WO2014048263A1 - 气压式萃取核酸的方法及其装置 - Google Patents

气压式萃取核酸的方法及其装置 Download PDF

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Publication number
WO2014048263A1
WO2014048263A1 PCT/CN2013/083610 CN2013083610W WO2014048263A1 WO 2014048263 A1 WO2014048263 A1 WO 2014048263A1 CN 2013083610 W CN2013083610 W CN 2013083610W WO 2014048263 A1 WO2014048263 A1 WO 2014048263A1
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Prior art keywords
purification
nucleic acid
tube
purification tube
membrane
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PCT/CN2013/083610
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English (en)
French (fr)
Inventor
李德政
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精专生医股份有限公司
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Publication of WO2014048263A1 publication Critical patent/WO2014048263A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Definitions

  • the invention relates to a method for extracting nucleic acid and a device thereof, in particular to a method and device for extracting nucleic acid by pneumatic pressure, and an automatic machine designed by using the method. Background technique
  • the extraction process using the column extraction method can be further divided into two types, one is a column centrifugal extraction method, and the other is a column vacuum extraction method.
  • 1 is a conventional column centrifugal extraction method.
  • a treated sample contained in a microcentrifuge tube 10 for example, an anionic detergent is used to break cells to release nucleic acid and denature proteins
  • the purification tube 20 is divided into three parts, an upper neck portion 201, an intermediate tube portion 202, and a lower tip portion 204; the bottom portion of the intermediate tube portion 202 has a purification membrane 203, and the lower tip portion 204 has a lower portion 204.
  • the passage of the passage allows the liquid to flow out.
  • the sample is sucked from the sample in the microcentrifuge tube 10 by a pipette, and then injected into the purification tube 20 from above the purification tube 20.
  • the purification tube 20 is provided with a waste liquid pipe 30, and the double tube which is integrated and integrated is centrifuged in a centrifuge. Since the nucleic acid is negatively charged, it is combined with the positively charged purification membrane 203 to be adsorbed thereon, and other impurities are adsorbed thereon.
  • the purified membrane 203 is penetrated by the centrifugal force and flows into the waste liquid pipe 30 through the passage of the lower tip of the purification pipe 20. This step is called a "binding step"; then, the cleaning pipe 20 is added for cleaning.
  • the purification tube 20 carrying the nucleic acid on the purification membrane 203 is transposed into the collection tube 40.
  • the electrophoresis of the purified membrane 203 is changed by adding a specific salinity and pH, and the nucleic acid is separated from the purification membrane 203 to be separated, and the nucleic acid is collected in the collection tube 40.
  • This step is called a "collection step”. .
  • Column centrifugation requires centrifugation at least three times. The process is complicated and the extraction time is lengthened by the subsequent centrifugation.
  • FIG. 2 Another type of column vacuum extraction method is shown in Figure 2.
  • the principle is similar to the column centrifugal extraction method, except that the steps of "binding step” and “cleaning step” are to use vacuum suction instead of centrifugation, which will directly
  • the purification tube 20 is inserted into the pressure cell 50, and under a gas pressure, a negative pressure is applied to cause the liquid to flow out of the purification tube 20 and directly into the liquid collection bottle (not shown), but in the final “collection step", the nucleic acid must be collected by centrifugation. To the collection tube 40. Therefore, even for an automated machine, a centrifugal device must be placed in it.
  • the inventors have unexpectedly thought that the pressure can be directly used in the purification tube by using a conventional purification tube. Use only the lower end of the purification tube to pump and discharge the sample, the cleaning solution, the rinse solution, etc., without using the centrifuge at all, and it is not necessary to inject the sample, the cleaning solution or the extract from the neck of the purification tube. , the nucleic acid can be efficiently extracted. Accordingly, the inventors have provided a method for pneumatically extracting nucleic acid and a device thereof, and an automated machine designed by the method.
  • An aspect of the invention provides a method for pneumatically extracting nucleic acid, comprising: (a) hermetically bonding an upper end of a purification tube to a pressure device capable of applying a positive or negative gas pressure, wherein the purification tube has a half a through-hole tube through which the membrane is permeable; (b) opening the pressure device to apply a negative air pressure to the purification tube, and using the lower end of the purification tube to absorb the sample containing the nucleic acid, and passing the sample through the semi-permeable membrane, the nucleic acid Combining with the semi-permeable membrane electrical or polar; (c) turning on the pressure device to apply positive pressure to the purification tube, and passing the residual sample through the semi-permeable membrane in the opposite direction to the step 0), from the purification tube The lower end is discharged, and wherein the steps (b) and (c) can be performed only once or repeatedly; (d) the pressure device is turned on to apply a negative air pressure to the purification tube, and the lower
  • purification tube is a column for purifying nucleic acid as known in the art, and generally comprises an upper neck portion, an intermediate tube portion, a half-permeable membrane and a lower tip portion, the lower tip portion Having a through hole for discharging liquid, but the purification tube in the present invention is not limited to this structure, as long as the purification tube is a through-hole tube having a semi-permeable membrane, that is, within the scope of the present invention, the material of the tube body may be It is a plastic such as polypropylene, polyphenylacetyl, polycarbonate or polyvinyl chloride, or a biodegradable material.
  • the semipermeable membrane has also been in the technical field It is known in the art that the material can be silicon dioxide, the surface charge is positively charged, can be electrically coupled with the negatively charged nucleic acid in the sample, and the nucleic acid is released by the extract liquid, thereby releasing the nucleic acid;
  • the "semi-permeable membrane” is also referred to as “purified membrane” in order to allow the solution to pass through the interior thereof, that is, between the space in contact with one side of the semipermeable membrane and the other surface of the semipermeable membrane.
  • the solution When the pressure is different, the solution may flow from the high pressure space to the low pressure space to penetrate the semipermeable membrane; or, when the semipermeable membrane is subjected to centrifugal force, the solution may penetrate the semipermeable membrane along the direction of the centrifugal force, but the nucleic acid is The semipermeable membrane is adsorbed on the membrane; and the semipermeable membrane used in the present invention transmits nucleic acid through the interaction between the membrane and the nucleic acid, wherein the nucleic acid and the semipermeable membrane are transmitted through polarity or electricity.
  • the semipermeable membrane is a porous membrane having a hydrophilic group (also referred to as a hydrophilic membrane), and it is presumed that the hydrophilic group and the semipermeable membrane of the nucleic acid are semipermeable.
  • the hydrophilic groups of the membrane attract each other by changing the polarity of the surroundings
  • the semipermeable membrane having a hydrophilic group as referred to herein means that the porous membrane material itself has a hydrophilic group (such as polyhydroxyethyl acrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol, poly A vinylpyrrolidone, a polyacrylic acid, a polymethacrylic acid, a polyoxyethylene, a cellulose acetate, a cellulose acetate saponified product, a cellulose acetate mixture or the like, preferably an organic polymer having a hydroxyl group, or a porous film is formed.
  • a hydrophilic group such as polyhydroxyethyl acrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol, poly A vinylpyrrolidone, a polyacrylic acid, a polymethacrylic acid, a polyoxyethylene, a cellulose acetate, a cellulose a
  • the material is treated or coated to introduce a hydrophilic group, and any organic or inorganic material is suitable as a material of the porous film, or a combination of a plurality of porous films; in the porous film,
  • the pore diameter of the film is preferably 0.2 ⁇ m or more, and the thickness is preferably ⁇ ⁇ to 500 ⁇ , more preferably 50 ⁇ to 250 ⁇ .
  • the upper end and the lower end of the purification tube are defined as a lower end of the general purification tube structure closer to the purification membrane position, and an upper end away from the purification membrane position, but the present invention is not limited thereto.
  • the one that joins the pressure device is called the upper end of the purification tube, and the one that is used to suck the liquid is called the lower end of the purification tube.
  • pressure equipment capable of applying positive or negative gas pressure includes any device that provides positive or negative gas pressure.
  • the simplest device such as a cartridge, can be provided by pumping or pressing a push rod in the cylinder. Or a negative gas pressure; or a syringe, a pipette, a pump or a pneumatic cylinder as in the prior art, comprising a pneumatic valve and a pressure regulator, which can be controlled to generate a positive or negative gas pressure, wherein the solution is made
  • the pressure that can penetrate the semipermeable membrane is from 10 kPa to 300 kPa, preferably from 40 kPa to 200 kPa, and the most preferred pressure value is 60 kPa.
  • the extracted nucleic acid can be purified in order to further wash off impurities on the semipermeable membrane, and therefore, steps (C) and (d) of the above method of the present invention may further comprise a cleaning step, the cleaning step comprising: (c-1) turning on the pressure device to apply a negative air pressure to the purification tube, and sucking the cleaning liquid through the lower end of the purification tube to pass the cleaning liquid through the semipermeable membrane; and (c- 2) The pressure device is turned on to apply a positive air pressure to the purification tube, and the cleaning solution is passed through the semipermeable membrane in a direction opposite to (c-1), and then flows out from the lower end of the purification tube.
  • a cleaning step comprising: (c-1) turning on the pressure device to apply a negative air pressure to the purification tube, and sucking the cleaning liquid through the lower end of the purification tube to pass the cleaning liquid through the semipermeable membrane; and (c- 2)
  • the pressure device is turned on to apply a positive air pressure to the purification
  • cleaning solution includes any liquid that can be used to clean a purification tube, including but not limited to water, alcohol or other buffers that do not alter the electrical or polar properties of the semipermeable membrane, ie, the cleaning
  • the liquid is capable of washing out impurities in the nucleic acid mixture solution, and the impurities are adsorbed on the semipermeable membrane together with the nucleic acid, In this respect, the cleaning solution only causes impurities to be eluted from the semipermeable membrane without flushing the nucleic acid.
  • a suitable aqueous organic solution is used. Solutions, all within the scope of the invention, such as alcohols, include methanol, ethanol, isopropanol, n-propanol or butanol, preferably ethanol.
  • extraction solution encompasses any liquid of the prior art which can be used to dissociate a nucleic acid from the semipermeable membrane, which comprises altering the polarity or electrical properties of the semipermeable membrane from which the nucleic acid is placed.
  • the dissociated liquid for example, a buffer containing 1.2 M NaCl, 50 mM 3-(N-morpholine)propanesulfonic acid (MOPS), 15% ethanol, and pH 8.0; or as containing 0.5 M ammonium acetate, 10 mM acetic acid a buffer of magnesium (pH 7.5) and 1.5 mM EDTA; or a buffer containing 1.25 M NaCl, 50 mM Tris-HCl pH 8.5, and 15% isopropanol.
  • a buffer containing 1.2 M NaCl, 50 mM 3-(N-morpholine)propanesulfonic acid (MOPS), 15% ethanol, and pH 8.0 or as containing 0.5 M ammonium acetate, 10 mM acetic acid a buffer of magnesium (pH 7.5) and 1.5 mM EDTA; or a buffer containing 1.25 M NaCl, 50 mM Tris-HCl pH 8.5, and 15% isopropanol.
