KR20020028387A - PURITY SEPARATION METHOD OF mtDNA FROM ANIMALS OF A LARGE QUANTITY - Google Patents

Abstract

PURPOSE: Provided is a method for separating mitochondrial DAN(mtDNA) from a small amount of animal tissues, thereby separating pure mtDNA from a large amount of animal tissues or cells without using chemicals, such as phenol and the like. CONSTITUTION: The separation method of mtDNA from a large amount of animal tissues comprises the steps of: adding a first buffer to a sample tissue in a mortar; transferring the first buffer to a homogenizing tube with a gauze then homogenizing it; dividing the supernatant into two test tube then followed by centrifuging them and transferring supernatants to another test tubes each; removing the supernatant then adding a second buffer to the each test tube with mixing; transferring the resultants to another test tubes each and centrifuging them; leaving a small amount of the supernatant each with stirring; adding a third buffer thereto with stirring then leaving them on ice; adding a forth buffer to the resultants with stirring then leaving them on ice; centrifuging them and transferring supernatants to another test tubes each; adding a fifth and a sixth buffer to the resultants with stirring then centrifuging them to remove supernatants; adding the fifth buffer to the resultants with stirring then removing supernatants; adding seventh buffer to the resultants with stirring then removing supernatants; drying the resultants with a vacuum pump; and adding an eighth buffer the resultant then followed by centrifuging and transferring supernatants to new clean test tubes each.

Description

Pure separation of mitochondrial D.N.A from a large amount of animal tissue {PURITY SEPARATION METHOD OF mtDNA FROM ANIMALS OF A LARGE QUANTITY}

The present invention relates to a pure separation method of mitochondrial D. N. (hereinafter referred to as mtDNA) from a large amount of animal tissue. In particular, the present invention relates to a pure separation method of mtDNA which can extract purely mtDNA from a large amount of animal tissue or animal 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. Since DNA obtained from living organisms or tissues and cells is present, as well as nuclear DNA, mtDNA and chloroplast DNA, as well as parasitic or symbiotic DNA, specific DNA can be amplified from these by PCR.

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 the mtDNA has an A + T rich part. When this part is amplified by the polymerase chain reaction (PCR) method, the pure mtDNA must be extracted to perform the amplification. Or to reduce failures, it was necessary to purely separate mtDNA.

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 tube, 1 ml of a homolytic buffer, and then 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).

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

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.

Accordingly, an object of the present invention is to solve the above-mentioned problems, and an object of the present invention is to purely separate mtDNA from a large amount of animal tissue, which allows to extract only pure mtDNA excluding this substance such as RNA from a large amount of animal tissue or animal cells. To provide a method.

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

A first step of putting the sample tissue in a mortar and a predetermined amount of a first buffer and then crushing it; Using a gauze to transfer the first buffer to the homogenizer tube and then homogenizing at a predetermined rate; Dividing the synergistic liquid into two tubes and centrifuging, then transferring the supernatant to another tube and centrifuging; A fourth step of removing all of the supernatant, adding a predetermined amount of the second buffer, flowing and mixing the same, and then centrifuging each of them to another tube; A fifth process of removing all the remaining amount of the supernatant and shaking it, putting a predetermined amount of the third buffer and shaking it a predetermined number of times, and leaving it on ice for a predetermined time; A sixth step of inserting a predetermined amount of the fourth buffering agent and shaking it a predetermined number of times, leaving it on ice for a predetermined time, and then centrifuging to transfer a predetermined amount of the supernatant to another tube; A seventh process of adding a predetermined amount of the fifth buffer and the sixth buffer and shaking the mixture, then centrifuging and removing the supernatant; An eighth step of inserting the fifth buffer and shaking and centrifuging to remove the supernatant; A ninth step of inserting a predetermined amount of the seventh buffer and shaking and centrifuging to remove the supernatant; A tenth step of completely drying using a vacuum pump; A pure separation method of mtDNA from a large amount of animal tissue is provided, comprising an eleventh step of inserting a predetermined amount of the eighth buffer and shaking, centrifuging, and transferring the supernatant to a clean tube.

Hereinafter, the pure separation method of mtDNA from a large amount of animal tissue according to the present invention will be described in more detail.

<Step 1>

: Add 1 mg of sample to mortar and add 20 ml of buffer and crush.

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

<Step 2>

: Using four layers of gauze, transfer the buffer of Procedure 1 to a homogenizer tube and homogenize at a speed of about 1,300 to 1,500 rpm.

