KR20110035476A - Method and apparatus for isolating nucleic acids - Google Patents

Abstract

PURPOSE: A method for isolating nucleic acids is provided to effectively elute nucleic acid of high concentration. CONSTITUTION: A method for isolating nucleic acid comprises: a step of contacting nucleic acids with a solid support(120); a step of adding an effluent to a nucleic acid-solid support to dispersing the nucleic acid-solid support complex to the effluent; a step of eluting the nucleic acid from the nucleic acid-solid support; and a step of washing unbound sample.

Description

Method and apparatus for isolating nucleic acids

A method for efficiently separating nucleic acids and a nucleic acid separation apparatus.

Nucleic acid separation devices currently on the market can be divided into a method using magnetic beads and a method using a filter column. The devices can separate nucleic acids in amounts of several tens to several tens of micrograms, and the concentration of the extracted nucleic acids is in the order of several tens to ng / μl. In the process of molecular diagnosis in which nucleic acid is used, a high concentration of nucleic acid of several hundred ng / μl or more may be required, but the apparatus has a limitation in obtaining a high concentration of nucleic acid eluate.

In order to obtain a high concentration of nucleic acid, the volume of the eluate should be about several to several tens of microliters. However, in the case of a nucleic acid separation apparatus using magnetic beads, when the magnetic beads using the magnetic field are collected during the nucleic acid separation process, the beads move together with the dispersed eluate. Therefore, it is difficult to recover the eluate and the elution efficiency is lowered. In addition, in a nucleic acid separation apparatus using a filter column, the nucleic acid is bound to the entire surface area of the filter in the column during the elution process. Therefore, it is difficult to elute the whole bound nucleic acid with only a small amount of eluate, and it is difficult to elute a high concentration of nucleic acid.

Therefore, even with the prior art, there is still a need for a method for more efficiently separating nucleic acids and related devices.

It provides a method for separating nucleic acid comprising the step of dispersing the nucleic acid-solid support complex in the eluate.

Provided is a nucleic acid separation apparatus comprising a dispersion unit disposed in a filter column into which a solid support or nucleic acid-solid support complex having a property of binding to a nucleic acid can be filled.

One aspect comprises contacting a sample comprising nucleic acid with a solid support having the property of binding a nucleic acid to bind the nucleic acid to the solid support; Adding an eluate to the nucleic acid-solid support complex and dispersing the nucleic acid-solid support complex in the eluate; And it provides a nucleic acid separation method comprising the step of eluting the nucleic acid from the nucleic acid-solid support.

The method includes contacting a sample comprising nucleic acid with a solid support having the property of binding to the nucleic acid to bind the nucleic acid to the solid support.

The term "nucleic acid" is used to mean DNA (gDNA and cDNA) and RNA molecules. Nucleotide, the basic structural unit of a nucleic acid, is interpreted to include not only natural nucleotides but also analogs in which sugar or base sites are modified.

In this step, the contact with the solid support having the property of binding the sample containing the nucleic acid to the nucleic acid may be carried out by, for example, by adding a sample containing the nucleic acid to the solid support directly mixing. .

The sample used in the contacting step may be any sample containing a nucleic acid. Samples containing nucleic acids may be biological or non-biological samples. Such biological samples include, for example, viruses, bacteria, tissues or cells, and the like. The non-biological sample includes, but is not limited to, for example, a synthesized nucleic acid product or an amplification product by PCR. In addition, the biological sample may be applied to the method by extracting the extract of the biological sample according to various methods known in the art to facilitate the binding of the nucleic acid contained in the sample with the solid support. .

The solid support that can be used in the contacting step can be selected from the group consisting of slides, wafers, beads, membranes and plates, for example, beads can be used . The beads may be selected from the group consisting of magnetic beads, silica beads, polystyrene beads, glass beads, and cellulose beads, but are not limited thereto.

The solid support may have a property of binding to a nucleic acid. That is, the solid support has a property of binding to a nucleic acid through non-covalent bonds such as ionic bonds and adsorption with the nucleic acid. For example, the solid support may be coated with a functional group capable of binding a nucleic acid to itself or the surface of the solid support. The functional group that can be coated on the solid support may be, for example, an amine-based or carboxyl-based functional group, but is not limited thereto.

