Patent Application
20050186607
The present invention relates to technology for eluting and extracting nucleic acid included in a biological sample, for example, from coexisting materials.
In gene-related researches and technologies, the purification of nucleic acid is necessary by which nucleic acid included in a biological sample, such as blood, is extracted by elution from coexisting materials. Until today, various nucleic acid purification methods and apparatuses used for the methods have been proposed.
JP Patent Publication (Kohyo) No. 2003-501644 A discloses a syringe type apparatus for processing samples. In this apparatus, a syringe filled with solid-phase bead groups capable of binding biological molecules, such as DNA, is used. Nucleic acid is captured by the solid-phase bead groups by allowing a sample that includes nucleic acid to pass through the solid-phase bead groups.
The size of clearance of solid-phase bead groups has an influence on the efficiency of binding nucleic acid by the solid-phase beads, namely the probability of contact between the solid-phase beads and nucleic acid. However, in the aforementioned syringe type apparatus for processing samples, the clearance of the solid-phase bead groups varies by the passage of a sample when the sample that includes nucleic acid is allowed to pass the solid-phase bead groups disposed in a movable manner inside the syringe. Thus, the clearance of the solid-phase bead groups cannot be maintained in a state that is optimum for binding nucleic acid, so that the efficiency of isolating nucleic acid is not stable.
It is an object of the present invention to further stabilize the efficiency of isolating nucleic acid concerning an apparatus for isolating nucleic acid.
The present invention relates to an apparatus for isolating nucleic acid, the apparatus being provided with a meshed solid substance for binding nucleic acid. By employing the meshed solid substance for binding nucleic acid, fluid resistance can be reduced upon allowing a sample that includes nucleic acid to pass the solid substance for binding nucleic acid, while securing solid-phase volume that is sufficient for binding a large amount of nucleic acid. Consequently, even when the sample is allowed to pass the solid substance for binding nucleic acid at a high aspiration/dispense speed, force added to the solid substance for binding nucleic acid is small and the solid substance for binding nucleic acid is almost undistorted. The average pore size of the mesh that has an influence on the efficiency of binding nucleic acid by the meshed solid substance for binding nucleic acid is also almost unchanged. Therefore, the optimum state of the efficiency of isolating nucleic acid can be maintained.
According to the present invention, the efficiency of isolating nucleic acid concerning an apparatus for isolating nucleic acid can be improved.
The above and further objects and novel features of the invention will be more fully appear from the following detailed description when the same is read in connection with the accompanying drawings. It is to be expressly understood, however, that the drawing is for purpose of illustration only and is not intended as a definition of the limits of the invention.
(Embodiment)
A syringe is described as an apparatus for isolating nucleic acid according to the present embodiment with reference to
The syringe body 10 has a cylindrically-shaped cylindrical portion 101, an open portion 102 at the upper end, a bottom portion 103 at the lower end, a brim-shaped holding position 104 disposed on the periphery of the open portion 102, and a connecting portion 105 for connecting the nozzle disposed on the bottom portion 103. The bottom portion 103 may be formed in a conical manner.
The plunger 20 has a plunger body 201 and a seal piece 203. The seal piece 203 is formed as a separate member of the plunger body 201, and is attached to an attachment portion 202 at the lower end of the plunger body 201. The seal piece 203 has a conical protrusion 204 at its lower end. In the present invention, the plunger body 201 and the seal piece 203 are separate members. However, they may be formed by a single component so long as the sealability of the member is maintained.
The nozzle 30 has a connecting portion 301 at the upper end and a cylindrical portion 302 extending downward thereof. The lower end of the cylindrical portion 302 may be thin like a knife so as to promote sharp aspiration/dispense of liquid. The connecting portion 301 of the nozzle and the connecting portion 105 of the syringe body are connected by press fitting, screws, adhesion by adhesive, welding, and the like.
The syringe body 10 and the plunger body 201 are formed by resin that has high chemical resistance and is readily subjected to a forming process. Preferably, the syringe body and the plunger body are formed by transparent materials, such as polypropylene, for example. Scales are formed or written on the outer surface of the cylindrical portion 101 of the syringe body 10. The seal piece 203 is formed by elastic materials, such as rubber. The nozzle 30 may be formed by resin that has high chemical resistance and is readily subjected to a forming process, or the nozzle may be formed by metal, such as stainless.
The dimensions of the syringe body, plunger, and nozzle are different in accordance with the volume of sample to be handled. In the example, a syringe for 30 ml is described. In the syringe for 30 ml, the inside diameter of the nozzle is 2 mm.
