JP2611985B2 - Sample purification method and device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention is suitable for automating the purification operation of DNA (deoxyribonucleic acid) samples. In particular, the protein is denatured using phenol and centrifuged to separate the phenol layer ( The present invention relates to a sample purification method suitable for an operation of collecting an aqueous layer, which is performed after the separation into an (lower layer) and an aqueous layer (upper layer).

[Conventional technology]

The process of purifying a DNA sample involves first injecting and mixing phenol into a solution in which the DNA sample and proteins such as enzymes are mixed to denature and insolubilize the protein.
Separate the protein at the interface between the phenol and aqueous layers, leaving the DNA behind. Then, in order to obtain the target DNA, only the aqueous layer containing the DNA was collected, and ethanol was added to the precipitate to precipitate the DNA dissolved in the aqueous layer, and the precipitate was collected by centrifugation.
Wash with 0-80% ethanol to remove salts. In a conventional method for obtaining an aqueous layer containing DNA, an experimenter aspirates an aqueous solution containing DNA in the upper layer of the liquid separated into two layers by pipetting to obtain a target DNA sample. As a method of automating such a similar operation, Japanese Patent Publication No. 56-51299 is disclosed, in which light is incident from one of the branch ends and reflected light from a two-layer boundary surface is received from the other. In this method, the upper layer liquid is sucked and collected by suction noise separately provided while inserting and inserting the liquid into the liquid to directly detect the boundary surface.

[Problems to be solved by the invention]

When the amount of the sample to be handled is small, such as when purifying and extracting a DNA sample solution on the order of μl using phenol, a small container having a capacity of about 1.5 ml is used for convenience of various operations. In order to automate such a purification process, aspiration of a DNA sample by an experimenter's pipetting operation cannot be adopted. If the above-mentioned conventional technique (Japanese Patent Publication No. 56-51299) is used as one method of automation, there are the following problems. That is, it is not only dimensionally difficult to use the light detecting means in terms of the size of the sample container, but also the method of controlling the operation of the apparatus becomes complicated,
There has been a problem that both the hardware and software of the apparatus increase the burden.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems and to provide a DN of the order of μl.
It is an object of the present invention to provide a method suitable for purifying A sample using phenol.

[Means for solving the problem]

A feature of this process is that the amount of phenol to be injected is preset.

The above-mentioned object adopts a method of mixing a sample solution and phenol in a container, centrifuging the mixture, and then aspirating the phenol located at the lower portion of the container to an amount equal to or greater than the amount of phenol supplied using the dispensing device. Is achieved by doing

[Action]

The protein is first denatured and insolubilized by injecting and mixing phenol into a solution in which a DNA sample and a protein such as an enzyme are mixed. Next, the DNA is centrifuged to leave the DNA in the aqueous layer, and the protein is separated at the interface between the phenol layer and the aqueous layer. Where DNA
It is a problem to collect only the aqueous layer containing phenol. However, in the method disclosed in the present invention, the tip (dispensing tip) of the dispensing device is positioned at the lower end of the container, and phenol is injected first in this state. The same amount or more of the liquid is sucked, discarded at an appropriate place, and the target DNA is purified from the remaining aqueous solution.

〔Example〕

FIG. 2 is a schematic diagram of an automatic sample processing apparatus according to the present invention. The apparatus comprises a dispensing device 15 for transferring a liquid sample between a plurality of containers 8, a dispensing tip 1 (FIG. 1), a transfer table 9a for transferring the containers 8, a transfer mechanism 9b, 9c, and a container 8. A centrifuge 16 for centrifuging the solution in the container, a mixer 17 for mixing the solution in the container 8, a controller 10 for controlling the operation of the above mechanical elements, a dispensing tip 1, a container 8, and a supply of sample reagents It consists of part 18.

