KR101406347B1 - Microfluidic chip for extracting nucleic acid, device comprising the same, and method for extracting nucleic acid using the same - Google Patents

Abstract

The present invention relates to a microfluidic chip for nucleic acid extraction, a nucleic acid extracting apparatus including the same, and a nucleic acid extracting method using the same, which, unlike existing nucleic acid extracting apparatuses and nucleic acid extracting methods, can be miniaturized, In addition, reliable nucleic acid extraction efficiency can be maintained and / or improved.

Description

TECHNICAL FIELD The present invention relates to a microfluid chip for nucleic acid extraction, a nucleic acid extracting apparatus including the same, and a nucleic acid extracting method using the microfluid chip,

The present invention relates to an apparatus and a method for extracting a nucleic acid from a biological sample such as a cell, a bacterium, or a virus.

Recently, techniques for extracting nucleic acids from biological samples such as cells, bacteria, or viruses have been widely used in connection with nucleic acid amplification reaction techniques in order to diagnose, treat, or prevent disease at a gene level. In addition to the diagnosis, treatment, or prevention of diseases, technologies for extracting nucleic acids from biological samples in various fields such as customized drug development, forensic medicine, and environmental hormone detection are required.

As an example of a conventional nucleic acid extraction technique, there is a method of treating a sample containing cells with SDS or proteinase K, followed by solubilization, and denaturing the protein with phenol to purify the nucleic acid. However, since the phenol extraction method requires a lot of processing steps, it takes a lot of time and the efficiency of the nucleic acid extraction depends greatly on the experience and the skill of the researcher. In recent years, a kit using silica or glass fiber that specifically binds to nucleic acid has been used to solve this problem. Since the silica or glass fiber has a low binding ratio with proteins and cellular metabolites, a relatively high concentration of nucleic acid can be obtained. Such a method is advantageous compared with the phenol method, but since it uses a chaotropic reagent or ethanol which strongly inhibits the enzyme reaction such as PCR, it is necessary to completely remove these substances, This is a very cumbersome and time-consuming disadvantage. A method of directly purifying nucleic acids using recent filters is disclosed in WO 00/21973, which involves passing a sample through a filter to adsorb the cells to the filter, dissolving the adsorbed cells in the filter, After filtration, the nucleic acid adsorbed on the filter is washed and eluted. However, in order to elute the nucleic acid after adsorbing the cells on the filter, there is a problem in that a filter must be selected according to the type of the cell, and there are disadvantages that the apparatuses are large and complex and can not be used easily by researchers.

In order to solve the problems of the conventional nucleic acid extraction technology,

The present invention relates to a microfluidic chip which can be miniaturized and ultra-high-speed unlike conventional nucleic acid extraction devices and nucleic acid extraction methods, can maintain and / or improve reliable nucleic acid extraction efficiency, A nucleic acid extracting apparatus, and a nucleic acid extracting method using the same.

One embodiment of the invention is for extracting nucleic acid from a biological sample, comprising: an inlet; A heating unit disposed in a first channel region connected to the inlet unit and configured to transfer heat obtained from the outside to the biological sample introduced through the inlet unit; A first filter disposed in a second channel region connected to the heating unit and capable of passing a substance having a size corresponding to a nucleic acid; A nucleic acid separator disposed in a third channel region connected to the first filter and having a nucleic acid binding substance capable of specifically binding to the nucleic acid; A second filter disposed in a fourth channel region connected to the nucleic acid separation unit and capable of passing a substance having a size corresponding to the nucleic acid; And an outlet connected to the second filter. The microfluidic chip for extracting nucleic acid may be provided.

According to another embodiment of the present invention, there is provided a microfluidic chip according to one embodiment of the present invention. A chip mounting module for mounting the microfluidic chip; A heating module configured to apply heat to the heating unit of the microfluidic chip mounted on the chip mounting module; And an inlet and / or outlet of the microfluidic chip mounted on the chip mounting module to introduce a solution for nucleic acid extraction into the microfluidic chip and / or to introduce a solution present in the microfluidic chip An apparatus for extracting nucleic acid from a biological sample can be provided, including a fluid control module adapted to be discharged to the outside.