The method may further comprise washing the unbound sample after the contacting step. The washing can be done using washing solutions well known in the art. The washing liquid may be any substance as long as it has a property of removing a substance such as nucleic acid not attached to the solid support without leaving the nucleic acid bound to the solid support. For example, the wash liquid is a solution having the above characteristics and capable of removing impurities that may adversely affect subsequent processes. For example, after the contacting step, 70% of ethanol can be added to the solid support, and the sample not bound to the solid support can be washed by centrifugation or drying. Through the washing step, it is possible to remove impurities contained in the sample containing the nucleic acid. Thus, by removing impurities that may be present in the nucleic acid and the solid support complex, it is possible to more efficiently elution of the nucleic acid in a later step, it is possible to increase the purity of the nucleic acid.

The method also includes adding an eluate to the nucleic acid-solid support complex and dispersing the nucleic acid-solid support complex in the eluate.

In this step, the addition of the eluate is carried out to separate the nucleic acid bound to the nucleic acid-solid support complex formed in the contacting step from the solid support. The term "nucleic acid-solid support complex" refers to a complex in which the nucleic acid and the solid support are formed in the contacting step through non-covalent bonds such as hydrogen bonds, van der Waals forces, ionic bonds or hydrophobic interactions. Means.

The eluate may preferably have a pH of 6 to 10, and the nucleic acid-solid support complex may be dispersed using an eluent known in the art within the pH range, for example, water or Tris-HCl. have. In addition, in order to isolate nucleic acids at high concentration, the amount of the eluate may be used to be 10 μl to 500 μl, 10 μl to 200 μl or 10 μl to 100 μl.

The addition can be made with the application of pressurized gas or ultrasonic waves. The nucleic acid-solid support complex may be dispersed in the eluate by pressurized gas or ultrasonic application. When using the pressurized gas in the adding step, the pressurized gas may be used to have a pressure of 0.01 Pa to 150 kPa, 0.1 Pa to 100 kPa or 1 Pa to 50 kPa relative to atmospheric pressure. In addition, when using ultrasonic waves, the ultrasonic waves may be applied at a frequency of 15 to 400 kHz, 30 to 200 KHz or 50 to 100 KHz.

The method also includes eluting nucleic acid from the nucleic acid-solid support.

In this step, elution of the nucleic acid means that the nucleic acid is separated into the eluate from the nucleic acid-solid support complex through the dispersion process. In addition, it is interpreted to mean including the step of separating the eluted eluate from the solid support. As described above, by using an external physical force generated from the pressurized gas or ultrasonic waves, the nucleic acid bound to the solid support can be separated more efficiently with chemical separation by the eluate. The eluate containing the isolated nucleic acid can be separated from the solid support through a separation method well known in the art, for example, centrifugation, pressurized gas, vacuum, and the like.

Another aspect includes a filter column into which a solid support or nucleic acid-solid support complex having the property of binding to a nucleic acid can be filled; And a dispersion unit disposed in the filter column for dispersing the solid support or the nucleic acid-solid support complex in a liquid.

The description of the solid support having the property of binding to the nucleic acid is as described above. The filter column in which the solid support can be filled may be filled directly with a commercially available empty filter column, or a commercially available filter column filled filter support may be used according to methods well known in the art. have. In addition, the solid support that can be filled in the filter column may be already bound to the nucleic acid. That is, the filter column may be filled in the form of the nucleic acid-solid support complex.

The filter column may be, for example, a spin column of Qiagen. In addition, the material of the filter column may be an ultrasonically transmissive material, for example, but may be selected from the group consisting of metal, ceramic, glass and plastic, but is not limited thereto. The shape of the filter column may be, for example, a body having a cylindrical shape and a lower portion having an open cone shape. In addition, the solid support or the nucleic acid-solid support complex may be filled at the bottom of the filter column. The lower opening of the filter column may include a filter including a pore having a size smaller than that of the solid support or the nucleic acid-solid support complex so that the solid support or the nucleic acid-solid support complex does not flow out.

The dispersion unit may be a unit capable of dispersing the solid support or the nucleic acid-solid support complex in a liquid. The dispersion unit may comprise, for example, a pressurizing device for applying pressure to the solid support or the nucleic acid-solid support complex or a sonicator for providing ultrasonic waves. In addition, the pressurizing device or the ultrasonic wave generating device may be connected in fluid communication with the upper or lower opening in the filter column. For example, in the filter column, the nucleic acid eluate may be fluidly connected to the end of the opening in the elution direction or the opposite direction. In addition, the pressurizing device or the ultrasonic wave generating device may be mounted to be detachable. For example, the pressurizing device may be connected to the bottom opening of the filter column through a rubber tube.