The structure of the nucleic acid binding unit 40 is described with reference to
As shown in
The inside diameter of a second protrusion 444 is smaller than the outside diameters of the holding members 42 and 43. Thus, the nucleic acid binding member 41 and the holding members 42 and 43 are held in a sandwich manner between the two protrusions 442 and 444. A taper or a chamfer 445 is formed at the lower end of the outer surface of the holder 44.
The nucleic acid binding member 41 comprises a multitude of fine fiber mesh that includes silica. In other words, the nucleic acid binding member 41 is composed of a multitude of fine fibers disposed in random directions along a substantial plane. The fibers that constitute the nucleic acid binding member may be any materials, such as glass wool, sintered quartz, and the like, as long as silica is included.
The nucleic acid binding member may be solidified by a binder. In other words, the fibers that constitute the nucleic acid binding member are adhered to one another by the binder, thereby maintaining suitable strength and preventing the fibers from crumbling. Consequently, an operation of incorporating the nucleic acid binding member into the holder can be readily conducted. Also, since a sample that includes whole blood generally has high viscosity, a large force is added to the nucleic acid binding member when the sample is passed through the nucleic acid binding member. However, by solidifying the nucleic acid binding member using the binder, the shape of the nucleic acid binding member is not changed significantly when the high-viscosity sample is passed through the nucleic acid binding member, and the average pore size of the nucleic acid binding member can be maintained in a state that is optimum for binding nucleic acid. As a matter of course, the solidification by the binder can be omitted if such strength is not necessary. The nucleic acid binding member may be manufactured by punching out a sheet comprising fibers that includes silica using a punch-like cutter, or by press cutting the sheet.
The shape of the nucleic acid binding member is represented by an aspect ratio defined as a ratio of thickness to diameter. For example, if the nucleic acid binding member is disk shaped, the aspect ratio is the thickness of the disk/the diameter of the disk. If the nucleic acid binding member is platy, the aspect ratio is the thickness of the plate/the length of the diagonal. Preferably, the aspect ratio of the nucleic acid binding member in the present example is not more than 1. Preferably, the thickness of the nucleic acid binding member is not more than 0.5 mm. In the embodiment, the thickness of the nucleic acid binding member is 0.4 mm.
Silica binds to nucleic acid in the presence of chaotropic material. The binding efficiency of nucleic acid to silica can be improved by increasing the contact ratio of nucleic acid to the surface of silica. In the inside of the nucleic acid binding member, a multitude of paths are formed in order to pass a sample (sample solution) that includes nucleic acid. The larger the total value of the inner surface area of such paths is, the higher the contact ratio of nucleic acid to the surface of silica becomes. The total value of the inner surface area of the paths can be increased by adding the number of the paths and reducing the average pore size of the paths.
However, by reducing the average pore size of the paths, fluid resistance when the sample is passed through the nucleic acid binding member is increased. If the average pore size of the paths is too small, clogging is caused by biological materials, such as blood corpuscles, nucleic acid, and the like included in the sample. Preferably, the maximum pore size of the nucleic acid binding member is 60 μm. The average pore size of the nucleic acid binding member is 0.2 to 30 μm, preferably 10 to 20 μm.
The maximum pore size of the nucleic acid binding member is measured by the bubble point method (JIS K 3832). According to the bubble point method, the nucleic acid binding member is immersed in a solution so as to wet the nucleic acid binding member completely, and then the minimum pressure when the nucleic acid binding member starts to release bubbles is measured. The maximum pore size is obtained on the basis of the minimum pressure. The average pore size can be obtained from the maximum pore size. For example, it may be assumed that the average pore size is a half of the maximum pore size.
The nucleic acid binding member 41 has an outside diameter slightly larger than the inside diameter of the holder 44. Inside the holder 44, the nucleic acid binding member 41 is disposed along the inner wall of the holder 44 such that the circumferential outer edge of the nucleic acid binding member 41 is brought into contact with the inner wall. The nucleic acid binding member 41 is held inside the holder 44 in a state where the nucleic acid binding member 41 is sandwiched between the holding members 42 and 43. The circumferential outer edge of the nucleic acid binding member 41 is compressed by being sandwiched between the holding members 42 and 43, and adhered to the inner wall of the holder 44. Therefore, sealability between the nucleic acid binding member 41 and the holder 44 can be increased.
The holding members 42 and 43 have fluid resistance at least smaller than that of the nucleic acid binding member. In other words, the holding members are constructed such that a sample is flown at least more readily than in the nucleic acid binding member. The average pore size of the holding members 42 and 43 is 100 μm, for example. The holding members may be constructed by porous materials that have no function of binding nucleic acid. For example, holding members may be formed by heat forming a multiple of resin beads. Examples of resin include polypropylene.