According to another embodiment of the present invention, there is provided a method of manufacturing a microfluidic chip, comprising: providing a microfluidic chip according to an embodiment of the present invention; Introducing a biological sample selected from the group consisting of cells, bacteria, and viruses through the inlet of the microfluidic chip; Transferring the introduced biological sample to a heating section of the microfluidic chip, and applying heat to the heating section of the microfluidic chip to lyse the biological sample; Moving the substance obtained from the dissolving step to a first filter of the microfluidic chip, passing the substance through the first filter, and removing a substance not passing through the first filter; The method comprising: moving a substance passing through the first filter to a nucleic acid separating unit of the microfluidic chip, binding a nucleic acid in the substance that has passed through the first filter to the nucleic acid binding substance, ; Separating the nucleic acid from the nucleic acid binding material, transferring the separated nucleic acid to the second filter, and passing the separated nucleic acid through a second filter; And extracting the nucleic acid from the biological sample through the outlet after moving the material passed through the second filter to the outlet.

Meanwhile, in one embodiment of the present invention,

The channel including the first channel region to the fourth channel region may be implemented in fluid communication, and the width and depth of the channel may be 0.001 to 10 millimeters (mm), respectively. .

The first filter and the second filter may have pores having a diameter in the range of 0.1 to 0.4 micrometers (μm), and may have a thickness in the range of 0.01 to 10 millimeters (mm).

In addition, the first filter and the second filter may have a pore having a diameter of 0.2 micrometer (mu m), and may have a thickness of 0.01 to 0.5 millimeter (mm).

In addition, the nucleic acid separation unit may be a nucleic acid-binding material having a bead having a nucleic acid-binding functional group attached thereto.

In addition, the nucleic acid-bonded functional beads may have a diameter within the range of 0.001 to 20 millimeters (mm).

In addition, the nucleic acid separating unit may include beads having a nucleic acid binding functional group within a range of 1 microgram (μg) to 200 milligram (mg).

The microfluidic chip may be formed of a plastic material.

The microfluidic chip may further include a first plate; A second plate disposed on the first plate and including a channel including the first to fourth channel regions; And a third plate disposed on the second plate, wherein the inlet and the outlet are disposed.

In addition, the first and third plates may be formed of a material selected from the group consisting of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate (PMMA), polycarbonate ), A material selected from the group consisting of polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof, The second plate is made of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), polyamide (PA), polyethylene (PE) polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyether ether ketone (p polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT), fluorinated ethylene A thermoplastic resin or a thermosetting resin material selected from the group consisting of fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), and combinations thereof.

Wherein the inlet of the third plate is implemented within a range of 0.1 to 5.0 millimeters (mm) in diameter, the outlet is implemented within a range of 0.1 to 5.0 millimeters (mm) in diameter, and the thickness of the first and third plates Is realized within a range of 0.01 to 20 millimeters (mm), and the thickness of the second plate can be realized within a range of 30 micrometers (占 퐉) to 10 millimeters (mm).

According to the microfluid chip and the nucleic acid extracting apparatus including the microfluidic chip according to the present invention, unlike the conventional nucleic acid extracting apparatus, miniaturization and usability of the apparatus can be greatly improved while maintaining and / or improving nucleic acid extraction efficiency .

In addition, according to the nucleic acid extracting method using the microfluidic chip according to the present invention, unlike the conventional nucleic acid extracting method, the nucleic acid extracting efficiency and the process efficiency can be greatly improved while maintaining and / or improving the nucleic acid extracting efficiency .

Furthermore, the microfluid chip, the nucleic acid extracting apparatus including the microfluid chip, and the nucleic acid extracting method using the microfluid chip according to the present invention can be efficiently linked to the polymerase chain reaction (PCR) It is highly utilized.

1 illustrates a microfluidic chip for nucleic acid extraction and its components according to an embodiment of the present invention.
2 is a cross-sectional view of a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention.
3 is a schematic diagram of a nucleic acid extracting apparatus equipped with a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention.
4 is a flowchart of a nucleic acid extraction method according to an embodiment of the present invention.
FIG. 5 shows a result of comparing a nucleic acid extraction method according to an embodiment of the present invention with a general nucleic acid extraction method.
FIG. 6 shows gel electrophoresis results obtained by amplifying the nucleic acid obtained by the nucleic acid extraction method according to an embodiment of the present invention in the third-party PCR apparatus and the applicant's PCR apparatus, respectively.

Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

The following description is only for the purpose of easy understanding of the embodiments of the present invention, and is not intended to limit the scope of protection of the present invention from such description.