  • a filter membrane may be further disposed between the upper end of the purification tube and the pressure device.
  • the joint is in airtight engagement with the upper end of the purification tube and the pressure device.
  • a pipette tip connected to the gas-tight joint may be further disposed at the lower end of the purification tube, whereby the purification tube can take a small amount by using the pipette pipette. Liquid or the purified sample is collected into a microcentrifuge tube for preservation or further experimentation.
  • the upper end structure of the pipette is a set body, and the sleeve body can hermetically cover the intermediate pipe body of the purification pipe portion to the lower end to fit the purification pipe.
  • the structure of the junction of the pipette and the purification tube is versatile with the purification tube of different brands, and can be widely applied to various types of purification tubes, such as but not limited to the sleeve of the pipette and
  • the structure of the joint of the purification tube is made of rubber material to make it elastic structure, and can be joined with different purification tubes; or the sleeve portion has two different slopes, and the first layer is close to the middle tube of the purification tube. The slope is relatively gentle, and the slope of the second layer near the lower end of the purification tube is steep.
  • the two layers of different slopes are used to make it suitable for gas-tight fitting in various purification tubes of various brands on the market.
  • the method of the present invention when extracting the nucleic acid in the test body, it is not necessary to use a centrifuge to centrifuge the waste liquid or the extract liquid as in the prior art; on the contrary, it is only necessary to simply use the positive and negative air pressure to purify
  • the tube can be easily purified by the action of suction or discharge; in addition, it is not necessary to use the micro-dispenser to add the sample, the cleaning solution or the cleaning solution from the upper neck opening of the purification tube as in the prior art.
  • the extracting solution on the other hand, the method of the present invention can directly suck the sample, the cleaning solution or the extract from the lower end of the purification tube.
  • a device for pneumatically extracting nucleic acid comprising: a syringe comprising a barrel, a push rod disposed in the barrel, characterized by a push rod in the barrel Below is a semipermeable membrane that is capable of binding to the nucleic acid or polarity of the nucleic acid.
  • the device may further comprise a needle assembly detachably coupled to the barrel.
  • the user can use the negative air pressure generated when the push rod is pulled up, so that the needle can generate suction and suck the sample.
  • the negatively charged nucleic acid in the sample will be half
  • the membrane is electrically or polarly bonded, and the residual specimen can be discharged from the needle by pushing the positive air pressure generated by the push rod. This is a “combination step", and the “combination step” can be repeated one or more times.
  • the efficiency of binding the nucleic acid to the semipermeable membrane can be improved; then, the push rod is pulled up again, and the cleaning liquid is sucked by the needle, so that the impurities on the purified membrane are cleaned, thereby improving the purity of the nucleic acid, after washing Pushing the positive air pressure generated by the push rod and discharging the cleaning liquid from the needle. This step is called “cleaning step”. The cleaning step can be repeated one or more times according to requirements. Finally, the push rod is pulled up again and sucked by the needle.
  • the extract is made to change the electrical conductivity or polarity of the purified membrane by the extract of special salinity and pH.
  • the nucleic acid is flushed from the semipermeable membrane and dissolved in the extract.
  • the device of the present invention With the device of the present invention, the user can easily extract the nucleic acid from the sample without using a vacuum device or a centrifuge at all, and in the prior art, all the liquid materials are once passed through the membrane, and the liquid is It is absorbed or drained, and the semi-permeable membrane cannot be repeatedly passed through to improve the efficiency of nucleic acid extraction.
  • the present invention overcomes this drawback, and the apparatus of the present invention can easily and repeatedly pass the liquid material through the semipermeable membrane, and is effective. Increase the amount of nucleic acid extracted.
  • a further aspect of the present invention provides an automated machine for extracting nucleic acid pressure-extracting nucleic acid using the method of the present invention, comprising a mainframe, having an electronic component set required for operation, a mobile device, and the host station Connected, a cylinder, electrically connected to the mainframe, and a console electrically connected to the mainframe, the console comprising a purification kit.
  • the automated machine may further comprise a heating module and/or an oscillator to selectively oscillate or heat the sample during operation to improve nucleic acid purification. Yield.
  • the purification kit comprises: (a) a purification tube having a semipermeable membrane, (b) a filter adapter, (c) a pipette pipette, (d) - a well plate for accommodating the sample, (e) at least one orifice plate for accommodating the buffer; and (f) a orifice plate for accommodating the product after extraction.
  • the components (a) to (c) of the above kit may be integrally formed, and in a preferred embodiment of the invention, the purification kits are integrally arranged in a row of purified crucibles.
  • An automated machine for pneumatically extracting nucleic acids wherein the moving device is preferably a robotic arm, which can be used to move the cartridge and/or move the purification kit, and the tip of the cartridge has a tip for combining the adapters in the purification kit.
  • the moving device is preferably a robotic arm, which can be used to move the cartridge and/or move the purification kit, and the tip of the cartridge has a tip for combining the adapters in the purification kit.
  • the cartridge and the assembly are hermetically joined to provide a negative air pressure to pipette the pipette Purify the sample in the sample well plate of the kit to the purification tube, so that the nucleic acid in the sample reacts with the semi-permeable membrane in the sample or the polarity, and the adapter can avoid the specimen from rushing to the cartridge, and avoiding Contamination, when the reaction is completed, the cartridge is given positive air pressure, and the purification tube can directly discharge the residual sample into the sample well plate of the purification kit via the pipette pipette, and this step can be repeated one or more times to improve nucleic acid binding to The amount of semi-permeable membrane;
  • the robot arm mechanically drives the purification kit to fill the orifice plate with the cleaning liquid under the assembly on the cartridge, and the cartridge is lifted The negative pressure is applied to the pipette su
  • the cartridge After cleaning, the cartridge provides positive air pressure, and the cleaning liquid is discharged into the same orifice plate which is originally sucked out, and the cleaning step can be repeated one to several times;
  • the robot arm mechanically drives the upper end of the orifice plate filled with the flushing liquid to the lower part of the assembly on the cylinder, and provides a negative air pressure to allow the pipette pipette to absorb the extract liquid into the purification tube to make the electricity
  • the nucleic acid bound to the semipermeable membrane is eluted into the extracting liquid, and then the cartridge is supplied with positive air pressure, the eluent is discharged into the same orifice plate that was originally sucked out, or the robot arm can mechanically drive the purification kit.
  • the purified nucleic acid is collected in a collection tray under the assembly that is nested on the cartridge, and the step may be repeated one or more times to increase the amount of nucleic acid extracted.
  • the purification kit can be designed according to different requirements, for example, the orifice plate can increase the buffer plate of the buffer according to the number of times of cleaning required for the experiment, and the buffer is a cell. Lysates, cleaning solutions, alcohol, water or extracts, and the like, are within the scope of the invention.
  • the machine not only does not need the equipment of the centrifuge to be disposed therein, but only needs a simple moving device and a cylinder capable of providing positive or negative air pressure, that is, vacuum suction
  • the sample and the reaction liquid can not only omits the time required for centrifugation in time, but also the reaction stroke of the entire machine is simple, because the cartridge only needs simple suction, and only needs to take the straight line of the same column to be the front end.
  • the combination step is to the final stripping step, and the various steps in the middle need to be repeated multiple times, and the reaction can be performed in a purification kit integrated into a row of purified crucibles, unlike conventional automated centrifuge extraction.
  • Nucleic acid machine because of the centrifugal relationship, the robot arm may grab the sample in multiple directions to increase the pollution rate; moreover, the adapter and the pipette of the present invention have versatility, so it is not necessary to develop a new one.
  • the tubes are purified, and various conventional purification tubes can be used.
  • the automated machine for pneumatically extracting nucleic acids of the present invention improves the machine defects of the prior art and provides a new, simpler, and more efficient automation technique.
  • the present invention overcomes the technical bias, and before the purification tube is provided with the sample having the nucleic acid, the purification tube is first end (the upper end of the purification tube) Combining with pressure equipment, all liquid materials (including specimens with nucleic acids, residual specimens whose nucleic acids have been adsorbed by semipermeable membranes, cleaning fluids, waste cleaning fluids, eluents, or The extract containing the nucleic acid was only taken in and out of the other end of the purification tube (lower end of the purification tube).
  • An advantage of using the method of the present invention is that since all of the liquid material is only taken in and out of one end of the purification tube, in addition, since the sample having the nucleic acid is passed through the semipermeable membrane after being taken up from the lower end of the purification tube, the nucleic acid is first and the semipermeable membrane When the positive pressure is applied to the purification tube, and the sample is discharged, the sample is again passed through the semi-permeable membrane and discharged at the lower end of the purification tube. If there are residual nucleic acids not bound to the semipermeable membrane, There may be a chance to combine with the semipermeable membrane.
  • the bonding step of one operation in the present invention has actually repeated the bonding step twice; and the cleaning step and the collecting step are also the same principle, and are operated in the present invention.
  • the actual The cleaning and collection steps have been repeated twice, so the amount of extracted nucleic acid is increased and the efficiency is higher.
  • the positive/negative pressure provided by the pressure device can be repeatedly used in the extraction process, so that the liquid used for the extraction can repeatedly pass through the semipermeable membrane, further improving the efficiency of nucleic acid binding, washing or elution, and effectively increasing the extraction amount of the nucleic acid.
  • all of the reaction liquid passes through the semipermeable membrane at one time, and the liquid is drained or pumped away, but the method of the present invention can increase the extraction amount and improve the extraction efficiency.
  • all liquid materials used can be "absorbed/extracted in situ", that is, where the liquid material in the extraction process is taken up, and can be discharged back to where it was taken. Or other desired containers, without the use of special waste tanks for collection. Therefore, if a row of purified hydrazine is used, and after the extraction of the nucleic acid, all the effluent is returned to its original position in the purification sputum, and the purified hydrazine after use can be directly discarded, which is not only convenient but also It reduces the risk of cross-contamination.
  • Figure 1 Flow chart showing the extraction of nucleic acids by conventional column-centrifugal extraction.
  • Figure 2 Flow chart showing the extraction of nucleic acids by conventional column vacuum extraction.
  • Fig. 3 is a schematic view showing an embodiment of the pneumatic extraction nucleic acid device of the present invention.
  • Figure 4 is a schematic view showing another embodiment of the pneumatic extraction nucleic acid device of the present invention.
  • Figure 5 is a schematic representation of the purification kit used in the automated machine for the pneumatic extraction of nucleic acids of the present invention. detailed description
  • the pneumatic extraction nucleic acid device comprises a purification tube 20 and a pressure device 60 that can apply a positive or negative gas pressure.
  • the upper end of the purification tube 20 is hermetically joined to the pressure device 60, and the purification tube is an upper neck portion 201, an intermediate tube portion 202, a purification membrane 203 and a lower tip portion 204 from top to bottom.
  • the portion 204 has a through hole.