Meanwhile, the cover may further include a cover for opening and closing the opening of the filter column at an opposite end to which the pressurizing device or the ultrasonic wave generating device is connected to prevent the material in the filter column from flowing out. Therefore, a sample containing a nucleic acid, a nucleic acid-solid support complex, a washing solution or an eluate can be injected into the filter column manually with the cover open. In addition, the cover may further include an air vent (vent hole) through which air can be circulated.

As the dispersion unit in the apparatus, when the pressurizing device is mounted, the pressurizing device may be connected in fluid communication with the end of the opening in the elution direction of the nucleic acid eluate in the filter column. The pressurizing device may be a device capable of adjusting the pressure through the fourth adjusting unit. By adjusting the pressurizing device, for example, pressurized nitrogen gas may be injected into the liquid containing the nucleic acid-solid support complex present on the filter column within the above-mentioned pressure range to allow bubbling. Can be.

When the ultrasonic generator is mounted as the dispersion unit in the apparatus, the vibrator of the ultrasonic generator may be mounted inside the filter column. At this time, the vibrator vibrates the liquid containing the nucleic acid-solid support complex to disperse the nucleic acid-solid support complex in the liquid.

The nucleic acid separation device may further include a removable wash solution reservoir for supplying a wash solution to the filter column. The apparatus may further include a detachable eluent storage unit for supplying an eluent to the filter column. The nucleic acid separation apparatus may further include a removable sample storage unit for supplying a sample to the filter column.

The wash solution reservoir, the eluate reservoir or the sample reservoir may be respectively connected to an opening end in the elution direction or the opposite direction of the nucleic acid in the filter column. The wash solution reservoir, the eluate reservoir or the sample reservoir may be mounted by forming a passage that can be connected to the wash solution reservoir, the eluate reservoir or the sample reservoir at the end of the opening in the elution direction or the opposite direction of the nucleic acid of the filter column. Can be. In this case, the washing liquid storage unit, the eluent storage unit or the sample storage unit may be connected through a tube to be in fluid communication with the filter column. On the other hand, the washing liquid, eluent or sample supplied to the filter column from the washing liquid storage unit, eluent storage unit or the sample storage unit is the first control unit, the second control unit connected to the washing liquid storage unit, eluent storage unit or the sample storage unit Or it can be adjusted manually or automatically by the third control unit. In addition, the waste liquid storage portion capable of storing the waste liquid generated from the filter column may be in fluid communication with the end of the opening in the elution direction of the nucleic acid of the filter column.

According to the method for separating nucleic acid at a high concentration and the nucleic acid separation apparatus, a high concentration of nucleic acid can be efficiently eluted and separated from a sample containing the nucleic acid in a small amount of eluate.

Hereinafter, one or more embodiments will be described in more detail with reference to the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, but the scope of the present invention is not limited to these examples.

Example  1: nucleic acid separation device

1 to 3 is a schematic diagram of a nucleic acid separation apparatus according to an embodiment.

Hereinafter, an embodiment of a nucleic acid separation apparatus and a nucleic acid separation method using the same will be described with reference to FIGS. 1 to 3.

The apparatus for separating nucleic acids according to one embodiment includes a filter column 100 into which a solid support 120 or a nucleic acid-solid support complex 130 having a property of binding to a nucleic acid can be filled; And a dispersion unit 150 disposed inside or outside the filter column 100 to disperse the solid support 120 or the nucleic acid-solid support complex 130 in a liquid. In addition, the nucleic acid separation apparatus may further include a cover 140 for opening and closing the opening of the filter column to prevent the material inside the filter column 100 from flowing out during the dispersion process. The apparatus may further include a wash liquid storage unit 160, an eluent storage unit 170 or a sample storage unit 180 in fluid communication, and may be included in any combination thereof. In addition, the apparatus may further comprise a fourth adjusting unit 220 for adjusting the dispersion unit 150, wherein each of the storage units 160, 170, 180 is a first adjusting the amount of liquid stored therein. The adjusting unit 190 may further include a second adjusting unit 200 or a third adjusting unit 210.