1 illustrates a microfluidic chip for nucleic acid extraction and its components according to an embodiment of the present invention.

Referring to FIG. 1, a nucleic acid-extracting microfluid chip according to an embodiment of the present invention is for extracting nucleic acid from a biological sample, and includes an inlet 10; A heating unit 20 disposed in a first channel region connected to the inlet 10 and capable of transferring heat obtained from the outside to a biological sample introduced through the inlet 10; A first filter (30) disposed in a second channel region connected to the heating unit (20) and capable of passing a substance having a size corresponding to a nucleic acid; A nucleic acid separation unit 40 disposed in a third channel region connected to the first filter 30 and having a nucleic acid binding material 45 capable of specifically binding with the nucleic acid; A second filter (50) disposed in a fourth channel region connected to the nucleic acid separation unit (40) and capable of passing a substance having a size corresponding to the nucleic acid; And an outlet (60) connected to the second filter (50).

The microfluidic chip for nucleic acid extraction is a microfluidic chip for extracting nucleic acid. The microfluidic chip includes components for nucleic acid extraction, namely, an inlet, an outlet, a channel connecting the inlet and the outlet, And a second filter or the like is implemented in units of millimeter (mm) or micrometer (占 퐉).

The biological sample may be a biological sample containing a nucleic acid such as DNA or RNA and may be, but not limited to, a liquid sample including, for example, animal cells, plant cells, pathogens, fungi, bacteria, viruses and the like.

The inlet (10) is a portion where the biological sample or a solution for nucleic acid extraction is introduced into the microfluidic chip, and the outlet (60) is a portion for extracting the nucleic acid, the solution for nucleic acid extraction, And other waste is discharged to the outside of the microfluidic chip. In this case, the inflow section 10 and the outflow section 60 may serve as an outflow section and an inflow section, respectively, as needed. The solution for nucleic acid extraction includes all the solutions required for nucleic acid extraction, for example, distilled water, a nucleic acid binding buffer, an elution buffer, and the like. The inlet 10 and the outlet 60 are connected in fluid communication with a channel 70 and are connected to a heating unit 20, a first filter 30, The first filter 40, and the second filter 50 are arranged to be drivable on the channel 70 to perform respective functions. The channel 70 may be implemented in various standards, but the width and depth of the channel are preferably in the range of 0.001 to 10 millimeters (mm), respectively. The first, second, third, and fourth channel regions, which will be described below, refer to sequential placement from the inlet 10 to the outlet 60 and are limited to specific locations within the channel 70 It is not.

The heating unit 20 is disposed in a first channel region connected to the inlet 10, and is externally applied to the solution (including a biological sample) introduced through the inlet 10. For example, when a sample containing cells, bacteria, or viruses is introduced through the inlet 10, when the cell, bacteria, or virus reaches the heating unit 20, (° C), the outer wall of the cell, bacteria, or virus is destroyed and the cell material is released to the outside (cell lysis). The heating unit 20 can receive heat from a heating module 600 of a nucleic acid extracting apparatus, which will be described later, in a contact or non-contact manner.

The first filter 30 is a structure having pores of a predetermined size. The first filter 30 serves to distinguish a passing material from a non-passing material through the pores in a fluid flow direction. In one embodiment of the present invention, the first filter 30 is disposed in a second channel region connected to the heating unit 20, and is configured to pass a substance having a size corresponding to the nucleic acid. The first filter (30) collects a substance of a size larger than a nucleic acid in a soluble product generated by heating in the heating unit (20) in the heating unit (20), and the nucleic acid and a substance having a size corresponding thereto are filtered To the nucleic acid separation section 40 to be described below. The first filter 30 may be implemented in a variety of specifications, but it is preferable to have a pore having a diameter in the range of 0.1 to 0.4 micrometers (mu m), and having a thickness in the range of 0.01 to 10 millimeters (mm) desirable. More preferably, the first filter 30 has a pore having a diameter of 0.2 micrometers (mu m), and preferably has a thickness of 0.01 to 0.5 millimeters (mm).

The nucleic acid separation unit 40 is for selectively separating the nucleic acid from a nucleic acid or a substance having a size corresponding to the nucleic acid. 1, the nucleic acid separating unit 40 is a space between the first filter 30 and a second filter 50 to be described below. The nucleic acid separating unit 40 includes a nucleic acid binding material 45 (specifically, . The nucleic acid binding material 45 includes any substance capable of specifically binding to a nucleic acid. The nucleic acid binding material 45 may be a silica (SiO2) bead, a biotin or a strptavidin-attached bead to which a nucleic acid binding functional group is attached. The beads having the nucleic acid-binding functional groups may be implemented in various standards, but preferably have diameters within the range of 0.001 to 20 millimeters (mm). In addition, the nucleic acid separation unit 40 may include the nucleic acid-binding functional group-attached beads in various amounts, but it is preferable that the nucleic acid separation unit 40 is contained in the range of 1 microgram (μg) to 200 milligram (mg). After the nucleic acid is specifically bound to the nucleic acid binding material 45 and then the inside of the nucleic acid separating part 40 is washed to remove the foreign substance, the nucleic acid binding material 40 is immersed in the target nucleic acid- Only the complex remains. Then, when the elution buffer is provided to the nucleic acid separator 40, the target nucleic acid is separated from the complex.

The second filter 50 is a structure having a pore of a predetermined size, such as the first filter 30 already described. The second filter 50 is a structure having pores of a predetermined size, It performs a distinguishing role. In an embodiment of the present invention, the second filter 50 is disposed in a fourth channel region connected to the nucleic acid separator 40, and is capable of passing a substance having a size corresponding to the nucleic acid. The second filter 50 collects the nucleic acid binding material 45 in the nucleic acid separator 40 and the nucleic acid separated from the nucleic acid binding material 45 is filtered and transferred to the outlet 60 . The second filter 50 may be implemented in a variety of specifications, but it is preferred to have a pore having a diameter in the range of 0.1 to 0.4 micrometers (mu m), with a thickness in the range of 0.01 to 0.5 millimeters (mm) desirable. More preferably, the second filter 50 has a pore having a diameter of 0.2 micrometers (mu m), preferably a thickness of 0.3 millimeters (mm).

2 is a cross-sectional view of a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention.

FIG. 2 is a cross-sectional view of a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention. The microfluidic chip for nucleic acid extraction according to an embodiment of the present invention comprises a first silver plate 100; A second plate (200) disposed on the first plate and having a channel (70) including the first to fourth channel regions disposed therein; And a third plate 300 disposed on the second plate 200 on which the inlet 10 and the outlet 60 are disposed. The microfluidic chip for nucleic acid extraction according to an exemplary embodiment of the present invention may be formed of various materials, but it may be formed of a plastic material. As described above, when a plastic material is used, the heat transfer efficiency can be increased only by adjusting the thickness of the plastic, and the manufacturing process can be simplified, thereby greatly reducing the manufacturing cost. The first plate 100 and the third plate 300 may be formed of a material selected from the group consisting of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate (PMMA) A material selected from the group consisting of polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET) And the second plate 200 may be formed of a material selected from the group consisting of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), polyamide (PA) (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyether Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT) A thermoplastic resin or a thermosetting resin material selected from the group consisting of fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), and combinations thereof. Wherein the inlet of the third plate is implemented within a range of 0.1 to 5.0 millimeters (mm) in diameter, the outlet is implemented within a range of 0.1 to 5.0 millimeters (mm) in diameter, and the thickness of the first and third plates Is realized within a range of 0.01 to 20 millimeters (mm), and the thickness of the second plate can be realized within a range of 30 micrometers (占 퐉) to 10 millimeters (mm). In addition, the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention may be implemented with two or more inlet and outlet portions and a channel connecting the two or more inlet and outlet portions as necessary. In this case, two or more biological samples The nucleic acid can be extracted and the nucleic acid can be extracted quickly and efficiently.

3 is a schematic diagram of a nucleic acid extracting apparatus equipped with a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention.

3, the nucleic acid extracting apparatus according to an embodiment of the present invention includes the microfluidic chip 1 for nucleic acid extraction already described; A chip mounting module 500 configured to mount the microfluidic chip 1; A heating module 600 configured to apply heat to the heating unit 20 of the microfluidic chip 1 mounted on the chip mounting module 500; And a solution for nucleic acid extraction into the microfluidic chip 1 by being connected to the inflow part 10 and / or the outflow part 60 of the microfluidic chip 1 mounted on the chip mounting module 500, And / or a fluid control module 700 implemented to be able to discharge the solution present inside the microfluidic chip 1 to the outside.

The nucleic acid extracting apparatus is implemented to perform all steps for nucleic acid extraction in a state where the microfluidic chip 1 according to an embodiment of the present invention is mounted. The above-described chip mounting module 500, The heating module 600, and the fluid control module 700, as well as various modules required to extract other nucleic acids. In addition, the nucleic acid extracting apparatus according to an embodiment of the present invention can be implemented so that all the steps can be implemented in an automated manner, and the nucleic acid amplification reaction can be performed immediately after the nucleic acid extraction in conjunction with the PCR apparatus have.

The microfluidic chip 1 for nucleic acid extraction has already been described.

The chip mounting module 500 is a portion on which the microfluidic chip 1 is mounted. The chip mounting module 500 may be variously formed in correspondence with the shape of the contact surface of the microfluidic chip 1.

The heating module 600 is a module for supplying heat to the heating unit 20 of the microfluidic chip 1 when the microfluidic chip 1 is mounted on the chip mounting module 500. The heating module 600 may be variously implemented, but a contact heating block is preferred.

The fluid control module 700 is connected to the inlet 10 and / or the outlet 60 of the microfluidic chip 1 mounted on the chip mounting module 500, To introduce a solution for nucleic acid extraction into the microfluidic chip 1 and / or to discharge the solution present inside the microfluidic chip 1 to the outside. The fluid control module 700 may include various components including, for example, microchannels that are fluid passages, pneumatic pumps that provide the driving force of fluid movement, valves that can control the opening and closing of fluid movement, A storage chamber containing various solutions required for nucleic acid extraction such as binding buffer, elution buffer, silica gel, distilled water (DW), and the like.

Meanwhile, the nucleic acid extracting apparatus according to an embodiment of the present invention includes an electronic control module (not shown) for automatically controlling the microfluid chip 1, the heating module 600, and the fluid control module 700 ). ≪ / RTI > The electronic control module can precisely control the modules so that a predetermined amount of nucleic acid can be extracted in the microfluidic chip 1 according to a program stored in advance. The pre-stored program includes, for example, a program for a series of steps relating to a nucleic acid extraction method, which will be described in detail below.

4 is a flowchart of a nucleic acid extraction method according to an embodiment of the present invention.

4, the nucleic acid extraction method according to an embodiment of the present invention is based on the microfluidic chip 1 for nucleic acid extraction according to an embodiment of the present invention already described.

Specifically, a method for extracting nucleic acid from a biological sample according to an embodiment of the present invention includes

Providing a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention (providing a microfluidic chip);

Introducing a biological sample selected from the group consisting of cells, bacteria, and virus (biological sample introduction step) through the inlet of the microfluidic chip;

Moving the introduced biological sample to a heating section of the microfluidic chip and then applying heat to the heating section of the microfluidic chip to lyse the biological sample (biological sample dissolution step);

Moving the material obtained from the dissolving step to a first filter of the microfluidic chip, passing through the first filter, and removing material that has not passed through the first filter (filtration through a first filter );

The method comprising: moving a substance passing through the first filter to a nucleic acid separating unit of the microfluidic chip, binding a nucleic acid in the substance that has passed through the first filter to the nucleic acid binding substance, (Nucleic acid separation step);

Separating the nucleic acid from the nucleic acid binding material, transferring the separated nucleic acid to the second filter, and passing the separated nucleic acid through a second filter (filtering step through a second filter); And

Extracting the nucleic acid through the outlet after moving the substance passed through the second filter to the outlet (nucleic acid extracting step);

. ≪ / RTI >

Hereinafter, in Examples 1 to 3, the amount and time of the nucleic acid extracts were determined while extracting the nucleic acid from the biological sample, and the resultant reliability of the nucleic acid extract was confirmed again through the PCR (polymerase chain reaction).

Example  1. Identification of nucleic acid extraction yield and run time

First, DNA was extracted from a tubercle bacilli cell using a general tube and a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention, respectively, and then the amount and time of the production of the DNA were determined.

Common nucleic acid extraction steps are as follows.