  • a "binding step” is first performed: the pressure device 60 is turned on to apply a negative air pressure to the purification tube 20, and the lower tip portion 204 of the purification tube 20 sucks a sample containing nucleic acid to pass the sample through the purification membrane.
  • the nucleic acid is electrically or polarly bonded to the purification membrane 203; the pressure device 60 is turned on to apply positive pressure to the purification tube 20, and the residual sample is passed through the purification membrane 203 in the opposite direction, and then purified.
  • the lower tip 204 of the tube 20 flows out, and this step can be repeated one to many times as needed.
  • a "collection step” is performed: the pressure device 60 is turned on to apply a negative air pressure to the purification tube 20, and the extract liquid is taken up by the lower tip portion 204 of the purification tube 20, and the extract liquid is passed through the purification membrane 203, and then the solution is changed.
  • the purified membrane 203 is electrically or polarized to separate the nucleic acid from the purification membrane 203 and dissolved in the extract; the pressure device 60 is turned on to apply a positive pressure to the purification tube 20, so that the nucleic acid-dissolved solution is in the opposite direction. After passing through the purification membrane 203 again, it is discharged and collected from the lower tip portion 204 of the purification tube 20, and the collection step can be repeated one to many times as needed.
  • the purification tube 20 can utilize purification tubes existing in various conventional techniques, and purification tubes of different brands on the market can be utilized in the present invention. Furthermore, in order to prevent the sample in the purification tube 20 from being sucked by the negative air pressure, it is flushed onto the pressure device 60 to cause pollution. It can be seen that the filter tube 901 can be further disposed between the upper end of the purification tube 20 and the pressure device 60. The adapter 90 is in airtight engagement with the upper end of the purification tube 20 (i.e., the upper neck 201) and the pressure device 60.
  • the tip 204 of the purification tube 20 may further be provided with a pipette pipette 80 that is in airtight engagement therewith, whereby the purification tube 20 can utilize the pipette straw 80 Aspirate a small amount of liquid or collect the purified sample into a microcentrifuge tube.
  • the upper end of the pipetting pipette 80 can further comprise a set of bodies that can be airtightly from the intermediate pipe body 202 to the lower tip portion 204 of the purification pipe portion.
  • the pipetting pipette 80 can be widely applied to various types of purification pipes; in addition, the structure and the different plant for the joint of the adapter 90 and the pipette 80 and the purification pipe are
  • the purification tube of the card has versatility, and the structure of the adapter 90 and the pipette pipe 80 and the purification pipe 20 can be made of a rubber material to have an elastic structure, and can be joined to the purification pipe 20 of different specifications.
  • the nucleic acid can be extracted by a pneumatic method because the components must be hermetically bonded.
  • the manner in which the components are hermetically joined can be any conventional technique, such as the addition of an O-ring, and is not limited.
  • FIG. 4 is a schematic diagram of another embodiment of the pneumatic extraction nucleic acid device of the present invention.
  • the pneumatic extraction nucleic acid device can be a syringe 70, which is a pressure element that can apply a positive or negative gas pressure.
  • the syringe 70 includes a barrel 701, a push rod 702 disposed in the barrel 701, and a needle assembly 703 detachably engaged with the barrel 701, wherein the barrel 701 is below the push rod 702.
  • a purification membrane 203 is provided which is electrically coupled to the nucleic acid.
  • the "synthesis step”, the “cleaning step” and the “collection step” can be performed by directly using the syringe 70 by the suction and pushing of the push rod 702 according to the above method; in detail, first
  • the needle assembly 703 sucks the sample by the negative air pressure generated when the push rod 702 is pulled up.
  • the sample enters the barrel 701 through the needle, the sample passes through the purified film 203 from bottom to top, and the sample body is negatively charged.
  • the nucleic acid is electrically or polarly bound to the purification membrane 203, and the residual sample is discharged from the needle through the purification membrane 203 by pushing the positive air pressure generated by the push rod 702, thereby completing the "binding step”.
  • the push rod 702 is pulled up again, and the cleaning liquid is sucked by the needle assembly 703, and the cleaning liquid passes through the purification film 203 from bottom to top, so that the impurities on the purification film 203 are cleaned, thereby improving the purity of the nucleic acid.
  • the cleaning step can be repeated one or more times according to requirements;
  • the pusher 702 is pulled up again, and the extract is sucked by the needle, and the extract passes through the purified membrane 203 from bottom to top, so that the extract of the special salinity and pH changes the electrical properties of the purified membrane 203 or Polarity, the nucleic acid is flushed from the semi-permeable membrane and dissolved in the extract, and the push rod 702 is pushed to collect the nucleic acid-dissolved extract from the top to the bottom through the purified membrane 203 and then discharged through the needle. In the collection tube, complete the "collection step".
  • Another aspect of the invention is an automated machine for pneumatically extracting nucleic acids designed using the method of the invention
  • the automated machine may further comprise a heating module and/or an oscillator to selectively oscillate or heat the sample during operation to improve nucleic acid purification. Yield.
  • FIG. 5 is a schematic view of a purification kit 150 according to an embodiment of the present invention.
  • the purification kit 150 includes: a purification tube 20 having an upper neck portion, an intermediate tube portion, a semi-permeable membrane, and a lower tip portion. a filter connector 90, a pipette pipette 80, a hole plate for accommodating the sample (referred to as the sample well plate 100), and at least one orifice plate for accommodating the buffer solution (referred to as a buffer hole) A tray 120); and a well plate for accommodating the product after extraction (abbreviated as product orifice plate 110).
  • An automated machine for pneumatically extracting nucleic acids having a cylinder on a robot arm having a tip in front of the cartridge for combining the adapter 90, the purification tube 20, and the pipette pipette 80 in the purification kit 150 (from above)
  • the cartridge when the three are combined (hereinafter referred to as the assembly), the cartridge can be airtightly engaged with the assembly, and the negative air pressure is supplied to cause the pipette pipette 80 to suck into the sample hole plate 100 in the purification set 150.
  • the sample is transferred to the purification tube to make the nucleic acid in the sample react with the semi-permeable membrane in the electrical or polar phase, and the adapter 90 can prevent the sample from rushing to the cartridge to avoid contamination.
  • the cartridge is completed.
  • the positive pressure is given, and the purification tube 20 directly discharges the residual sample into the sample well plate 100 in the purification set 150 via the pipette pipette 80 (this is a "binding step"), wherein in the combining step, the repeating step can be repeated.
  • the sample passes through the purification tube 20 to a plurality of times to increase the amount of nucleic acid bound to the semipermeable membrane; then, the robot arm mechanically drives the assembly to the upper end of the purification tray filled with the cleaning liquid (ie, the buffer)
  • the liquid orifice disk 120 provides a negative air pressure to cause the pipette pipette 80 to suck the cleaning liquid into the purification pipe 20.
  • the cylinder After cleaning, the cylinder provides positive air pressure, and the cleaning liquid is discharged into the same hole plate originally sucked out (this is " a washing step"), the washing step may be repeated one to many times; and when the washing step is finished, the purification tube 20 may be further heated by a heating mechanism to evaporate the ethanol in the washing liquid, and/or through the plurality of tubes
  • the positive/negative air pressure is provided to dry the semipermeable membrane; finally, the robot arm mechanically drives the assembly to the upper end of the purification tray (the buffer hole tray 120) filled with the extracting liquid, and the cylinder provides a negative air pressure.
  • the pipette pipette is sucked into the purification tube 20, and the nucleic acid which is electrically/polarly bound to the semipermeable membrane is eluted into the extract liquid, and then the cylinder is supplied with positive air pressure, and the extract liquid is discharged into the original suction.
  • the robot arm can mechanically drive the assembly to the product orifice plate 110 of the purification kit 150, and collect the purified nucleic acid (this is a "collection step"), and the collection step
  • the extract can effectively increase the amount of nucleic acid.
  • the purification kit 150 can be two separate components, and can also form an integrally formed component, which is not limited, and is preferably a one-piece molding.
  • the buffer wells 120 in the purification kit 150 can be different in number according to requirements, but the arrangement is optimal according to the steps in the process of extracting nucleic acids, followed by cell lysate, washing solution, alcohol, water or Effervescent liquid, whereby, when the dispenser drives the assembly to move, there may be a step in sequence The step is moved straight to the rear for nucleic acid extraction.
  • the nucleic acid-containing sample usually contains a lysis buffer to break the cell membrane of the cells contained in the sample to release the nucleic acid
  • the buffer contains a large amount of surfactant, so After the completion of the process, when the residual sample is discharged, if it is discharged into the empty orifice plate, a large amount of air bubbles are easily generated and the operation is unfavorable.
  • the purification kit 150 may include a An orifice disk (not shown) for accommodating the residual sample, wherein the orifice disk contains 100% EtOH, the EtOH can absorb bubbles, and the residual sample is discharged to the orifice plate without generating The bubble; wherein the amount of EtOH is not limited, but is preferably from 100 to 500 ⁇ , more preferably from 200 to 400 ⁇ , and most preferably from 300 ⁇ l.
  • the purification kit is improved.
  • 150 may include: a well plate (not shown) containing 100% EtOH before the buffer plate 120, wherein the amount of EtOH is not limited, but is preferably 100 to 500 ⁇ 1, more preferably 200 to 400 ⁇ . , the best is 300 ⁇ 1.
  • reagents were used in the QIAamp DNA Reagent Kit (Qiagen Taiwan Co. Ltd.), containing tissue lysate, cell lysate, cleaning solution I, cleaning solution II, rushing a liquid extracting protein and a proteinase K, and a purification tube, wherein the semipermeable membrane in the purification tube is a silica membrane.
  • the fish liver of about 5x5mm size was placed in a 2ml screw cap test tube, and 800 ⁇ 1 phosphate buffer saline (PBS) and a 5mm steel ball were added to homogenize the tissue homogenizer for 30 seconds.
  • PBS phosphate buffer saline
  • 800 ⁇ l of cell lysate mix with a shaker
  • 80 ⁇ l of proteolytic enzyme and mix using an oscillating shaker.
  • 800 ⁇ l of ethanol was added and shaken with a shaker for 30 seconds. After centrifugation at 13,000 rpm for 3 minutes, 200 ⁇ l of the supernatant was taken into each of four sample tubes, which were the sample of Example 1, the sample of Example 2, the sample of Comparative Example 1, and the sample of Comparative Example 2.
  • the upper neck of the purification tube is hermetically joined to a syringe, and the lower tip of the purification tube is hermetically joined to a pipette suction tube.
  • Washing step Pulling the push rod of the syringe, sucking 500 ⁇ 1 of the cleaning solution I through the pipette pipette, passing the lower tip of the purification tube through the purification membrane, sucking it to the middle tube body portion, and then, pushing the needle tube Push down to discharge the cleaning solution I that has been cleaned of the purified membrane.
  • the cleaning step is performed again, and 750 ⁇ l of the cleaning solution II is sucked through the pipette, and the lower tip of the purification tube is passed through the purification membrane, and then taken up to the intermediate tube body. Then, the syringe is pushed down. Pushing out the cleaning solution II that has been cleaned of the purified membrane.
