracting DNA required for experiments during genetic manipulation is one of the important processes. The DNA obtained from living organisms or tissues and cells contains not only nuclear DNA, but also mtDNA and chloroplast DNA, as well as parasitic or symbiotic DNA. PCR can be used to amplify.

However, some experiments require that only certain genes be extracted. For example, it was difficult to obtain satisfactory results with the above-described DNA extraction method in experiments in which a distinction between groups or species by the restriction enzyme map of mtDNA was desired. In this case, there was a need for pure separation of mtDNA.

In addition, most of mtDNA has A + T rich part. When this part is amplified by PCR method, pure mtDNA must be extracted. Therefore, only mtDNA must be purely separated in experiment or to reduce failure. The need to do so.

Although there is a pure separation method of mtDNA that has been developed so far, it takes a lot of time, and when mtDNA was separated, it was troublesome to process RNA to remove RNA by processing RNA enzyme (hereinafter referred to as RNAse).

Thus, a lot of existing mtDNA pure separation methods have been developed that can shorten the time and obtain clean mtDNA without RNAse treatment.

Each procedure below is a more advanced mtDNA pure separation method as a prior art.

Procedure 1) 50 mg of the sample is placed in a 1.5 ml centrifuge tube, 1 ml of tissue crushing buffer (homo buffer), crushed and centrifuged (speed: 3,000 rpm, temperature: 4 ° C., time: 2 minutes).

At this time, the tissue grinding buffer is 0.25 M sucrose (sucrose); 10 mM EDTA; 30 mM Tris-HCl, ph 7.5 is used.

Procedure 2) Transfer the supernatant to a new 1.5 ml tube and centrifuge (speed: 12,000 rpm, temperature: 4 ° C., time: 10 minutes).

Step 3) Remove all supernatant and add 100 μl of SET buffer at 4 ° C.

At this time, the SET buffer is 10 mM Tris; 10 mM EDTA; 0.15 M NaCl, pH 8.0 is used.

Procedure 4) Add 0.2 N NaOH 9: 1 10% SDS to 200 μl per 100 μl of the sample, mix slowly 2 to 3 times, and place on ice for 5 minutes.

Step 5) Add 150 μl of 3 M potassium 5 M acetate (acetate) at 4 ° C. per 100 μl of the sample, mix slowly 2 to 3 times, and place on ice for 5 minutes.

Procedure 6) Centrifuge (speed: 12,000 rpm, temperature: 4 ° C., time: 10 minutes) and transfer the supernatant to a new 1.5 ml tube.

Step 7) Saturated phenol-chloroform was added to the TE at 4 ° C., mixed in the same amount, and immediately centrifuged (speed: 12,000 rpm, temperature: 4 ° C., time: 5 minutes).

At this time, phenol 25: chloroform 24: isoamyl alcohol 1 (saturated) is saturated with TE, and then the lower layer of the mixed solution is used.

Step 8) Transfer the supernatant to a sterile 1.5 ml tube and add twice the ethanol (EtOH) or equivalent isopropanol and mix well.

Step 9) After allowing to stand at room temperature for 30 minutes, centrifugation (speed: 12,000 rpm, temperature: 4 ° C., time: 5 minutes).

Process 10) Remove the supernatant, add 800 μl of 70% EtOH and mix well.

Procedure 11) Centrifuge (speed: 12,000 rpm, temperature: 4 ° C, time: 5 minutes), remove all supernatant and dry thoroughly.

Process 12) 10 μl of TE (pH 8.0) was added to dissolve the mtDNA and finally stored at −20 ° C. to complete the pure mtDNA separation process.

At this time, the buffer was 10 mM Tris-HCl, ph 8.0; 20 μg / ml of 1 mM EDTA containing RNAse is used.

As described above, the extraction of mtDNA using a more advanced conventional mtDNA pure separation method also exposes the following problems.

First, when mtDNA is extracted, not only purely mtDNA is extracted, but also RNA is extracted together, resulting in the need for additional RNAse treatment to remove RNA.

Second, when mtDNA is extracted by the conventional method, chemicals such as phenol are used, so the more the experiment is conducted, the easier it is to be exposed to environmental pollution.

Third, when mtDNA was extracted by the conventional method, a considerable amount of time was required because DNA should be precipitated using drugs such as alcohol or isopropanol.

Therefore, the present invention is to solve the above problems, an object of the present invention is a pure separation method of mtDNA from a small amount of animal tissue to extract only pure mtDNA excluding foreign substances such as RNA from a small amount of animal tissue or animal cells To provide.

As the technical idea for achieving the above object of the present invention

A predetermined amount of sample tissue is placed in a centrifuge tube, a predetermined amount of the first buffer is added, the tissue is crushed and centrifuged, the supernatant is transferred to another centrifuge tube, centrifuged, and all the supernatant is removed. A first process of doing; A second step of mixing a predetermined amount of the second buffering agent, then adding a predetermined amount of the third buffering agent and shaking it a predetermined number of times and leaving it on ice; A third step of putting a predetermined amount of the third buffer and shaking it a predetermined number of times and leaving it on ice; Inserting a predetermined amount of the fourth buffer agent, shaking a predetermined number of times, leaving it on ice, and centrifuging to transfer a predetermined amount of supernatant to a new centrifuge tube; A fifth step of adding a predetermined amount of the fifth buffer and the sixth buffer and shaking and centrifuging to remove the supernatant; A sixth step of inserting a predetermined amount of the fifth buffer and shaking and centrifuging to remove the supernatant; A seventh step of inserting a predetermined amount of the seventh buffer and shaking and centrifuging to remove the supernatant; An eighth step of completely drying using a vacuum pump; A pure separation method of mtDNA from a small amount of animal tissue is provided, which includes a ninth step of adding a predetermined amount of the eighth buffer and shaking and centrifuging the supernatant to a clean centrifuge tube.

Hereinafter, a method for purely separating mtDNA from a small amount of animal tissue according to the present invention will be described in more detail.

<Step 1>

: Sample 100 mg of tissue was placed in 1.5 ml centrifuge tube, 300 μl of buffer was added, the tissue was crushed and centrifuged (rate: 3000 rpm, temperature: 4 ° C., time: 2 minutes) to supernatant fresh 1.5. Transfer to ml centrifuge tube, centrifuge (speed: 13000 rpm, temperature: 4 ° C., time: 5 minutes) and remove all supernatant.

At this time, the buffer used is 0.25 M Sucrose (Sucrose), 30 mM Tris-HCl (pH 7.5), 10 mM EDTA.

<Step 2>

: Add 50 μl of buffer (10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.15 M NaCl), shake, add 100 μl of buffer (0.2 N NaOH, 0.1% SDS), and invert 3-4 times. Shake and leave on ice for 5 minutes.

<3rd course>

: Add 100 μl of buffer (0.2 N NaOH, 0.1% SDS), shake 3-4 times upside down and leave on ice for 5 minutes.

At this time, precautions should never be over 5 minutes on ice.

<Step 4>

: Add 75 μl of buffer (3M Potassium 5M Acetate), shake 3-4 times upside down, leave on ice for 5 minutes, centrifuge (speed: 13000 rpm, temperature: 4 ° C, time: 5 minutes) 400 μl of supernatant is transferred to a new 1.5 ml tube.

<Step 5>

: Add 1200 μl of buffer (4M Guanidine Thiocyanate, 100 mM Tris-HCl pH <7.0) and 10 μl of buffer (Silica Gel (5μ Diameter)), shake, and centrifuge Speed: 13000 rpm, temperature: room temperature (hereafter; RT), time: 30 seconds) and the supernatant is removed.

<Step 6>

: Add 800 μl of buffer (Silica Gel (5 μ Diameter)), shake, centrifuge (speed: 13000 rpm, temperature: R.T., time: 30 seconds), and remove supernatant.

<Step 7>

: Add 1000 μl of buffer (10 mM Tris-HCl (pH = 7.5), 100 mM NaCl: Ethanol = 1: 4), shake, and centrifuge (speed: 13000 rpm, temperature: RT, time: 30 seconds) Remove the liquid. At this time, repeat step 7 twice.

<Step 8>

: Dry completely using a vacuum pump. At this time, care should be taken not to dry too long.

<Course 9>

: Add 20 μl of buffer (10 mM Tris-HCl, ph 8.0, 1 mM EDTA), shake, centrifuge (speed: 13000 rpm, temperature: R.T., time: 30 seconds), and transfer the supernatant to a clean tube. At this time, the procedure is terminated by repeating step 9 twice.

As described above, according to the pure separation method of mtDNA from a small amount of animal tissue according to the present invention, there are advantages as follows.

First, when mtDNA is extracted by the conventional method, foreign substances such as RNA are extracted together, but when mtDNA is extracted according to the present invention, only pure mtDNA is extracted, and thus better results can be expected in the following process.

Secondly, when mtDNA is extracted by the conventional method, Phenol is used, so the more the experiment is conducted, the more easily it is exposed to environmental pollution. However, when mtDNA is extracted according to the present invention, Phenol is not used at all. It is possible to extract mtDNA much faster than conventional methods.

Claims (7)

In the pure separation method of mtDNA which purely separates mitochondrial D.A. (mtDNA) from a small amount of animal tissue, A predetermined amount of sample tissue is placed in a centrifuge tube, a predetermined amount of the first buffer is added, the tissue is crushed and centrifuged, the supernatant is transferred to another centrifuge tube, centrifuged, and then the total amount of supernatant is removed. A first process of doing; A second step of mixing a predetermined amount of the second buffering agent, then adding a predetermined amount of the third buffering agent and shaking it a predetermined number of times and leaving it on ice; A third step of putting a predetermined amount of the third buffer and shaking it a predetermined number of times and leaving it on ice; Inserting a predetermined amount of the fourth buffer agent, shaking a predetermined number of times, leaving it on ice, and centrifuging to transfer a predetermined amount of supernatant to a new centrifuge tube; A fifth step of adding a predetermined amount of the fifth buffer and the sixth buffer and shaking and centrifuging to remove the supernatant; A sixth step of inserting a predetermined amount of the fifth buffer and shaking and centrifuging to remove the supernatant; A seventh step of inserting a predetermined amount of the seventh buffer and shaking and centrifuging to remove the supernatant; An eighth step of completely drying using a vacuum pump; The method of pure separation of mtDNA from a small amount of animal tissue, comprising the ninth step of inserting a predetermined amount of the eighth buffer and shaking and centrifugation, and then transfer the supernatant to a clean centrifuge tube. The method of claim 1, wherein the sample tissue of the first process is 100 mg, and the supernatant of the fourth process is 300 μl pure separation method of mtDNA from a small amount of animal tissue. The method of claim 1, wherein the centrifugation of another centrifugal tube of the first step and the centrifugal separation of the centrifugal tube of the fourth step are performed at a speed of 13000 rpm, a temperature of 4 ° C., and a time of 5 minutes, respectively. A method for purely separating mtDNA from a small amount of animal tissues. The method according to claim 1, wherein the 1,2,3,4 in the first, 1,2,3,4 buffer is 300 μl, 50 μl, 100 μl, 75 μl, respectively, the second, 3, 4 buffer A pure separation method of mtDNA from a small amount of animal tissues, which is placed in each centrifugal tube and shaken upside down 3-4 times and left on ice. The method according to claim 1, wherein the 5,6,7,8 in the process of the 5,6,7,8 buffer mtDNA from a small amount of animal tissue, characterized in that each of 1200 μl, 10 μl, 1000 μl, 20 μl Pure separation method. The method of claim 1, wherein the centrifugation of the centrifugal tube in the fifth, sixth, seventh, and ninth steps is performed in a range of speed: 13000 rpm, temperature: room temperature (RT), and time: 30 seconds. Method for pure separation of mtDNA from animal tissues. The method of claim 1, wherein in the seventh and ninth processes, the seventh and ninth processes are repeated twice in succession.