The Mycobacterium tuberculosis main cell was prepared and mixed with 6% NaOH and 4% NaLC at a ratio of 1: 1: 1 to prepare a sample solution. The sample solution was then centrifuged to remove supernatant (20 min, 4300 rpm, 4 캜). Thereafter, 1 ml of distilled water (DW) was added to the sample solution, vortexed, and the sample solution was transferred to another tube. Thereafter, the sample solution was centrifuged again and the supernatant was removed (3 minutes, 12000 rpm, room temperature). Thereafter, 1 ml of distilled water (DW) was added to the sample solution, followed by vortexing. Thereafter, 500 μl of distilled water (DW) was added to the sample solution to obtain a genomic DNA (in this case, a commercially available QIAamp DNA Kit was used). As a result, about 100 μl of the final DNA product was obtained, and it took about one hour or more to obtain the final DNA product.

Subsequently, nucleic acid was extracted from the same M. tuberculosis main cell using a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention. The detailed procedure is as follows.

The Mycobacterium tuberculosis main cell was prepared and mixed with 6% NaOH and 4% NaLC at a ratio of 1: 1: 1 to prepare a sample solution. Thereafter, the sample solution was introduced using a syringe at the inlet of the microfluidic chip for nucleic acid extraction (25 × 72 × 2 mm, silica bead (OPS Diagnostics, LLC), filter (Whatman) 1 minute required). Thereafter, 300 ㎕ of silica gel and 1X DNA binding buffer were introduced into the inlet of the microfluidic chip according to the instant embodiment of the present invention, and then the microfluid chip was heated The portion was rapidly heated to 95 캜 (takes about 1 minute and 30 seconds). Thereafter, wastes were removed from the sample solution through the inlet of the microfluidic chip according to the instant embodiment of the present invention, and 100 μl of an elution buffer was introduced (takes about 30 seconds). Thereafter, a final product was obtained through the outlet of the microfluidic chip according to the instant embodiment of the present invention, and as a result, about 100 μl of the final DNA product was obtained and about 7 minutes was required to obtain the final DNA product . In this case, if the above-mentioned experiment is conducted by an automated nucleic acid extracting apparatus instead of a manual operation, it will be obvious that the required time will be reduced to about 5 minutes or less.

As a result of the above experiment, it was confirmed that the microfluidic chip according to an embodiment of the present invention can maintain the total amount of nucleic acid extraction product, unlike the conventional nucleic acid extraction method, .

Example  2. General nucleic acid extraction method and work of the present invention In the embodiment  Obtained by the nucleic acid extraction method according to DNA  Result of polymerase chain reaction

In order to ensure the reliability of the DNA product obtained in Example 1, PCR was carried out based on the DNA product. The PCR was performed using a PCR apparatus including two column blocks described in Korean Patent Application No. 2011-0037352 of the present applicant and a commercially available PCR apparatus (Roche, Light cycler). The applicant's PCR apparatus is a real-time PCR apparatus comprising: a first column block disposed on a substrate; A second column block disposed on the substrate and spaced apart from the first column block; And a chip holder mounted on the first column block and the second column block and capable of moving left and right and / or up and down by a driving means and having a PCR chip of a light-transmitting plastic material. The driving means includes a rail extending in the left-right direction, and a connecting member slidably movable in the left-right direction through the rail and slidable in the vertical direction, and one end of the connecting member is connected to the chip holder Is disposed. Further, a light source is further disposed between the first column block and the second column block, and a light detecting portion for detecting light emitted from the light source is further disposed on the chip holder, or the first column block and the second column block A light detecting unit for detecting light emitted from the light source is further disposed between the thermal blocks, and a light source is further disposed on the chip holder. By using the PCR chip and the PCR device, the PCR execution time can be greatly shortened to within about 5 to 15 minutes. The PCR chip and the PCR device can be used as a microfluid chip for nucleic acid extraction and nucleic acid extraction In conjunction with the device, the nucleic acid extraction time can be shortened to within about 5 to 7 minutes and can be shortened to within at least about 20 minutes prior to obtaining the final nucleic acid amplification product. In order to perform the PCR, 8 microliters (쨉 l, final concentration 1X) of a real-time PCR mixed solution (NBS SYBR Green I real-time PCR mixture 2X) (1 μM), 1.6 μL of forward primer (10 μM), 1.6 μL of the reverse primer (10 μM), 3 μL of template DNA (μL) 16 μl total of PCR reagents including 1.8 μl of distilled water (DW) (adjusted to 16 μl) were prepared. When using a third-party PCR device, a real-time PCR mixed solution (Takara SYBR Green I 2 μl of a forward primer (10 μM), 2 μl of a reverse primer (10 μM), 1 μM of a final concentration, 1 μM of a forward primer, 10 μM of a real-time PCR mixture 2X) , Final concentration 1 [mu] M), template DNA (Template DNA) A total of 20 microliters (占 퐇) of PCR reagent including 3 microliters (占 퐇) and 3 microliters (占 퐇, adjusted to 20 占 퐇) of distilled water (DW) were prepared.