Referring to the nucleic acid separation process with reference to Figures 1 and 3, by providing a sample containing a nucleic acid to the filter column 100 filled with the solid support 120, when contacted with the solid support 120, And the nucleic acid-solid support conjugate to which the solid support 120 is bound is formed. Thereafter, the nucleic acid-solid support conjugate is washed using a wash solution to remove impurities contained in the nucleic acid. In this case, the washing solution may be directly injected into the filter column 100 by the experimenter, or may be supplied from the removable washing solution storage 160 which may be connected to the opening end of the nucleic acid in the elution direction or the opposite direction in the filter column 100. . In addition, the washing liquid supplied from the washing liquid storage unit 160 to the filter column may be controlled by the first adjusting unit 190 connected to the washing liquid storage unit 160.

Then, the eluate is added to the filter column 100 to disperse the nucleic acid-solid support conjugate. The eluate may be directly injected into the filter column 100 by the experimenter, or may be supplied from the detachable eluate storage unit 170 which may be connected to the end of the opening in the nucleic acid elution direction or the opposite direction of the filter column 100. The eluate supplied from the eluent storage unit 170 to the filter column may be controlled by the second adjusting unit 200 connected to the eluent storage unit 170. In addition, dispersion of the eluate and the nucleic acid-solid support conjugate is performed by the dispersion unit 150, wherein the dispersion unit 150 is an opening in the elution direction of the nucleic acid in the filter column 100 or the opposite direction thereof. It may be a detachable pressurizing device or a sonicator, which may be in fluid communication with the distal end. The eluate and the nucleic acid-solid support conjugate are sufficiently dispersed in the filter column 100 by the pressurized gas generated from the pressurization device or the ultrasonic wave generated from the ultrasonic generator, and during this process, the nucleic acid is separated from the nucleic acid-solid support conjugate. It elutes in this eluate. In the case of using the pressurizing device as the dispersion unit 150, the pressure may be adjusted through the fourth adjusting unit 220. On the other hand, separation of the eluate from the solid support can be made according to methods known in the art.

In addition, in the case of using the sample providing unit in the nucleic acid separation apparatus as shown in Figure 2, the formation of the nucleic acid-solid support complex 130 has a characteristic of binding the sample containing the nucleic acid to the nucleic acid in the sample providing unit 180 It can be achieved by contact with a solid support having. The nucleic acid-solid support complex 130 is supplied to the filter column 100 from the removable sample reservoir 180 which may be connected to an opening end in the elution direction or the opposite direction of the nucleic acid in the filter column 100. The filter column 100 may be filled. In addition, the sample supplied from the sample storage unit 180 to the filter column may be controlled by a third control unit 210 connected to the sample storage unit 180. The nucleic acid-solid support complex filled in the filter column is washed and eluted in the same manner as described above, and as a result, the nucleic acid can be eluted with the eluent.

In the following examples, the nucleic acid is separated by using the nucleic acid separation apparatus according to the one or more embodiments, and the results of confirming the efficiency of the apparatus. The results of the following examples are the results of five repeated experiments.

Example  2: binding and elution of a sample comprising nucleic acid to a solid support in a filter column

An AN25 filter (Millipore) having a pore size of 2.5 μm in the column (Qiagen) was mounted in the lower opening of the inside of the column and used as a filter column. Amplified nucleic acid by PCR with beads (30 μl of carboxyl-coated polystyrene beads (4 μm in diameter) (Bangs Lab) in the presence of PEG (20% polyethylene, 2.5 M NaCl) solution in a microtube, and with PCR removed from dNTP and primers ( 203 μg of DNA) were combined and the bead-nucleic acid complexes were charged to the filter column under reduced pressure (˜−75 kPa) in vacuo. Thereafter, 2 mL of 70% ethanol was added to the filter column, and vacuum was removed to remove unbound DNA and impurities. The mixture was depressurized to vacuum again, and the beads were dried for 15 minutes, and then 80 µl of the nucleic acid eluate (dH ₂ O) was added. Thereafter, pressurized nitrogen gas was generated from a pressurization device connected to a rubber tube at the bottom of the filter column, and injected into the nucleic acid eluate containing the DNA-bound beads at a pressure of ˜20 kPa for 15 minutes (FIG. 3). Thereafter, the DNA-bound beads were sufficiently dispersed in the nucleic acid eluate, and then the filter column was placed in a centrifuge and centrifuged at 13,200 rpm for 30 seconds to obtain a DNA eluate.