  • the cleaning step pulling the plunger of the syringe, sucking 800 ⁇ l of 100% alcohol through the pipette pipette, passing the lower tip of the purification tube through the purification membrane, sucking it to the middle tube body, and then, the syringe The pusher is pushed down to discharge the alcohol that has been cleaned of the purified membrane; this step is repeated twice. Next, the syringe was aspirated and pushed at least 15 times to volatilize the alcohol remaining in the purification tube.
  • the collecting step pulling the push rod of the syringe, sucking the 200 ⁇ l extract liquid through the pipette pipette, passing the lower tip of the purification tube through the purification membrane, sucking it to the middle tube body portion, and letting it stand for 3 minutes, then , push down the syringe plunger and drain the nucleic acid-dissolved extract into a 1.5 ml microcentrifuge tube.
  • Example 2 The procedure of Example 2 was repeated as in Example 1, except that the procedure of Example 1 was repeated, and only the specimen was replaced with the specimen of Example 2.
  • Nucleic acid extraction was performed according to the QIAamp DNA kit kit product manual. Combining step: The sample of Comparative Example 1 was pipetted into a purification tube using a micropipette, and the sample was injected from above the purification tube. Next, a waste liquid tube is installed under the purification tube, and the two tubes are centrifuged in a centrifuge at a rotational speed of 13000 rpm for 2 minutes, and the residual sample is centrifuged through the purification membrane to the waste liquid tube through the channel of the lower tip of the purification tube. After centrifugation, the waste liquid in the waste liquid tube is drained, and the nucleic acid is electrically coupled to the purification membrane, and the purification tube is then placed back into the waste liquid tube.
  • Washing step Using a micro-dispenser, 500 ⁇ l of the cleaning solution I is sucked into the purification tube, and the cleaning solution I is injected from above the purification tube.
  • the two tubes were centrifuged at a speed of 13,000 rpm for 1 minute in a centrifuge.
  • the cleaning solution I was centrifuged through the passage of the lower end of the purification tube to the waste liquid tube through the purification membrane. After the centrifugation, the waste liquid in the waste liquid tube was drained.
  • the purification tube is then placed back into the waste tube.
  • the cleaning step is again performed again, and 750 ⁇ l of the cleaning solution II is sucked into the purification tube, and the cleaning solution is injected from above the purification tube.
  • the two tubes are centrifuged at 13,000 rpm for 1 min in a centrifuge, and the washing solution II is centrifuged through the passage of the lower end of the purification tube to the waste liquid tube through the purification membrane. After the centrifugation is completed, the waste liquid in the waste liquid tube is drained, and the purification tube is further set. Return to the waste pipe.
  • Post-cleaning procedure Centrifuge the two kits at 13000 rpm for an additional three minutes to ensure complete removal of the cleaning solution.
  • Collection procedure Remove the waste tube and replace it with a 1.5 ml microcentrifuge tube.
  • the 200 ⁇ l extract was pipetted into the purification tube by a micro-dispenser, and the extract was injected from the top of the purification tube to the middle tube body and allowed to stand for 3 minutes. Then, the two sets were placed in a centrifuge and centrifuged at a rotational speed of 13,000 rpm. 1 minute to get The nucleic acid-dissolved extract was transferred to a 1.5 ml microcentrifuge tube.
  • Comparative Example 2 was repeated in the same manner as in Comparative Example 1, except that the sample was replaced with the sample of Example 2.
  • the concentration of DNA extracted using the method and apparatus of the present invention is similar to the concentration of DNA collected using conventional methods, and the method is relatively simple.
  • the method and apparatus of the present invention are applicable to purification kits for extracting nucleic acids from different brands.
  • the method and apparatus of the present invention are applicable to purification kits for extracting nucleic acids from different brands.
  • Example 3 The procedure and the sample of Example 3 were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic silica membrane (product name: Glass Fiber Media) of PALL Corporation, and the membrane pore diameter was ⁇ .
  • the procedure of Example 1 was repeated, but nucleic acid extraction was carried out in the apparatus of the present invention, using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step was repeated twice.
  • Example 4 The procedure of Example 4 was the same as that of Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic nitrocellulose membrane of PALL (product name: BioTrace), and the membrane pore diameter was 0.2 ⁇ m; Example 1 was repeated.
  • the procedure performs nucleic acid extraction in the apparatus of the present invention using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step is repeated twice.
  • Example 5 The procedure and the sample of Example 5 were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic nitrocellulose membrane (product name: Mixed Cellulose Esters) of Advantec Co., Ltd., and the membrane pore diameter was 0.2 ⁇ ;
  • Example 6 The procedure and the sample of Example 6 were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic cellulose acetate membrane (product name: Cellulose Acetate) of Advantec Co., Ltd., and the membrane pore diameter was 0.45 ⁇ ;
  • Comparative Example 5 The procedure and the sample of Comparative Example 5 were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 5.
  • Comparative Example 6 The procedure and the sample of Comparative Example 6 were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube was replaced with the semipermeable membrane used in Example 6.
  • the amount of nucleic acid extracted by using the apparatus of the present invention is higher than that of the prior art, because the liquid material is only used by the lower end of the purification tube when the apparatus of the present invention is used, and therefore,
  • the positive/negative air pressure provided by the pressure device can be repeatedly used to repeatedly pass the liquid through the semi-permeable membrane, thereby improving the efficiency of nucleic acid extraction and effectively increasing the extraction amount of the nucleic acid.
  • all of the reaction liquid passes through the semipermeable membrane at one time, and the liquid is eliminated.
  • the method and apparatus of the present invention can increase the extraction amount and improve the extraction efficiency.
  • Comparative Example 7A The procedure and sample of Comparative Example 7A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with PCAR Corporation's hydrophobic carboxylated polyvinylidene fluoride (product name: Fluoro Trans G), and the membrane pore size was 0.2 ⁇ .
  • the procedure of Example 1 was repeated, but nucleic acid extraction was carried out in the apparatus of the present invention, using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step was repeated twice.
  • Comparative Example 8A The procedure and sample of Comparative Example 8A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with the hydrophobic nylon product name of PALL: Hydrolon), and the membrane pore diameter was 1.2 ⁇ m; the procedure of Example 1 was repeated, but Nucleic acid extraction was carried out in the apparatus of the present invention using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step was repeated twice.
  • Comparative Example 9A The procedure and sample of Comparative Example 9A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with the hydrophobic polyethersulfone of PALL Company (product name: Supor-450PR), and the membrane pore diameter was 0.45 ⁇ ;
  • Comparative Example 10A The procedure and sample of Comparative Example 10A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with
  • Advantec's hydrophobic polytetrafluoroethylene product name: Supported PTFE with a membrane pore size of 0.45 ⁇ ; repeat the procedure of Example 1, but perform nucleic acid extraction in the apparatus of the present invention using a positive and negative gas pressure of 60 kPa/- 60kpa, and the collection step is repeated twice.
  • Comparative Example 7B The procedure and the sample of Comparative Example 7B were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane in the purification tube used was replaced with the semipermeable membrane used in Example 7A.
  • Comparative Example 8B The procedure and sample of Comparative Example 8B were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane in the purification tube used was replaced with the semipermeable membrane used in Example 8A.
  • Comparative Example 9B The procedure and sample of Comparative Example 9B were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane in the purification tube used was replaced with the semipermeable membrane used in Example 9A.
  • Comparative Example 10B The procedure and the sample of Comparative Example 10B were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane in the purification tube used was replaced with the semipermeable membrane used in Example 10A.
  • the present invention overcomes the technical bias.
  • the purification tube is first (purification tube).
  • the upper end) is combined with the pressure device so that all liquid materials used to extract the nucleic acid are only taken in and out of the other end of the purification tube (lower end of the purification tube).
  • all steps in the extraction of the nucleic acid can be easily utilized to provide positive and negative pressure, and the liquid is sucked/discharged from the same end of the purification tube, which not only simplifies the extraction.
  • the step, and the liquid can be repeatedly passed through the semi-permeable membrane to achieve the result of improving the extraction efficiency, and the automated machine can be configured without a centrifuge, and only needs to be equipped with a device capable of providing a pressure difference, thereby achieving the effect of reducing the volume.