FIG. 5 shows a result of comparing a nucleic acid extraction method according to an embodiment of the present invention with a general nucleic acid extraction method. Specifically, FIG. 5A is a graph showing the results of real-time PCR using the PCR apparatus of the present applicant, and FIG. 5B is a photograph of gel electrophoresis of the final PCR product.

5A, the curve (1) is a PCR result curve (X axis: cycle, Y axis: fluorescence) of a DNA product by a nucleic acid extraction method in Example 1, and a curve (2) The curve of the PCR product of the DNA product by the nucleic acid extraction method according to an embodiment of the present invention and the curve (3) is the negative control curve using the solution containing no DNA. According to FIG. 5A, curve 1 and curve 2 start to rise from about 20 cycles as compared to curve 3, and therefore, it can be seen that Example 2 is proceeding with an appropriate polymerase chain reaction And the fluorescence intensity of about 350 and 250, respectively, when the curves (2) and (3) were about 30 cycles, it was confirmed that the nucleic acid extraction method according to one embodiment of the present invention accurately extracted the DNA product And further, the amount of the polymerase chain reaction product was improved compared to a general nucleic acid extraction method. In addition, the nucleic acid extraction method according to an embodiment of the present invention took about 15 minutes to complete. In addition, in FIG. 5B, column 1 shows the result of PCR of the DNA product by the general nucleic acid extraction method in Example 1, and column 2 shows the result of DNA product by the nucleic acid extraction method according to one embodiment of the present invention PCR, and column 3 is a negative control result using a solution containing no DNA. According to the gel electrophoresis image of FIG. 5B, the final result of FIG. 5A can be confirmed again. Meanwhile, the real-time PCR results using the PCR apparatus of the third-party were measured by fluorescence of each PCR cycle, and the photographs of the gel electrophoresis of the final PCR products were confirmed. As a result, the results were almost the same as those of FIGS. 5A and 5B But it took about 30 minutes to complete.

Example  3. The present invention In the embodiment  Obtained by the nucleic acid extraction method according to DNA  Result of polymerase chain reaction by product

The DNA product obtained by the nucleic acid extraction method according to one embodiment of the present invention was amplified using a third-party PCR device (Roche, LightCycler) and the applicant's PCR device (same as in Example 2). In this case, the PCR device of the other company was subjected to 30 cycles of 1 cycle for 2 minutes at the pre-denaturation step (95 ° C), 10 seconds for the denaturation step (95 ° C) and 10 seconds for the annealing and expansion step (72 ° C) The PCR apparatus of the present applicant performed one cycle for 8 seconds at the pre-denaturation step (95 DEG C), 8 seconds for the denaturation step (95 DEG C) and 72 seconds for the annealing and expansion step (PCR) PCR was carried out for 30 seconds each for 14 seconds.

FIG. 6 shows gel electrophoresis results obtained by amplifying the nucleic acid obtained by the nucleic acid extraction method according to an embodiment of the present invention in the third-party PCR apparatus and the applicant's PCR apparatus, respectively. Specifically, FIG. 6A is a photograph of a gel electrophoresis of the final PCR product using a third-party PCR device, and FIG. 6B is a photograph of gel electrophoresis of the final PCR product using the applicant's PCR device. In FIG. 6, in this case, column 1 is a negative control result for a solution containing no DNA, and columns 2 to 4 are DNAs obtained by using the nucleic acid extraction method according to one embodiment of the present invention from 200 μl of Mycobacterium germ cells And column 5 is the result of the final PCR product using a commercially available kit (QIAamp DNA Kit) from 1 ml of Mycobacterium tuberculosis cells. As a result, according to FIG. 6, even if the PCR device is used, the PCR result is almost close to that of the PCR device. Thus, although the time required for the nucleic acid extraction method according to one embodiment of the present invention is greatly shortened Was completed in about 30 minutes to complete, whereas Applicant's PCR device takes about 15 minutes to complete), again confirming that reliable nucleic acid extraction results can be obtained.