<3rd course>

: Split into two 50 ml tubes and centrifuge (speed: 3,000 rpm, temperature: 4 ° C., time: 10 minutes), transfer the supernatant to a new 50 ml centrifuge tube and centrifuge (speed: 12,000 rpm, temperature: 4). ℃, time: 20 minutes).

<Step 4>

: Remove all supernatant, add 1000 μl of buffer, flow, mix, transfer to each new 2.0 ml tube and centrifuge (speed: 12,000 rpm, temperature: 4 ° C., time: 10 minutes).

At this time, the buffer used is 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.15 M NaCl.

<Step 5>

: Leave 100 μl of the supernatant and remove all. Vortex and add 200 μl of buffer (0.2 N NaOH, 0.1% SDS). Shake upside down 3-4 times and leave on ice for 5 minutes.

<Step 6>

: Add 150 μl of buffer (3M potassium, 5M acetate), shake upside down 3-4 times, stand on ice for 5 minutes, and centrifuge (speed: 13000 rpm, temperature: 4 ° C., time: 5 Min) transfer 400 μl of supernatant to a new 1.5 ml tube.

<Step 7>

: 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 8>

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

<Course 9>

: 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 supernatant. At this time, process 9 is repeated twice.

<10th course>

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

<Eleventh course>

: 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 11 twice.

As described above, according to the pure separation method of mtDNA from a large amount of animal tissue according to the present invention has the following advantages.

First, when mtDNA is extracted by the conventional method, this substance such as RNA is extracted together, but when mtDNA is extracted according to the present invention, since only mtDNA is extracted purely from a large amount of animal tissue, better results can be expected in the following process. have.

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 large amount of animal tissue, A first step of putting the sample tissue in a mortar and a predetermined amount of a first buffer and then crushing it; Using a gauze to transfer the first buffer to the homogenizer tube and then homogenizing at a predetermined rate; Dividing the supernatant into two tubes and centrifuging, then transferring the supernatant to another tube and centrifuging; A fourth step of removing all of the supernatant, adding a predetermined amount of the second buffer, flowing and mixing the same, and then centrifuging each of them to another tube; A fifth process of removing all the remaining amount of the supernatant and shaking it, putting a predetermined amount of the third buffer and shaking it a predetermined number of times, and leaving it on ice for a predetermined time; A sixth step of inserting a predetermined amount of the fourth buffering agent and shaking it a predetermined number of times, leaving it on ice for a predetermined time, and then centrifuging to transfer a predetermined amount of the supernatant to another tube; A seventh process of adding a predetermined amount of the fifth buffer and the sixth buffer and shaking the mixture, then centrifuging and removing the supernatant; An eighth step of inserting the fifth buffer and shaking and centrifuging to remove the supernatant; A ninth step of inserting a predetermined amount of the seventh buffer and shaking and centrifuging to remove the supernatant; A tenth step of completely drying using a vacuum pump; The method of pure separation of mtDNA from a large amount of animal tissues comprising the eleventh step of inserting a predetermined amount of the eighth buffer, shaking and centrifuging, and then transferring the supernatant to a clean tube. The method according to claim 1, wherein the sample tissue of the first process is 1 mg, a large amount of animal tissue, characterized in that the homogenizing at 1,300 ~ 1,500 rpm after transferring to the homogenizer tube through the first buffer in the second process Method for pure separation of mtDNA from The method of claim 1, wherein the centrifugation range in the third process is speed: 12000 rpm, temperature: 4 ° C., time: 20 minutes, and the centrifugation range in the fourth process is speed: 12000 rpm, temperature: 4 ° C., time. : 10 minutes, the centrifugation range in the sixth process is a speed: 13000rpm, temperature: 4 ℃, time: 5 minutes, characterized in that the pure separation of mtDNA from a large amount of animal tissue. The method according to claim 1, wherein the first 1,2,3,4 in the first, 1,2,3,4 buffer is 20 μl, 1000 μl, 200 μl, 150 μl, respectively, and the third, fourth buffer A pure separation method of mtDNA from a large amount of animal tissues, which is placed in a centrifuge tube and shaken upside down 3-4 times for 5 minutes 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 large 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 centrifuge tubes in the 7,8,9 process is a large amount of animal, characterized in that carried out within the range of speed: 13000rpm, temperature: room temperature (RT), time: 30 seconds Pure separation of mtDNA from tissues. The method of claim 1, wherein in the ninth and eleventh processes, the seventh and ninth processes are repeated twice in succession.