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Abstract

本发明有关一种新颖的抽取核酸的方法及其装置，其利用气压式萃取核酸的方法，在纯化管上端与一可施以正或负气体压力装置接合，即可由纯化管下尖部吸取/排出检体、清洗液及冲提液，达到不需要离心机即可简便抽取核酸的效果。

Description

气压式萃取核酸的方法及其装置
技术领域
本发明有关一种抽取核酸的方法及其装置， 特别是关于一种气压式抽取核酸 的方法及其装置， 以及利用该方法所设计的自动化机台。 背景技术
随着生物科技的发展以及遗传物质的解码， 越来越多的生物相关实验室或医 院甚至法医检验等等， 皆频繁的使用抽取检体内的核酸来进行实验或检查。 抽取 并纯化核酸的方法很多， 而目前最常见的方法分成三类： 管柱萃取法、 磁珠萃取 法以及试剂萃取法， 而试剂萃取法又分成两类， 分别是有机溶剂萃取法和非有机 溶剂萃取法。 各种萃取方法皆有其优缺点， 然而， 管柱萃取法是目前操作上最安 全简便且效果最佳的方式。
而利用该管柱萃取法的萃取流程又可分成两种， 一种是管柱离心萃取法， 另 一种则为管柱真空萃取法。 图 1为习知的管柱离心萃取法， 首先， 装于微量离心管 10内的处理过的检体 (如利用阴离子清洁剂将细胞打破释放出核酸， 并使蛋白质变 性)转置于一纯化管 20中， 该纯化管 20通成可分成三部分， 上颈部 201、 中间管体部 202， 以及下尖部 204; 中间管体部 202的底部具有纯化膜 203， 而下尖部 204则具通 道可使液体流出， 通常检体是利用微量分注器 (pipette)将微量离心管 10内的检体吸 取后， 由纯化管 20的上方注入至纯化管 20中。 纯化管 20外套有一废液管 30， 将套 合而为一体的双管置于离心机中离心， 由于核酸带负电， 会与带正电的纯化膜 203 结合而吸附于其上， 而其它杂质则会因为离心力的关系而穿透纯化膜 203且通过纯 化管 20的下尖部的通道而流到废液管 30内， 此步骤称为 「结合步骤」 ； 接着， 于 纯化管 20内加入清洗液后再次离心， 使纯化膜 203上的杂质被离心出来提高核酸的 纯度， 此步骤称为 「清洗步骤」 ； 最后， 将纯化膜 203上带有核酸的纯化管 20转置 到收集管 40中，加入特殊盐度及 pH值的冲提液改变纯化膜 203的电性，并再度离心， 使核酸与纯化膜 203分离而流出，收集核酸于收集管 40中，此步骤称为「收集步骤」。 管柱离心萃取法需要经过至少三次的离心， 过程较为繁复且萃取时间会因等后离 心而拉长。 虽然市面上已有自动化的管柱离心萃取机台， 但由于机台内必须设有 离心设备， 因此机台通常体积较大， 且其萃取较为耗时的缺点并未克服。
另外一种管柱真空萃取法如图 2所示， 其原理也与管柱离心萃取法雷同， 只是 其 「结合步骤」 以及 「清洗步骤」 的步骤是用真空吸引取代离心方式， 其直接将 纯化管 20插入压力盒 50中， 利用气体压力， 给予负压使液体流出纯化管 20而直接 进入液体收集瓶 (未图示)， 但是在最后的 「收集步骤」仍然必须利用离心方式将核 酸收集至收集管 40中。 因此， 即便是自动化机台， 也必须要有离心设备设置于其 中。
综上所述， 目前市面上虽然已有许多厂商研发出各种的管柱萃取套组及自动 化机台， 但其原理及萃取流程皆与上述的习知技术雷同， 其存在的体积大且耗时 较久的缺陷尚未被克服， 因此， 本技术领域中尚需一种新的方式及机台来克服这 些缺点。 发明内容
为了解决上述萃取核酸所产生耗时且耗费人力的问题， 以及自动化机台体积 过大等的缺陷， 本发明人出人意料地发想， 利用习知的纯化管， 直接在纯化管中 利用气压的方式， 完全仅利用纯化管的下端抽吸及排放检体、 清洗液、 冲提液等， 完全不需要利用离心机， 且不需要从纯化管上颈部处注入检体、 清洗液或冲提液， 即可有效地萃取核酸。 因此， 本发明人提供了一种气压式抽取核酸的方法及其装 置， 以及利用该方法所设计的自动化机台。
本文中术语 「一」 及 「一种」 代表于本文中的语法对象有一个或多于一个 (即 至少一个)。
本发明的一方面提供一种气压式萃取核酸的方法， 其包含： （a)将一纯化管上 端与一可施以正或负气体压力的压力设备气密接合， 其中该纯化管为具有半透膜 的通孔管体； （b)开启该压力设备对该纯化管施以负气压， 利用该纯化管的下端吸 取含有核酸的检体， 使该检体通过该半透膜后， 该核酸与该半透膜电性或极性结 合； （c)开启该压力设备对该纯化管施以正气压， 使残余检体以相反于步骤 0?)的方 向通过半透膜后， 由纯化管的下端流出， 且其中该步骤 (b)及 (c)可仅进行一次或重 复进行多次； （d)开启该压力设备对该纯化管施以负气压， 利用该纯化管的下端吸 取冲提液， 使该冲提液通过半透膜后， 改变该半透膜电性或极性使核酸与半透膜 分离并溶于冲提液中； 及 (e)开启该压力设备对该纯化管施以正气压， 使该溶有核 酸的冲提液相反于步骤 (d)的方向通过半透膜后， 由该纯化管的下端排出并收集， 且其中该步骤 (d)及 (e)可仅进行一次或重复进行多次。
本文中术语 「纯化管」 为技术领域中习知的用于纯化核酸的管柱， 通常而言 其包含一上颈部、 一中间管体部、 一半透膜以及一下尖部， 该下尖部具有一通孔 而可用于排出液体， 但本发明中的该纯化管并不限于此结构， 只要该纯化管为具 有半透膜的通孔管体即在本发明的范围内， 管体的材料可为塑料如聚丙烯、 聚苯 乙酰、 聚碳酸酯或聚氯乙烯等， 或生物可降解性材料。 该半透膜亦已为技术领域 中所习知， 该材质可为二氧化硅， 表面电荷带有正电， 可与检体中带负电的核酸 电性结合， 并受冲提液改变其电性后， 释放核酸； 于本文中， 该 「半透膜」 又称 「纯化膜」 ， 为可使溶液由其内部通过， 亦即， 当在与半透膜的一面接触的空间 与该半透膜的另一面空间之间存有压力差时， 溶液可由高压空间往低压空间流动 而穿透该半透膜； 或者， 当该半透膜受到离心力时， 溶液可沿着离心力的方向穿 透该半透膜， 但核酸则与该半透膜作用而吸附于该膜上； 而本发明中所使用的半 透膜透过该膜与核酸之间的相互作用来吸附核酸， 其中， 核酸与半透膜是透过极 性或电性而互相吸引， 于本发明的较佳实施例中， 该半透膜为具有亲水性基团的 多孔膜 (又称亲水性膜)，且推测核酸的亲水性基团与半透膜的亲水性基团透过改变 周围的极性而互相吸引； 本文中所称的具有亲水性基团的半透膜是指构成多孔膜 材料本身具有亲水性基团 (如聚丙烯酸羟乙酯、 聚甲基丙烯酸羟乙酯、 聚乙烯醇、 聚乙烯吡咯垸酮、 聚丙烯酸、 聚甲基丙烯酸、 聚氧乙烯、 醋酸纤维素、 醋酸纤维 素皂化物、 醋酸纤维素混合物等， 优选可使用具有羟基的有机聚合物)， 或者是对 构成多孔膜的材料进行处理或涂敷， 从而引进亲水性基团， 而任意的有机或无机 材料均适合做为该多孔膜的材料， 也可以是多种多孔膜的组合； 所述的多孔膜中， 为了能使溶液由内部通过， 因此该膜的孔径优选为大于或等于 0.2μηι， 厚度优选为 ΙΟ μηι至 500 μηι， 更佳为 50 μηι至 250 μηι。 又本文中， 纯化管的上端及下端的定义 为， 一般的纯化管结构中， 较靠近该纯化膜位置者为下端， 而远离该纯化膜位置 者为上端， 但本发明并不限于此， 而以与压力设备接合处者称为纯化管的上端， 而用于吸取液体者称为纯化管的下端。
本文中术语 「可施以正或负气体压力的压力设备」 包含任何装置可提供正或 负气体压力者， 最简易的装置如唧筒， 可藉由抽或压该唧筒体内的推杆而提供正 或或负气体压力； 抑或如习知技术中的针筒、 移液器、 泵或气压缸， 其包含一气 压阀以及一压力器， 可受控制而产生正或负气体压力， 其中， 使溶液可穿透半透 膜的压力值为 lOkPa至 300kPa， 优选为 40kPa至 200kPa， 最优选的压力值为 60kPa。
在本发明的一实施例中， 为了可进一步洗掉半透膜上的杂质， 而可纯化萃取 出的核酸， 因此， 本发明上述的方法的步骤 (C)及 (d)之间可进一步包含清洗步骤， 该清洗步骤包含： （c-1)开启该压力设备对该纯化管施以负气压， 利用该纯化管的 下端吸取清洗液， 使该清洗液通过该半透膜； 及 (c-2)开启该压力设备对该纯化管 施以正气压， 使该清洗液以相反于 (c-1) 的方向通过半透膜后， 由纯化管的下端流 出。
本文中术语 「清洗液」 包含习知技术中任何可用于清洗纯化管的液体， 其包 含但不限于水、 酒精或其他不会改变半透膜电性或极性的缓冲液， 亦即该清洗液 能够洗出核酸混合物溶液中的杂质， 该杂质是与核酸一起吸附于该半透膜上， 就 此方面而言， 该清洗液仅使杂质由半透膜上被冲提， 而不会使核酸被冲提， 为达 到此目的， 由于核酸难溶于水性的有机溶液， 因此使用适合的水性有机溶液， 皆 在本发明的范围当中， 例如醇类， 包含甲醇、 乙醇、 异丙醇、 正丙醇或丁醇， 优 选为乙醇。
本文中术语 「充提液」 包含习知技术中任何可用于将核酸由该半透膜上解离 的液体， 其包含可将半透膜的极性或电性改变， 而使核酸由其上解离下来的液体， 例如为含有 1.2 M NaCl、 50mM 3- (N-吗啉） 丙磺酸 (MOPS)、 15%乙醇且 pH 8.0的 缓冲液； 或如为含有 0.5M乙酸铵、 10mM乙酸镁 (pH7.5)及 1.5mM EDTA的缓冲液； 或如为含有 1.25M NaCl、 50 mM Tris-HCl pH 8.5及 15% 异丙醇的缓冲液等。
为了避免纯化管内的检体受到负气压的吸取， 冲到压力设备上造成污染， 在 本发明的一较佳实施例中， 该纯化管上端与该压力设备中间可进一步设有一具滤 膜的转接头， 当然， 该转接头与纯化管上端以及压力设备气密接合。 此外， 为了 使吸取液体或收集液体时更方便操作， 在纯化管下端可进一步设有与其气密接合 的移液器吸管 (tip), 藉此， 纯化管可利用该移液器吸管吸取少量的液体或是将纯化 的检体收集到微离心管中， 以利保存或进一步的实验。 在本发明较佳的实施例中， 该移液吸管上端结构为一套体， 该套体可气密地包覆该纯化管部分的中间管体至 下端处， 使其套合于该纯化管， 且该移液吸管与该纯化管接合处的结构与不同厂 牌的纯化管具有通用性， 而可广泛应用于各类型的纯化管， 该手段例如但不限于 该移液吸管的套体与该纯化管接合处的结构使用橡胶材质， 使其为弹性结构， 而 可与不同规格的纯化管接合； 或该套体部分具有两层不同的斜度， 第一层靠近纯 化管中间管体的斜度较平缓， 第二层靠近纯化管下端的斜度较陡， 利用此两层不 同的斜度而使其可适用并气密地套合于市面上各种厂牌的纯化管。
依据本发明的方法， 在萃取检体内的核酸时， 完全不需要如同先前技术一样， 必须使用离心机离心掉废液或是冲提液； 反之， 仅需要简单地利用正负气压， 而 使纯化管抽吸或排放的动作， 就可简便地纯化出检体内的核酸； 此外， 也不需要 如同先前技术一般必须利用微量分注器由纯化管的上颈部开口加入检体、 清洗液 或是冲提液， 反之， 本发明的方法都是从纯化管的下端直接吸取检体、 清洗液或 是冲提液即可。
本发明的另一方面提供一种气压式萃取核酸的装置， 其包含： 一针筒， 该针 筒包含一筒体、 设置于该筒体内的一推杆， 其特征在于该筒体内的推杆下方设有 一可与核酸电性或极性结合的半透膜。 其中， 该装置可进一步包含一可拆卸式地 与筒体结合的一针头组件。
藉由本发明的装置， 使用者可利用推杆上拉时所产生的负气压， 使针头产生 吸力而可吸取检体， 当检体通过针头进入体内时， 检体内带有负电的核酸会与半 透膜电性或极性结合， 而残余的检体此时可藉由推动推杆产生的正气压， 而由针 头排出， 此为 「结合步骤」 ， 该 「结合步骤」 可重复一至多次， 而可提高检体内 核酸结合至该半透膜的效率； 接着， 再次将推杆上拉， 并由针头吸取清洗液， 使 纯化膜上的杂质被受清洗后而可提高核酸的纯度， 清洗后再推动推杆产生的正气 压， 将清洗液由针头排出， 此步骤称为 「清洗步骤」 ， 该清洗步骤可依照需求重 复一到多次； 最后， 再次将推杆上拉， 并由针头吸取冲提液， 使特殊盐度及 pH值 的冲提液改变纯化膜的电性或极性， 让核酸由半透膜上冲提出来而溶于冲提液中， 此时， 即可将推杆推动使溶有核酸的冲提液经由针头排出而收集于收集管中， 此 步骤称为 「收集步骤」 ， 该 「收集步骤」 亦可重复一至多次， 而可提高检体内核 酸回收的效率。 藉由本发明的装置， 使用者完全不需要使用真空装置或离心机， 即可简便地由检体中抽取出核酸， 且先前技术中， 所有的液体材料均为一次性通 过膜后， 液体即被吸走或排掉， 而无法反复通过该半透膜来提高核酸萃取的效率， 但本发明克服了该缺陷， 透过本发明的装置， 可简便地将液体材料反复通过半透 膜， 而有效地提高核酸萃取量。
本发明的再一方面提供一种使用本发明的方法萃取核酸的气压式萃取核酸的 自动化机台， 其包含一主机台， 具有运作所需的电子零件组、 一移动装置， 与该 主机台电性相连、 一唧筒， 与该主机台电性相连以及一操作台， 与该主机台电性 相连， 该操作台包含一纯化套组。 在本发明的一较佳实施例中， 该自动化机台可 进一步包含一加热模组及 /或一振荡器， 使检体在操作过程中可选择性地受振荡或 加热， 以提高核酸纯化的产量。
于本发明一实施例中， 该纯化套组包含： （a)—具有半透膜的纯化管、 （b)—具 滤膜的转接头、 （c)一移液器吸管、 （d)—用于容置检体的孔盘、 （e)至少一个用于容 置缓冲液的孔盘； 及 (f)一用于容置萃取后产物的孔盘。 其中， 上述套组中的 (a)至 (c)组件可为一体成形者， 于本发明的一较佳实施例中， 该纯化套组一体排列为一 行纯化匣的形式。
根据本发明的气压式萃取核酸的自动化机台， 其中该移动装置较佳为机械手 臂， 可用于移动唧筒及 /或移动纯化套组， 唧筒前方具有尖端用于组合该纯化套组 内的转接头、 纯化管以及移液器吸管 (由上往下的顺序)， 当此三者组合完毕 (以下 简称组合件)， 唧筒与组合件气密接合后， 即可提供负气压使移液器吸管吸取纯化 套组内检体孔盘中的检体至纯化管中， 使检体中的核酸与其内的半透膜电性或极 性反应， 且转接头可避免检体上冲至唧筒， 而避免污染， 当反应完成后， 唧筒给 予正气压， 纯化管可将残余检体经由移液器吸管直接排入纯化套组内检体孔盘， 而此步骤可重复一至多次， 以提高核酸结合至半透膜的量； 接着， 机器手臂机械 式地带动组纯化套组中装填有清洗液的孔盘至唧筒上套合的组合件下方， 唧筒提 供负气压使移液器吸管吸取清洗液至纯化管中， 经清洗后， 唧筒提供正气压， 将 清洗液排入原先吸取出来的同一孔盘中， 此清洗步骤可重复一至多次； 最后， 机 器手臂机械式地带动纯化套组中装填有冲提液的孔盘上端至该唧筒上套合的组合 件下方， 提供负气压使移液器吸管吸取冲提液至纯化管中， 使电性结合于半透膜 上的核酸溶出至冲提液中， 接着唧筒提供正气压， 将冲提液排入原先吸取出来的 同一孔盘中， 或是机器手臂可机械式地带动纯化套组的另一收集孔盘中至该唧筒 上套合的组合件下方， 收集所纯化的核酸， 此步骤亦可重复一至多次， 以提高核 酸萃取量。 依据本发明的气压式萃取核酸的自动化机台， 其纯化套组可依照不同 的需求而设计， 如该孔盘可依照实验所需的清洗次数， 增加缓冲液的孔盘， 该缓 冲液为细胞溶解液、 清洗液、 酒精、 水或冲提液等等， 皆在本发明的范围之内。
藉由本发明的气压式萃取核酸的自动化机台， 该机台不仅不需要离心机的设 备设置于其中， 且仅需要简便的移动装置以及可提供正或负气压的唧筒， 即可以 真空方式抽吸检体以及反应液体， 不仅时间上可省略离心所需的时间， 且在配置 整个机台的反应行程亦较为简单， 因为唧筒仅需要简单的抽吸， 只需要走同一列 的直线即可由最前端的结合步骤到最后的冲提步骤， 且中间各种步骤须要重复多 次也均可执行， 且反应可在一体排列为一行纯化匣的纯化套组内完成， 而不像传 统的自动化离心机萃取核酸机台， 因为离心的关系， 机械手臂可能会横越多个方 向抓取检体， 提高污染率； 更有甚者， 本发明的转接头和移液吸管具有通用性， 因此不必再开发新的纯化管， 而可利用习知的各种厂牌纯化管即可。 因此， 本发 明的气压式萃取核酸的自动化机台改良了习知技术的机台缺陷， 且提供了一种新 颖、 简便但效率更高的自动化技术。
在本发明的技术领域中存在有一种技术偏见， 亦即本发明所属技术领域的技 艺人士通常认为， 须要将具有核酸的检体， 由纯化管上端提供， 而使其中的核酸 与半透膜结合后， 残余检体由纯化管的另外一端 (纯化管下端)排出； 然而， 本发明 克服了技术偏见， 在对该纯化管提供具有核酸的检体之前， 便先将纯化管一端 (纯 化管上端)与压力设备结合，而使萃取核酸时所须用到的所有液体材料 (包含具有核 酸的检体、 核酸已被半透膜吸附的残余检体、 清洗液、 废弃清洗液、 充提液或溶 有核酸的充提液)均仅由纯化管的另一端 (纯化管下端)进出。 使用本发明的方法的 优点在于， 由于所有液体材料仅由纯化管的一端进出， 此外， 由于当该具有核酸 的检体由纯化管下端吸取而通过半透膜后， 核酸先与该半透膜结合， 而当对该纯 化管施以正气压， 将该检体排出时， 检体又会再通过一次半透膜并于纯化管下端 排出， 若有残余未与该半透膜结合的核酸， 可有再次的机会与半透膜结合， 因此， 于本发明中操作一次的结合步骤， 实际上已经重复了两次的结合步骤； 而清洗步 骤以及收集步骤亦为相同原理， 于本发明中操作一次的清洗或收集步骤中， 实际 上已经重复了两次的清洗和收集步骤， 故而萃取核酸的量会增加， 且效率更高。 此外， 在萃取过程中还可反复利用压力装置提供的正 /负气压， 使萃取所用的液体 可反复通过半透膜， 又进一步增进核酸结合、 清洗或冲提的效率， 有效提升核酸 的萃取量； 相较于先前技术中， 所有反应液体均是一次性地通过半透膜， 而液体 即被排掉或抽走， 但本发明的方法则可增进萃取量且提高萃取效率。
再者， 使用本发明的方法以及装置， 所有使用的液体材料均可 「原位吸取 /排 除」 ， 亦即， 萃取过程中的液体材料由何处被吸取， 即可排回其被吸取之处或其 他所欲的容器中， 而不须使用特出的废液桶进行收集。 因此， 若使用一行的纯化 匣时， 而当萃取核酸结束后， 所有的废液均还是回到其原本于纯化匣中的位置， 而可直接将该使用过后的纯化匣丢弃， 不仅方便且亦可减少交叉污染的风险。 附图说明
图 1 : 显示习知的管柱离心萃取法萃取核酸流程图。
图 2: 显示习知的管柱真空萃取法萃取核酸流程图。
图 3 : 为本发明的气压式萃取核酸装置的一实施例示意图。
图 4: 为本发明的气压式萃取核酸装置的另一实施例示意图。
图 5 : 为本发明的气压式萃取核酸的自动化机台中所使用的纯化套组示意图。 具体实施方式
首先， 参见图 3， 其发明的气压式萃取核酸装置的一实施例示意图。 该气压式 萃取核酸装置包含一纯化管 20以及一可施以正或负气体压力的压力设备 60。 该纯 化管 20上端与该压力设备 60气密接合， 而该纯化管由上而下分别为一上颈部 201、 一中间管体部 202、 一纯化膜 203以及一下尖部 204， 该下尖部 204具有一通孔。 藉 由该装置， 首先进行「结合步骤」 ： 开启该压力设备 60对该纯化管 20施以负气压， 该纯化管 20的下尖部 204吸取含有核酸的检体， 使检体通过该纯化膜 203， 而使该 核酸与该纯化膜 203电性或极性结合； 开启该压力设备 60对该纯化管 20施以正气 压， 使残余检体以相反方向再度通过该纯化膜 203后， 由纯化管 20的下尖部 204流 出， 此步骤可视需要重复一至多次。
接着进行 「收集步骤」 ： 开启该压力设备 60对该纯化管 20施以负气压， 利用 该纯化管 20的下尖部 204吸取冲提液， 使该冲提液通过纯化膜 203后， 改变该纯化 膜 203电性或极性使核酸与纯化膜 203分离并溶于冲提液中； 开启该压力设备 60对 该纯化管 20施以正气压， 使该溶有核酸的冲提液以相反方向再度通过该纯化膜 203 后， 由该纯化管 20的下尖部 204排出并收集， 此收集步骤可视需要重复一至多次。 而使用者可依据需要， 在结合步骤完成后进一步进行一至多次的清洗步骤。 根据本发明的气压式萃取核酸装置， 该纯化管 20可利用各种习知技术中已存 在的纯化管， 市面上不同厂牌的纯化管均可利用于本发明。 再者， 为了避免纯化 管 20内的检体受到负气压的吸取， 冲到压力设备 60上造成污染， 图中可见， 该纯 化管 20上端与该压力设备 60中间可进一步设有一具滤膜 901的转接头 90， 该转接头 90与纯化管 20上端 (即上颈部 201)以及压力设备 60气密接合。 此外， 为了使吸取液 体或收集液体时更方便操作， 在纯化管 20下尖部 204可进一步设有与其气密接合的 移液器吸管 80， 藉此， 纯化管 20可利用该移液器吸管 80吸取少量液体或将纯化的 检体收集到微离心管中。
由图中可见， 在本发明较佳的实施例中， 该移液吸管 80上端可进一步具备一 套体， 该套体可气密地由该纯化管部分的中间管体 202至下尖部 204处套合， 据此， 使该移液吸管 80可广泛应用于各种不同类型的纯化管； 此外， 为使该转接头 90与 该移液吸管 80与该纯化管接合处的结构与不同厂牌的纯化管具有通用性， 可将该 转接头 90与该移液吸管 80与该纯化管 20接合处的结构使用橡胶材质， 使其为弹性 结构， 而可与不同规格的纯化管 20接合。 在本发明中， 由于元件间皆必须气密接 合， 才可利用气压式方式萃取核酸。 各个元件间气密接合的方式可使用任何习知 的技术， 如增设 O型环等方式， 并未有所限制。
接着， 请参见图 4， 其为本发明的气压式萃取核酸装置的另一实施例示意图。 可见该气压式萃取核酸装置可为一针筒 70， 该针筒 70即系一种可施以正或负气体 压力的压力元件。 该针筒 70包含一筒体 701、 设置于该筒体 701内的一推杆 702及可 拆卸式地与筒体 701接合的一针头组件 703， 其特征在于该筒体内 701的推杆 702下 方设有一可与核酸电性结合的纯化膜 203。藉由本发明的装置，可依循上述的方法， 直接使用该针筒 70利用推杆 702的抽吸及推排， 进行 「结合步骤」 、 「清洗步骤」 以及 「收集步骤」 ； 详言之， 先利用推杆 702上拉时所产生的负气压， 使针头组件 703吸取检体，当检体通过针头进入筒体 701内时，检体由下往上通过该纯化膜 203， 检体内带有负电的核酸会与纯化膜 203电性或极性结合， 而残余的检体藉由推动推 杆 702产生的正气压， 由上往下再度通过该纯化膜 203后由针头排出， 完成 「结合 步骤」 ； 接着， 再次将推杆 702上拉， 并由针头组件 703吸取清洗液， 该清洗液由 下往上通过该纯化膜 203， 使纯化膜 203上的杂质被受清洗后而可提高核酸的纯度， 并再度推动推杆 702产生的正气压， 将清洗液由上往下通过该纯化膜 203后由针头 排出， 完成 「清洗步骤」 ， 该清洗步骤可依照需求重复一到多次； 最后， 再次将 推杆 702上拉， 并由针头吸取冲提液， 该冲提液由下往上通过该纯化膜 203， 使特 殊盐度及 pH值的冲提液改变纯化膜 203的电性或极性，让核酸由半透膜上冲提出来 而溶于冲提液中， 并将推杆 702推动使溶有核酸的冲提液由上往下通过该纯化膜 203后经由针头排出而收集于收集管中， 完成 「收集步骤」 。 本发明的另一方面利用本发明的方法所设计的气压式萃取核酸的自动化机台
(未图示)， 其包含一机械手臂、 一组或一组以上的唧筒以及一操作台， 且该操作台 包含一纯化套组 150。 在本发明的一较佳实施例中， 该自动化机台可进一步包含一 加热模组及 /或一振荡器， 使检体在操作过程中可选择性地受振荡或加热， 以提高 核酸纯化的产量。
图 5为于本发明一实施例中的纯化套组 150示意图， 该纯化套组 150包含： 一具 有一上颈部、 一中间管体部、 一半透膜以及一下尖部的纯化管 20、 一具滤膜的转 接头 90、 一移液器吸管 80、 一用于容置检体的孔盘 (简称检体孔盘 100)、 至少一个 用于容置缓冲液的孔盘 (简称缓冲液孔盘 120); 及一用于容置萃取后产物的孔盘 (简 称产物孔盘 110)。
根据本发明的气压式萃取核酸的自动化机台， 其机械手臂上具有唧筒， 唧筒 前方具有尖端用于组合该纯化套组 150内的转接头 90、 纯化管 20以及移液器吸管 80(由上往下的顺序)， 当此三者组合完毕 (以下简称组合件)， 唧筒可与组合件气密 接合后， 提供负气压使移液器吸管 80吸取纯化套组 150内检体孔盘 100中的检体至 纯化管中， 使检体中的核酸与其内的半透膜电性或极性反应， 且转接头 90可避免 检体上冲至唧筒， 而避免污染， 当反应完成后， 唧筒给予正气压， 纯化管 20将残 余检体经由移液器吸管 80直接排入纯化套组 150内检体孔盘 100(此为「结合步骤」）， 其中， 于该结合步骤中， 可重复将该检体通过该纯化管 20—至多次， 提高核酸结 合于半透膜的量； 接着， 机器手臂机械式地带动组合件至纯化套组 150中装填有清 洗液的孔盘上端 (即缓冲液孔盘 120)， 提供负气压使移液器吸管 80吸取清洗液至纯 化管 20中， 经清洗后， 唧筒提供正气压， 将清洗液排入原先吸取出来的同一孔盘 中 (此为 「清洗步骤」 ）， 该清洗步骤可重复一至多次； 而当清洗步骤结束后， 可进 一步将该纯化管 20以加热机构加热以蒸散清洗液中的乙醇， 及 /或透过多次的以唧 筒提供正 /负气压使该半透膜干燥； 最后， 机器手臂机械式地带动组合件至纯化套 组 150中装填有冲提液的孔盘上端 (即缓冲液孔盘 120)，唧筒提供负气压使移液器吸 管吸取冲提液至纯化管 20中，使电性 /极性结合于半透膜上的核酸溶出至冲提液中， 接着唧筒提供正气压， 将冲提液排入原先吸取出来的同一孔盘中， 或是机器手臂 可机械式地带动组合件至纯化套组 150的产物孔盘 110中， 收集所纯化的核酸 (此为 「收集步骤」 ）， 该收集步骤可重复一至多次， 亦即， 将同一冲提液重复通过该半 透膜上一至多次， 使核酸的萃取量可有效提高。
该纯化套组 150可为两个分开的组件， 亦可形成一体成型的组件， 并未限制， 较佳一体成型者为佳。 此外， 纯化套组 150中的缓冲液孔盘 120可根据需求而有不 同的数量， 但其排列方式最佳为根据萃取核酸时的步骤， 依序为细胞溶解液、 清 洗液、 酒精、 水或冲提液， 藉此， 当点胶机带动组合件移动时， 可有顺序地一步 步直线移往后方进行核酸萃取。
此外， 由于该含有核酸的检体通常包含溶解缓冲液 (lysis buffer)以打破检体中 所含细胞的细胞膜而使其释放出核酸， 该缓冲液中含有大量的界面活性剂， 故而 当 「结合步骤」 进行完毕后， 将残余检体排出时， 若排出至空的孔盘中， 容易产 生大量气泡而不利于操作， 因此， 于一较佳的实施例中， 该纯化套组 150可包含一 用于容置该残余检体的孔盘 (图中未示)， 且其中该孔盘中包含 100% EtOH, 则该 EtOH可吸收气泡， 使该残余检体排出至该孔盘时不会产生气泡； 其中， EtOH的量 并无限制， 但较佳为 100至 500μ1， 更佳为 200至 400 μΐ, 最佳为 300 μ1。
再者， 当 「结合步骤」 进行完毕后， 由于该半透膜上可能有残存的检体， 因 此， 若直接吸取清洗液， 容易产生较多气泡， 故而可先吸取一次 100% EtOH, 将 半透膜上的残余检体清除干净， 也增加该半透膜的通透性， 而能使下一清洗步骤 的清洗液较好通过该半透膜， 增进该清洗效率， 因此， 该纯化套组 150可包含： 含 有 100% EtOH的孔盘 (图中未示)于该缓冲液孔盘 120之前， 其中， EtOH的量并无限 制， 但较佳为 100至 500μ1， 更佳为 200至 400 μΐ, 最佳为 300 μ1。
以下实施例不应视为过度地限制本发明。 本发明所属技术领域中具有通常知 识者可在不背离本发明的精神或范畴的情况下对本文所讨论的实施例进行修改及 变化， 而仍属于本发明的范围。
[实施例]纯化鱼肝脏的 gDNA
前置作业
所有的试剂均使用 QIAamp DNA试剂套组 (凯杰生物科技有限公司 (Qiagen Taiwan Co. Ltd.))中所附者， 内含组织溶解液、 细胞溶解液、 清洗液 I、 清洗液 II、 冲提液及蛋白质分解酶 K (proteinase K)， 以及纯化管， 其中该纯化管中的半透膜为 二氧化硅 (silica)膜。
取约 5x5mm大小的鱼肝脏， 置入 2ml的螺盖试管， 加入 800μ1磷酸盐缓冲液 (phosphate buffer saline, PBS)以及一颗 5mm钢珠， 以组织均质机均质化 30秒。接着， 加入 800μ1细胞溶解液， 以振荡器混匀， 再加入 80μ1蛋白质分解酶 Κ， 并使用振荡 器混匀。 于 60Ό下培育 30分钟使检体溶解。 之后， 加入 800μ1的乙醇并以振荡器振 荡 30秒。 于 13000rpm下离心 3分钟， 各取 200μ1上清液至四个样品管中， 分别为实 施例 1检体、 实施例 2检体、 比较例 1检体及比较例 2检体。
实施例 1
首先， 先将纯化管上颈部与一针筒气密接合， 纯化管下尖部与一移液器吸管 气密接合。
结合步骤： 拉动针筒的推杆， 将实施例 1样品管中的实施例 1检体经由该移液 器吸管的吸取， 由纯化管的下尖部通过纯化膜后， 吸取至中间管体部， 接着， 将 针筒推杆下推， 排出未与纯化膜结合的残余检体， 而核酸则电性结合于纯化膜。 清洗步骤： 拉动针筒的推杆， 将 500μ1清洗液 I经由该移液器吸管的吸取， 由纯 化管的下尖部通过纯化膜后， 吸取至中间管体部， 接着， 将针筒推杆下推， 排出 该已清洗过纯化膜的清洗液 I。 同样的再一次进行清洗步骤， 将 750μ1清洗液 II由经 由该移液器吸管的吸取， 由纯化管的下尖部通过纯化膜后， 吸取至中间管体部， 接着， 将针筒推杆下推， 排出该已清洗过纯化膜的清洗液 II。
后清洗步骤：拉动针筒的推杆，将 800μ1的 100%酒精经由该移液器吸管的吸取， 由纯化管的下尖部通过纯化膜后， 吸取至中间管体部， 接着， 将针筒推杆下推， 排出该已清洗过纯化膜的酒精； 此步骤重复两次。 接着， 将针筒抽吸并推排至少 15次， 使残于在纯化管内的酒精挥发。
收集步骤： 拉动针筒的推杆， 将 200μ1冲提液经由该移液器吸管的吸取， 由纯 化管的下尖部通过纯化膜后， 吸取至中间管体部， 并静置 3分钟， 接着， 将针筒推 杆下推， 排出该溶有核酸的冲提液至 1.5ml的微量离心管中。
实施例 2
实施例 2的步骤与实施例 1一样， 重复实施例 1的步骤， 仅将检体换为实施例 2 检体。
比较例 1
依据 QIAamp DNA试剂套组产品说明书， 进行核酸萃取。 结合步骤： 利用微 量分注器 (pipette) 将比较例 1检体吸取至纯化管内，检体由纯化管上方注入。接着， 在纯化管下方装一废液管， 两管件一起至离心机中于转速 13000rpm下离心 2分钟， 残余检体将通过纯化膜而经由纯化管下尖部的通道离心至废液管中， 离心完后， 倒掉废液管中的废液， 而核酸电性结合于纯化膜上， 纯化管再套回废液管中。
清洗步骤： 利用微量分注器将 500μ1清洗液 I吸取至纯化管内， 清洗液 I由纯化 管上方注入。 两管件一起至离心机中于转速 13000rpm下离心 1分钟， 清洗液 I通过 纯化膜而经由纯化管下尖部的通道离心至废液管中， 离心完后， 倒掉废液管中废 液， 纯化管再套回废液管中。 同样的再一次进行清洗步骤， 将 750μ1清洗液 II吸取 至纯化管内， 清洗液 Π由纯化管上方注入。 两管件一起至离心机中 13000rpm离心 lmin，清洗液 II通过纯化膜而经由纯化管下尖部的通道离心至废液管中，离心完后， 倒掉废液管中废液， 纯化管再套回废液管中。
后清洗步骤： 将两套件于转速 13000rpm下离心额外的三分钟以确保将清洗液 完全移除。