Claims (13)

For extracting nucleic acid from a biological sample,
An inlet;
A heating unit disposed in a first channel region connected to the inlet unit and configured to transfer heat obtained from the outside to the biological sample introduced through the inlet unit;
A first filter disposed in a second channel region connected to the heating section, the first filter having a pore having a diameter in the range of 0.1 to 0.4 micrometers (占 퐉);
A nucleic acid separator disposed in a third channel region connected to the first filter and having a nucleic acid binding substance capable of specifically binding to the nucleic acid;
A second filter disposed in a fourth channel region connected to the nucleic acid separator, the second filter having a pore having a diameter in the range of 0.1 to 0.4 micrometers (占 퐉); And
An outlet connected to the second filter (50);
A microfluidic chip for nucleic acid extraction. 2. The method of claim 1, wherein the channels including the first to fourth channel regions are implemented in fluid communication, wherein the width and depth of the channels are in the range of 0.001 to 10 millimeters (mm) Wherein the microfluidic chip is implemented within a microfluidic chip. The microfluidic chip of claim 1, wherein the first and second filters have a thickness in the range of 0.01 to 10 millimeters (mm). 4. The microfluidic chip of claim 3, wherein the first filter and the second filter have pores having a diameter of 0.2 micrometers (m), and have a thickness of 0.01 to 0.5 millimeters (mm). [3] The microfluidic chip of claim 1, wherein the nucleic acid separator comprises a nucleic acid-binding material, and a bead having a nucleic acid-binding functional group attached to the surface thereof. 6. The microfluidic chip of claim 5, wherein the nucleic acid-binding functionalized bead has a diameter in the range of 0.001 to 20 millimeters (mm). 6. The microfluidic chip according to claim 5, wherein the nucleic acid separator comprises a bead having a nucleic acid binding functional group within a range of 1 microgram (μg) to 200 milligram (mg). The microfluidic chip according to claim 1, wherein the microfluidic chip is made of a plastic material. 2. The microfluidic chip of claim 1, wherein the microfluidic chip comprises: a first plate; A second plate disposed on the first plate and including a channel including the first to fourth channel regions; And a third plate disposed on the second plate, the third plate having the inlet and the outlet. The method of claim 9, wherein the first and third plates are made of a material selected from the group consisting of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate (PMMA), polycarbonate a material selected from the group consisting of polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET) And the second plate is made of a material selected from the group consisting of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), polyamide (PA), polyethylene ), Polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyether Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT) Characterized in that it comprises a thermoplastic resin or a thermosetting resin material selected from the group consisting of fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), and combinations thereof. . 10. The apparatus of claim 9, wherein the inlet of the third plate is implemented within a range of 0.1 to 5.0 millimeters (mm) in diameter, the outlet is implemented within a diameter of 0.1 to 5.0 millimeters (mm) Wherein the thickness of the third plate is realized within a range of 0.01 to 20 millimeters and the thickness of the second plate is within a range of 30 micrometers to 10 millimeters. 12. Microfluidic chip according to any one of the preceding claims.
A chip mounting module for mounting the microfluidic chip;
A heating module configured to apply heat to the heating unit of the microfluidic chip mounted on the chip mounting module; And
The microfluidic chip is connected to an inlet and / or an outlet of the microfluidic chip mounted on the chip mounting module to introduce a solution for nucleic acid extraction into the microfluidic chip and / A fluid control module implemented to be able to discharge the fluid;
Wherein the nucleic acid is extracted from the biological sample. Providing a microfluidic chip according to any one of claims 1 to 11;
Introducing a biological sample selected from the group consisting of cells, bacteria, and viruses through the inlet of the microfluidic chip;
Transferring the introduced biological sample to a heating section of the microfluidic chip, and applying heat to the heating section of the microfluidic chip to lyse the biological sample;
Moving the substance obtained from the dissolving step to a first filter of the microfluidic chip, passing the substance through the first filter, and removing a substance not passing through the first filter;
The method comprising: moving a substance passing through the first filter to a nucleic acid separating unit of the microfluidic chip, binding a nucleic acid in the substance that has passed through the first filter to the nucleic acid binding substance, ;
Separating the nucleic acid from the nucleic acid binding material, transferring the separated nucleic acid to the second filter, and passing the separated nucleic acid through a second filter; And
Moving the substance passing through the second filter to the outlet and extracting the nucleic acid through the outlet;
≪ / RTI > wherein the nucleic acid is extracted from the biological sample.

Images