According to the present example, load to the nucleic acid binding member due to the passage of a sample is sufficiently small, since the nucleic acid binding member 41 is formed in a mesh manner. The nucleic acid binding member 41 is sandwiched by the holding members 42 and 43, so that there is almost no clearance between the nucleic acid binding member 41 and the holding members 42 and 43. The nucleic acid binding member remains almost unmoved when the sample is passed through the nucleic acid binding member by moving the plunger 20. In other words, if the plunger 20 is reciprocated, the nucleic acid binding member is not moved synchronously. Thus, the nucleic acid binding member is not broken even if the nucleic acid binding member comprises fragile materials, such as glass wool, sintered quartz, and the like, and when a high-viscosity sample, such as whole blood, is passed through the nucleic acid binding member.
The assembling method of the syringe in the present example is described with reference to
In step S102, the nucleic acid binding member 41 is inserted into the holder 44. The nucleic acid binding member 41 is disposed on the holding member 43 on the lower side. In step S103, the holding member 42 on the upper side is inserted into the holder 44. The holding member 42 is press-fitted into the holder 44, since the outside diameter of the holding member 42 is slightly larger than the inside diameter of the protrusion 442 on the inner surface of the holder 44. Consequently, the nucleic acid binding member 41 is held in the holder 44 in a state where the nucleic acid binding member 41 is sandwiched between the two holding members 42 and 43, thereby forming the nucleic acid binding unit 40.
In step S104, appearance observation and liquid penetration inspection of the nucleic acid binding unit 40 are conducted. In the appearance observation, visual observation is conducted to confirm whether the nucleic acid binding member 41 is adhered to the inner surface of the holder 44, and whether there is no clearance between the nucleic acid binding member and the holding members 42 and 43. In the liquid penetration inspection, the sealability of the nucleic acid binding member 41 is inspected.
In step S105, the nucleic acid binding unit 40 is inserted into the syringe body 10. The nucleic acid binding unit 40 is held in the vicinity of the open portion 102 at the upper end of the syringe body 10. In step S106, the plunger 20 is inserted into the syringe body 10. The seal piece 203 of the plunger 20 is brought into contact with the nucleic acid binding unit 40. As shown by the broken line in
A method for extracting nucleic acid from a sample (sample solution) using the syringe of the present example is described with reference to
In step S205, nucleic acid is captured using the syringe of the present example. At first, the plunger 20 is inserted into the syringe body 10, such that the seal piece 203 is brought into contact with the nucleic acid binding unit 40. Thus, the plunger 20 is pulled up by a predetermined volume from the syringe body 10, in order to detach the seal piece 203 from the nucleic acid binding unit 40. Consequently, a space is formed between the seal piece 203 and the nucleic acid binding unit 40. Air held in the space is used in the end in order to dispense the sample from the syringe body 10 completely. Then, the tip of the nozzle 30 is inserted into the sample prepared in step S204. Preferably, only the lower end of the nozzle 30 is inserted into the sample so as to prevent the adhesion of a large quantity of the sample to the outer surface of the nozzle 30. By lifting the plunger 20, the sample is introduced into the inside of the syringe body 10. The sample is passed through the nucleic acid binding unit 40 and the binding of nucleic acid is initiated. The plunger 20 is stopped when it is lifted up to a predetermined location. When the stop of sample movement is confirmed, then the plunger 20 is pushed into the syringe body 10. By pushing into the plunger 20, the sample is passed through the nucleic acid binding unit 40 to the opposite direction, the binding of nucleic acid is continued. Such a reciprocating motion of the plunger 20 is repeated for several times. In the end, the plunger 20 is sufficiently pushed into the syringe body 10 and the sample is completely dispensed. As mention above, since air is held in advance inside the syringe body 10, the sample is completely dispensed by dispensing the air. After a lapse of a predetermined time, the plunger 20 is pulled up by a predetermined volume from the syringe body 10 so as to form a space between the seal piece 203 and the nucleic acid binding unit 40 for the following step.
It must be noted that air should not be entered into the syringe body 10 from the nozzle 30 along with the sample during the reciprocating motion of the plunger 20. If air is mixed, the passability of the sample to the nucleic acid binding unit 40 is reduced, so that the efficiency of nucleic acid binding is reduced. In the present example, a predetermined volume of air is introduced into the inside of the syringe body 10 before the sample is aspirated, as mentioned above. By sufficiently reducing the volume of air, the sample can be made to better track the motion of the plunger 20 as the sample is aspirated. In other words, the tracking ability of the liquid level can be improved. Especially, operating efficiency can be improved even when the viscosity of the sample is high, since the following ability of liquid level can be secured.
In step S206, a first washing of the syringe is conducted. The tip of the nozzle 30 is inserted into a fourth reagent and the plunger 20 is lifted to introduce the fourth reagent into the syringe body 10. The fourth reagent is a first washing buffer and is a solution that includes sterilized water. When the plunger 20 is lifted up to a predetermined location, then the plunger 20 is pushed into the syringe body 10, thereby reciprocating the first washing buffer in the nucleic acid binding unit 40. Foreign substances that adhered to the syringe body 10 and the nucleic acid binding unit 40 are washed out by the first washing buffer, so that only captured nucleic acid is left in the nucleic acid binding member 41. By sufficiently pushing the plunger 20 into the syringe body 10, the first washing buffer is completely dispensed. This operation is repeated using new first washing buffer. Although, the reciprocating motion of the plunger 20 may be repeated for several times using the same first washing buffer, washing effect is decreased, since used first washing buffer includes foreign substances. Thus, new first washing buffer is preferably used. When the first washing is ended, the plunger 20 is pulled up by a predetermined volume from the syringe body 10 so as to form a space between the seal piece 203 and the nucleic acid binding unit 40 for the following step.
In step S207, a second washing of the syringe is conducted. The second washing is conducted in order to wash out the first washing buffer left in the syringe. The tip of the nozzle 30 is inserted into a fifth reagent and the plunger 20 is lifted to introduce the fifth reagent into the syringe body 10. The fifth reagent is a second washing buffer and is a solution that includes ethanol. When the plunger 20 is lifted up to a predetermined location, then the plunger 20 is pushed into the syringe body 10, thereby washing out the first washing buffer adhered to the syringe body 10 and the nucleic acid binding unit 40. This operation is repeated using new second washing buffer. When the second washing buffer is completely dispensed, in the end, the plunger 20 is reciprocated at high speed in the air, thereby introducing air into the syringe body 10 at high speed and dispensing it at high speed. By repeating this, the second washing buffer is completely removed from the syringe. If the second washing buffer is left, it affects the following operation. When the second washing is ended, the plunger 20 is pulled up by a predetermined volume from the syringe body 10 so as to form a space between the seal piece 203 and the nucleic acid binding unit 40 for the following step.
In step S208, nucleic acid is eluted from the nucleic acid binding member 41. The tip of the nozzle 30 is inserted into a sixth reagent. The sixth reagent is tris buffer in order to elute nucleic acid. The plunger 20 is reciprocated, thereby reciprocating the tris buffer through the nucleic acid binding unit 40. Nucleic acid is eluted from the nucleic acid binding member 41 by the tris buffer. Although the reciprocating motion of the plunger 20 may be repeated using the same sixth reagent, the reciprocating motion of the plunger 20 may be repeated using new sixth reagent. When the elution of nucleic acid is conducted with replaced sixth reagent, a plurality of gained sixth reagent is mixed in a single container. This allows obtaining nucleic acid in a short time.
Although the furrows are disposed in the present embodiment, the thickness of the upper end of the holder 44 may be extremely thin instead. As a result, the sample or reagent is prevented from remaining on the top surface of the holder 44. However, in this case, a plurality of protrusions are preferably disposed at the upper end of the inner surface of the holder 44. The protrusions constitute a contact surface with an insertion jig upon inserting the nucleic acid binding unit 40 into the syringe. Thus, the nucleic acid binding unit 40 is inserted straightforwardly along an axis line direction without tilting upon inserting the nucleic acid binding unit 40 into the syringe.
Another example of the nucleic acid binding unit 40 is described with reference to
Another example of the holding member is described with reference to
Yet another example of the nucleic acid binding unit 40 is described with reference to
The nucleic acid binding body 48 of the present example is composed of porous materials that include silica. The nucleic acid binding body 48 is formed by sintering fine particles that include silica. The thickness of the nucleic acid binding body 48 in the axis line direction may be 10 mm, for example.
Another example of the syringe is described with reference to
The protrusions 511 and 512 function such that the nucleic acid binding unit 50 is capable of sliding inside the syringe body 10, while sealing the inner clearance between the nucleic acid binding unit 50 and the syringe body 10. The protrusions 511 and 512 may be any materials, such as resin, for example, as long as such a function is provided. Although the protrusions 511 and 512 may be formed in a unified manner with the cylindrical body of the holder 51, they may be formed by attaching rings separately formed to the cylindrical body.
A method of extracting nucleic acid from a sample (sample solution) using the syringe of the present example is described with reference to
In step S301, the nucleic acid binding unit 50 is disposed in the first location inside the syringe body, as shown in
If insufficiently dissolved sample is brought into contact with the nucleic acid binding member prior to a step for binding nucleic acid, foreign substances other than nucleic acid are adhered to the nucleic acid binding member 41, so that the contact area of nucleic acid is reduced. Thus, the agitation of a sample must be conducted such that the sample is not brought into contact with the nucleic acid binding member 41.
In step S303, the sample inside the syringe is heated. In the present example, the heating is conducted in a state where the sample is held inside the syringe. Step S303 corresponds to step S203 of
In step S305, the nucleic acid binding unit 50 is moved from the first location to the second location of the syringe body. The second location is the lower end of the syringe body. By pushing the plunger into the syringe body, the nucleic acid binding unit 50 is moved to the second location. In this case, it is necessary to prepare a container for storing a sample in advance, since the sample inside the syringe body is dispensed.
In step S306, nucleic acid is captured. The tip of the nozzle is inserted into the sample in the container and the plunger is lifted to aspirate the sample into the syringe body. The sample is passed through the nucleic acid binding unit 50, and then stored inside the syringe body. Then, the sample is dispensed from the syringe body by pushing into the plunger. This operation is repeated. Consequently, the sample is reciprocated through the nucleic acid binding unit 50, thereby binding nucleic acid by the nucleic acid binding member 41. Step S306 corresponds to step S205 of
In the present example, the number of containers for use can be reduced and operating efficiency can be improved, since steps from the adjustment of sample to the binding and elution of nucleic acid can be conducted by the syringe.
Yet another example of the syringe is described with reference to
Generally, if the volume of sample changes, it is necessary to prepare a plurality of capacities of syringes accordingly. However, in the present example, although it is necessary to prepare a plurality of capacities of syringe bodies 10 and plungers 20, it is sufficient to prepare one type of the nucleic acid binding unit 60. It is sufficient to change only the syringe body 10 and the plunger 20 in accordance with the volume of sample. Generally, if the types of sample change, it is necessary to prepare a plurality of types of syringes accordingly. However, in the present example, although it is necessary to prepare a plurality of nucleic acid binding units 60, it is sufficient to prepare one type of the syringe body 10 and the plunger 20. It is sufficient to change only the nucleic acid binding units 60 in accordance with the type of the sample.
Although the examples of the present invention are described, the present invention is not limited to the aforementioned examples. It must be noted that a person skilled in the art is capable of various modification concerning the range of the invention described in claims.
Also, some of the technologies disclosed in the present example can be applied to such an apparatus for refining nucleic acid as disclosed in Patent Application No. 10-70201 (1998). The apparatus for refining nucleic acid comprises a tip for binding nucleic acid that incorporates silica-included solid substance so as to be capable of contacting liquid, a movable nozzle for liquid aspiration/dispense that connects the tip for binding nucleic acid in a removable manner, a processing container capable of storing mixed solution of a substance that accelerates the binding of nucleic acid to solid substance and a sample that includes nucleic acid, means for supplying washing buffer to the processing container, means for supplying elution buffer to the processing container, a container for refined substance that accepts refined substance of nucleic acid, transfer means for allowing the tip for binding nucleic acid in an unused state to be connected to the movable nozzle for liquid aspiration/dispense and allowing the tip for binding nucleic acid in a connected state to be transferred to the locations of the processing container and the container for refined substance, means for a liquid aspiration/dispense process that allows the tip for binding nucleic acid connected to the movable nozzle for liquid aspiration/dispense to aspirate/dispense the mixed solution, then to aspirate/dispense the washing buffer, and then to aspirate/dispense the elution buffer, and means for tip removal that removes the tip for binding nucleic acid from the movable nozzle for liquid aspiration/dispense, after the elution buffer is discharged into the container for refined substance from the tip for binding nucleic acid. In this case, the tip for binding nucleic acid is a cylindrical member comprising transparent or translucent synthetic resin. The tip for binding nucleic acid has such an inside diameter that a head portion is fitted into the tip of the movable nozzle in an airtight manner and is formed such that the inside diameter of the lower portion gradually becomes small toward the tip.
Further, some of the technologies disclosed in the present example can be applied to such an portable-type apparatus for refining nucleic acid as disclosed in Patent Application No. 2003-139542. The portable-type apparatus for refining nucleic acid is provided with a tip for binding nucleic acid and a syringe unit. In this case, the syringe unit comprises a connecting portion for connecting the tip for binding nucleic acid, a body portion that also functions as a grip portion, and an operating portion disposed on the opposite side of the connecting portion via the body portion.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-48533 | Feb 2004 | JP | national |