收集步骤： 移除废液管， 并换上 1.5ml的微量离心管套于纯化管外。 利用微量 分注器将 200μ1冲提液吸取至纯化管内， 冲提液由纯化管上方注入至中间管体部， 并静置 3分钟， 接着， 将两套件置入离心机中于转速 13000rpm下离心 1分钟以获得 该溶有核酸的冲提液至 1.5ml的微量离心管中。
比较例 2
比较例 2的步骤与比较例 1一样， 重复比较例 1的步骤， 仅将检体换为实施例 2 检体。
以上实施例 1、 2以及比较例 1及 2的抽取出的 gDNA的结果汇整于下表 1中。
Figure imgf000013_0001
由上表 1可见，使用本发明的方法及装置所抽取出的 DNA浓度与使用习知方式 所收取出的 DNA浓度差不多， 且方法较为简便。
此外， 本发明的方法及装置可适用于不同厂牌的抽取核酸的纯化管套组。 接着， 为了比较使用亲水性膜与疏水性膜对于本发明的方法的影响， 且为了 比较使用本发明的装置与传统离心方式的影响， 进一步使用不同厂牌的半透膜并 利用本发明的装置以及使用传统离心方式， 进行核酸萃取试验。
实施例 3
实施例 3的步骤与样品皆同于实施例 1， 但将该纯化管的半透膜置换为 PALL公 司的一种亲水性二氧化硅膜 (品名： Glass Fiber Media), 膜孔径为 Ι .Ομηι; 重复实施 例 1的步骤， 但于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa, 且收集步骤重复两次。
实施例 4
实施例 4的步骤与样品皆同于实施例 1， 但将该纯化管的半透膜置换为 PALL公 司的亲水性硝化纤维膜 (品名： BioTrace), 膜孔径为 0.2 μηι; 重复实施例 1的步骤， 但于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa， 且收集步 骤重复两次。
实施例 5
实施例 5的步骤与样品皆同于实施例 1，但将该纯化管的半透膜置换为 Advantec 公司的亲水性硝化纤维膜 (品名： Mixed Cellulose Esters), 膜孔径为 0.2 μηι; 重复实 施例 1的步骤， 但于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa, 且收集步骤重复两次。
实施例 6
实施例 6的步骤与样品皆同于实施例 1，但将该纯化管的半透膜置换为 Advantec 公司的亲水性醋酸纤维素膜 (品名： Cellulose Acetate), 膜孔径为 0.45 μηι; 重复实 施例 1的步骤， 但于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa, 且收集步骤重复两次。
比较例 3
比较例 3的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用的 纯化管中的半透膜置换为实施例 3所使用的半透膜。
比较例 4
比较例 4的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用的 纯化管中的半透膜置换为实施例 4所使用的半透膜。
比较例 5
比较例 5的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用的 纯化管中的半透膜置换为实施例 5所使用的半透膜。
比较例 6
比较例 6的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用的 纯化管中的半透膜置换为实施例 6所使用的半透膜。
以上实施例 3至 6以及比较例 3至 6的抽取出的 gDNA的结果汇整于下表 2中。 表 2
Figure imgf000014_0001
由表 2中可见， 使用本发明的装置所萃取出的核酸萃取量较习知技术为高， 此 系因使用本发明的装置时， 由于液体材料仅由纯化管的下端进出， 因此， 在收集 步骤中可重复利用压力装置提供的正 /负气压， 使冲提液反复通过半透膜， 而增进 核酸冲提的效率， 而有效提升核酸的萃取量。 相较于使用离心的方式， 所有反应 液体均是一次性地通过半透膜， 而液体即被排除， 使用本发明的方法及装置可增 进萃取量且提高萃取效率。
比较例 7A
比较例 7A的步骤与样品皆同于实施例 1， 但将该纯化管的半透膜置换为 PALL 公司的疏水性羧化聚偏二氟乙烯 (品名： Fluoro Trans G)， 膜孔径为 0.2 μηι; 重复实 施例 1的步骤， 但于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa, 且收集步骤重复两次。
比较例 8A
比较例 8A的步骤与样品皆同于实施例 1， 但将该纯化管的半透膜置换为 PALL 公司的疏水性尼龙 品名： Hydrolon), 膜孔径为 1.2 μηι; 重复实施例 1的步骤， 但 于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa， 且收集步骤 重复两次。
比较例 9A
比较例 9A的步骤与样品皆同于实施例 1， 但将该纯化管的半透膜置换为 PALL 公司的疏水性聚醚砜 (品名： Supor-450PR)， 膜孔径为 0.45 μηΐ; 重复实施例 1的步 骤， 但于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa， 且收 集步骤重复两次。
比较例組
比较例 10A的步骤与样品皆同于实施例 1， 但将该纯化管的半透膜置换为
Advantec公司的疏水性聚四氟乙烯 (品名： Supported PTFE)， 膜孔径为 0.45 μηι; 重 复实施例 1的步骤， 但于本发明的装置中进行核酸萃取， 所使用的正负气压为 60kpa/-60kpa, 且收集步骤重复两次。
比较例 7B
比较例 7B的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用 的纯化管中的半透膜置换为实施例 7A所使用的半透膜。
比较例 8B
比较例 8B的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用 的纯化管中的半透膜置换为实施例 8A所使用的半透膜。
比较例 9B
比较例 9B的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用 的纯化管中的半透膜置换为实施例 9A所使用的半透膜。
比较例 10B 比较例 10B的步骤及检体均与比较例 1相同， 重复比较例 1的步骤， 仅将所使用 的纯化管中的半透膜置换为实施例 10A所使用的半透膜。
以上比较例 7A至 10A以及比较例 7B至 10B的抽取出的 gDNA的结果汇整于下 表 3中。
Figure imgf000016_0001
由表 3中可见， 使用疏水性半透膜由于无法与核酸的亲水性基团透过改变周围 的极性互相吸引， 因此无论是使用本发明的方法及装置， 或是使用习知的管柱离 心萃取法， 均无法有良好的萃取效能。
综上所述， 本发明克服了技术偏见， 不如习知技术中必须由纯化管的上端加 入检体， 而在对该纯化管提供具有核酸的检体之前， 便先将纯化管一端 (纯化管上 端)与压力设备结合， 而使萃取核酸时所须用到的所有液体材料均仅由纯化管的另 一端 (纯化管下端)进出。 如此一来， 使用本发明的方法、 装置或自动化机台， 于萃 取核酸中的所有步骤均可简易地利用提供正负压力的方式， 由纯化管的同一端吸 取 /排出液体， 不仅可简化萃取步骤， 且可使液体反复通过半透膜而达到增进萃取 效率的结果， 且该自动化机台可不必配置离心机， 仅须配置能够提供压力差的装 置， 而可达到缩小体积的功效。

Claims

权利要求书
1.一种气压式萃取核酸的方法， 其特征在于包含：
(a)将一纯化管上端与一施以正或负气体压力的压力设备气密接合， 其中该纯 化管为具有半透膜的通孔管体；
(b)开启该压力设备对该纯化管施以负气压， 利用该纯化管的下端吸取含有核 酸的检体， 使该检体通过该半透膜后， 该核酸与该半透膜电性或极性结合；
(C)开启该压力设备对该纯化管施以正气压， 使残余检体以相反于步骤 0?)的方 向通过半透膜后， 由纯化管的下端流出， 且其中该步骤 (b)及 (C)仅进行一次或重复 进行多次；
(d)开启该压力设备对该纯化管施以负气压，利用该纯化管的下端吸取冲提液， 使该冲提液通过半透膜后， 改变该半透膜电性或极性使核酸与半透膜分离并溶于 冲提液中； 及
(e)开启该压力设备对该纯化管施以正气压， 使该溶有核酸的冲提液相反于步 骤 (d)的方向通过半透膜后， 由该纯化管的下端排出并收集， 且其中该步骤 (d)及 (e) 仅进行一次或重复进行多次。
2.如权利要求 1 的方法， 其特征在于， 步骤 (c)及 (d)之间进一步包含至少一次 的清洗步骤， 该清洗步骤包含：
(c-1)开启该压力设备对该纯化管施以负气压， 利用该纯化管的下端吸取清洗 液， 使该清洗液通过该半透膜； 及
(C-2)开启该压力设备对该纯化管施以正气压， 使该清洗液以相反于 (C-1) 的方 向通过半透膜后， 由纯化管的下端流出。
3.如权利要求 1的方法， 其特征在于， 该纯化管上端与该压力设备中间进一步 设有一具滤膜的转接头。
4.如权利要求 1至 3中任一项的方法， 其特征在于， 该施以正或负气体压力的 压力设备为一唧筒。
5.如权利要求 1至 3中任一项的方法， 其特征在于， 该纯化管的下端进一步设 有一与其气密接合的移液器吸管。
6.—种气压式萃取核酸的装置， 其包含： 一针筒， 该针筒包含一筒体、 设置于 该筒体内的一推杆， 其特征在于， 该筒体内的推杆下方设有一与核酸电性或极性 结合的半透膜。
7.—种利用权利要求 1的方法萃取核酸的气压式萃取核酸的自动化机台，其特 征在于包含：
一主机台， 具有运作所需的电子零件组； 一移动装置， 与该主机台电性相连；
一唧筒， 与该主机台电性相连；
以及一操作台， 与该主机台电性相连， 且该操作台包含一纯化套组。
8.如权利要求 7的自动化机台， 其特征在于进一步包含一加热模组。
9.如权利要求 7或 8的自动化机台， 其特征在于该纯化套组包含：
(a) 一具有半透膜的纯化管、
(b)—具滤膜的转接头、
(c)一移液器吸管、
(d) 一用于容置检体的孔盘、
(e)至少一个用于容置缓冲液的孔盘； 及
(f) 一用于容置萃取后产物的孔盘。
10.如权利要求 9的自动化机台， 其特征在于， 该纯化套组一体排列为一行纯 化匣的形式。
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