Table 1 shows the DNA concentration, total amount and yield of DNA in the obtained DNA eluate.

sample Concentration (ng / μl) Elution volume (μl) Total amount (㎍) yield(%) 260/280 260/230 One 2166.8 75 162.5 80.1% 1.88 2.33 2 1995.6 76 151.7 74.7% 1.86 2.31 3 2126.7 75 159.5 78.6% 1.84 2.29 4 2180.6 75 163.5 80.6% 1.88 2.18 5 2033.2 79 160.6 79.1% 1.87 2.32 Average Concentration: 2100.6 ng / μl, Average Total Amount: 159.6 μg, Average Yield: 78.6%

As shown in Table 1, 80 μl of eluate was added in the above experiment to obtain a 75-79 μl volume of DNA eluate with an average concentration of 2100.6 ng / μl, with an average total amount of DNA of 159.6 μg, average yield. Was 78.6%. In addition, in Table 1, the absorbance ratio 260 nm / 280 nm (DNA / protein) is 1.84-1.88, and the absorbance ratio 260 nm / 230 nm (DNA / humic acid) is 2 or more. I could confirm it.

On the other hand, Qiagen's nucleic acid separation kit (QIAquick PCR purification kit) is 10 μg of the maximum processable nucleic acid and 800 μl of the maximum processable volume (Qiagen spin handbook), it was not possible to process the sample at a time.

1 is a schematic diagram of a nucleic acid separation apparatus according to an embodiment, which is equipped with a pressurizing apparatus as a dispersion unit.

2 is a schematic diagram of a nucleic acid separation apparatus according to an embodiment, wherein a pressurization apparatus is mounted as a dispersion unit, and a sample storage unit is added.

3 is a schematic diagram of a nucleic acid separation apparatus according to an embodiment, in which an ultrasonic wave generating apparatus is mounted as a dispersion unit.

4 shows a process of separating nucleic acids using a nucleic acid separation apparatus according to one embodiment. A is before bead dispersion, B is during bead dispersion, and C is after bead dispersion.

<Description of Major Symbols Used in Drawings>

100: filter column

110: filter

120: solid support

130: nucleic acid-solid support complex

140: cover

150: dispersion unit

160: washing liquid storage unit

170: eluent storage unit

180: sample storage unit

190: first adjusting unit

200: second adjusting unit

210: third adjustment unit

220: fourth adjusting unit

Claims (15)

Binding the nucleic acid to the solid support by contacting a sample comprising nucleic acid with a solid support having the property of binding to the nucleic acid; Adding an eluate to the nucleic acid-solid support complex and dispersing the nucleic acid-solid support complex in the eluate; And Eluting nucleic acid from the nucleic acid-solid support. The method of claim 1, further comprising washing the unbound sample after the contacting step. The method of claim 1, wherein the solid support is selected from the group consisting of magnetic beads, silica beads, polystyrene beads, glass beads, and cellulose beads. The method of claim 1, wherein the amount of the eluate added is 10 μl to 500 μl. The method of claim 1, wherein the adding step consists of applying a pressurized gas or ultrasonic waves. A filter column into which a solid support or nucleic acid-solid support complex having a property of binding to a nucleic acid can be filled; And And a dispersion unit disposed in said filter column for dispersing said solid support or nucleic acid-solid support complex in a liquid. 7. The apparatus of claim 6, wherein the dispersion unit is a pressurizing device for applying pressure to the solid support or an ultrasonic generator for providing ultrasonic waves. The device of claim 7, wherein the pressurizing device or the ultrasonic wave generating device is in fluid communication with a top or bottom opening in the filter column. The device of claim 7, wherein the pressurizing device or the ultrasonic wave generating device is in fluid communication with the opening end in the elution direction of the nucleic acid eluate or the opposite direction in the filter column. 7. The apparatus of claim 6, further comprising a cover for opening and closing the opening of the filter column at the opposite end to which the pressurizing device or the ultrasonic wave generating device is connected. The device of claim 6, wherein the vibrator of the ultrasonic wave generating device is included inside the solid support or the nucleic acid-solid support complex population. 7. The apparatus of claim 6, further comprising a removable wash solution reservoir connected to an opening end in the elution direction of the nucleic acid eluate in the filter column or the opposite direction, and for supplying the wash solution to the filter column. 7. The apparatus of claim 6, further comprising a detachable eluate reservoir connected to an opening end in the elution direction of the nucleic acid eluate in the filter column or the opposite direction, and for supplying the eluate to the filter column. The apparatus of claim 6, further comprising a detachable sample storage unit connected to an opening end in the elution direction of the nucleic acid eluate or the opposite direction in the filter column and supplying a sample including the nucleic acid to the filter column. The device of claim 6, wherein the material of the filter column is an ultrasonically transmissive material.

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