Next, the sample purification method according to the present invention will be described. In this method, after mixing the sample solution and phenol in the container 8 and centrifuging the mixture, the phenol layer 14 located below the container is removed using a dispenser, and the aqueous layer 13 containing DNA located above the container is removed. What you get. That is, in the operation state diagram of FIG. 1, the friction engagement part 2 for attaching and detaching the dispensing tip 1, the body part 3 located above the friction engagement part 2, the atmosphere communication hole 4 penetrating the friction engagement part 2 and the body part 3, Plunger 5, motor 6a, coupling 6b, ball screw 6c, which moves up and down in air communication hole 4,
Plunger driving mechanism 6, consisting of plunger holding member 6d, plunger vertical moving part 6e, bearing member 6f, motor 7a, rotational power conversion mechanism 7b, vertical driving mechanism consisting of bearing members 7c, 7d
7, using a dispensing device including a controller 10, a dispensing tip pushing member 11 sliding on the outside of the body 3 and a dispensing tip removing member 12, the tip of the dispensing tip 1 is placed on the bottom of the container 8. The phenol layer 14 is sucked into the dispensing tip 1 by raising the plunger 5 in this state. The set value of the phenol layer suction amount may be the same as the amount of the phenol injected beforehand, but may be set to a safe side according to the accuracy of the dispensing device 15. For example, consider the case where the amount of phenol injected is 50 μl. Assuming that the accuracy of the dispensing device 15 is about ± 1 μl, if the set value of the phenol layer suction amount xμl is set to be at least 2 μl larger, the residual amount of phenol = (50 ± 1) −x = (50 ± 1) ) − (52 ± 1) ≦ 0, and no phenol remains in the solution on the order of μl. After aspirating the phenol layer 14, the dispensing tip 1 is discarded at a predetermined position, and the container 8 in which the aqueous layer 13 containing DNA remains is used as it is for the next process. This method not only has the advantage of being easy to operate, but also has the advantage that it is not necessary to supply a new container as compared with the case where the upper layer 13 is collected and transferred to a new container.

The results of DNA cloning were compared using two methods: a method of removing the lower layer of phenol and a method of collecting an aqueous layer containing DNA in the upper layer and transferring it to a new container. It was revealed that the desired bound DNA can be produced by any of the phenol extraction methods. The method of removing phenol in the lower layer of the present invention is more effective than the method of collecting an aqueous solution containing DNA in the upper layer in that phenol can be easily removed from a DNA sample.

FIG. 3 is an operation state diagram of another sample automatic processing apparatus according to the present invention. Is shown when positioning is performed as follows. Elastic member 19 to container 8
When the tip of the dispensing tip 1 having a slight variation in its length collides with the bottom of the container 8, the elastic member 19 contracts and the container 8 moves downward. Therefore, there is no danger that the dispensing tip 1 is damaged, and there is an effect that precise positioning of the dispensing tip 1 is unnecessary.

In the simple operation method of directly removing the lower phenol layer by suction, it is conceivable that a little phenol may remain in the solution, but since the phenol is removed in the next ethanol precipitation operation, it may affect the next reaction (DNA binding reaction). Did not.

〔The invention's effect〕

As described above, by using the sample purification method of the present invention, phenol can be easily removed from an aqueous sample solution without performing complicated operations such as detection of a two-layer boundary surface, calculation, or positioning control of the tip of a dispenser. it can.

[Brief description of the drawings]

FIG. 1 is an operation state diagram of an apparatus used in the sample purification direction of the present invention, FIG. 2 is a schematic view of an automatic sample processing apparatus for implementing the sample purification method of the present invention, and FIG. 3 is an operation in another embodiment. It is a state diagram. 1 ... dispensing tip, 5 ... plunger, 6 ... plunger drive mechanism, 8 ... container, 9 ... container / dispensing tip transport mechanism, 13 ... aqueous layer containing DNA, 14 ... denaturation Phenol layer containing protein, 15: dispenser, 16: centrifuge, 17: mixer, 19: elastic member

 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-57-179119 (JP, A) JP-A-58-147652 (JP, A) JP-A-63-111437 (JP, A) 134486 (JP, U) JP-56-56299 (JP, B2)

Claims (2)

(57) [Claims] 1. A sample aqueous solution containing a protein and phenol are mixed in a container, and the mixture is centrifuged into two layers of an upper layer (sample aqueous layer) and a lower layer (phenol layer) by the action of centrifugal force. In a method for purifying a sample contained in an aqueous solution, a tip of a device for performing suction removal of the liquid is inserted to the bottom of the container, and an amount equal to or more than the supplied phenol is removed by suction to obtain a sample aqueous solution. Sample purification method. 2. A container for mixing a phenol solution with a sample aqueous solution containing a protein, a means for applying a centrifugal force to the container and centrifuging the container into two layers, an upper layer (sample aqueous solution layer) and a lower layer (phenol layer), in the container. In the apparatus for sucking and removing the liquid in the container, after inserting the tip of the apparatus for sucking and removing the liquid in the container to the bottom of the container, a controller for sucking and removing an amount equal to or more than the supplied phenol. A sample purification device having: