JP3337386B2 - Method for transferring DNA sequence sample to purification and separation detection system and plate used for the method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for simultaneously (at the same time) supplying a plurality of reaction products generated in the wells of a multi-reaction plate to an analyzer using a large number of capillaries represented by electrophoresis. The present invention relates to a plate for transferring a DNA sequence sample to a separation detection system. further,
The present invention relates to a method for transferring a DNA sequence sample using this plate, a method for purifying a DNA sequence sample using this plate, and a method for transferring DNA sequence using this plate.
The present invention relates to a method for purifying and transferring an NA sequence sample.

[0002]

2. Description of the Related Art Recently, a technique for detecting DNA using laser fluorescence has been developed, and a laser fluorescent DNA sequencer using the technique has been widely used as a very useful instrument. In addition, the development of such technology has made it possible to process a large amount of samples (about 100 lanes / 2 operations / day / unit or less) in genomic research and DNA diagnosis. Typical examples of the laser sequencer include a slab type gel and a capillary type gel.

[0003] Furthermore, in order to process a larger amount of a sample beyond the current status, more electrophoresis lanes (for example, 2
A device having (00 to 1000 lanes / driving) is also being devised. An example of such an instrument is a multi-capillary sequencer.

[0004]

However, as the number of lanes increases, the work of transferring the sample to the lane takes longer and the load becomes larger. That is, in order to load a large number of samples on each capillary gel, it is necessary to perform an operation of injecting the samples into the capillary by bringing the ends of the capillary into contact with the fine electrode wires for each sample. Therefore, there is an urgent need to develop a technology that can easily transfer a large number of samples to a capillary in a short time. Furthermore, the operation of filling the reaction solution into each well of the microtiter plate requires more time as the number of wells increases. Therefore, it is necessary to provide a means for filling each well with the reaction solution in a short time.

[0005] In a product obtained by the DNA sequencing reaction, fragments of various lengths labeled with a fluorescent substance or the like and an unreacted labeling substance coexist. Most of the co-existing unreacted labeling substance is released into the reaction solution without being used for the reaction, so if this is directly subjected to electrophoresis, a high-concentration fluorescent label is also electrophoresed, It produces a much stronger signal than the sequence band, making it impossible to decode the sequence.
Must be removed before separation. However, it takes a lot of manpower and time to remove the unreacted labeling substance for each of the hundreds of products. As a result, even if the efficiency of the DNA sequencing method itself is improved, the rate is limited in the preceding stage.

Accordingly, a first object of the present invention is to provide a means capable of filling a large number of reaction liquids in a short time and easily. A second object of the present invention is to provide a method for removing a labeling substance or the like from a plurality of DNA sequence samples containing an unreacted labeling substance or the like in a short time and easily. A third object of the present invention is to provide means for transferring a large number of DNA sequence samples to an electrophoresis capillary in a short time and easily. Further, a fourth object of the present invention is to remove a labeling substance and the like from a plurality of DNA sequence samples containing an unreacted labeling substance and the like in a short time and easily, and to remove a large number of DNA sequence samples in a short time. It is an object of the present invention to provide a method which can be easily transferred to an electrophoresis capillary.

[0007]

According to a first aspect of the present invention, there is provided:
The present invention relates to a reaction vessel precursor including a substrate having a plurality of through holes penetrating in a thickness direction. Further, in a second aspect of the present invention, the container precursor of the present invention is immersed in a reaction liquid to fill the reaction liquid into a through-hole, and then one opening of the through-hole is closed with a membrane. Wherein the through-hole is a reaction vessel.

A third aspect of the present invention is characterized by comprising a substrate having a plurality of through holes penetrating in the thickness direction and a film provided so as to close one opening of the through hole. The present invention relates to a plate for transferring a DNA sequence sample to a separation / detection system.

A fourth aspect of the present invention is a method for removing unreacted low-molecular compounds in a DNA sequence sample, wherein the DNA sequence sample is filled in the through-holes of the plate of the present invention. By applying a pressure difference between the opening of the through-hole and the outside of the membrane so that the outside of the membrane becomes a negative pressure, the unreacted low-molecular compound in the DNA sequence sample passes through the membrane through the sample. Moving out of the way.

A fifth aspect of the present invention is a method for removing unreacted low molecular weight compounds in a DNA sequence sample, wherein D is filled in the through hole of the plate of the present invention.
A voltage is applied between the NA sequence sample and the outside of the membrane that seals the through-hole filled with the DNA sequence sample, and unreacted low-molecular compounds in the DNA sequence sample are removed from the sample through the membrane. To a method characterized by moving to (1).

A sixth aspect of the present invention is a method for transferring a DNA sequence sample filled in the through hole of the plate of the present invention to an electrophoresis capillary for separation and detection, wherein one end of the electrophoresis capillary is provided. While immersed in the DNA sequence sample filled in the through-hole, applying a voltage between the membrane that seals the through-hole filled with the DNA sequence sample and the other end of the electrophoresis capillary, The present invention relates to a method for transferring a DNA sequence sample to a separation / detection system, which comprises transferring a sample in a DNA sequence sample to the electrophoresis capillary.

[0012] In a seventh aspect of the present invention, one end of the electrophoresis capillary is inserted into the through-hole of the plate of the present invention from the open side of the through-hole while the DNA sequence sample is filled in the through-hole. A DNA sequence characterized in that a sample in the DNA sequence sample is transferred into the electrophoresis capillary by immersing the sample in the DNA sequence sample and sucking the filler into the electrophoresis capillary from the other end of the electrophoresis capillary. The present invention relates to a method for transferring a sample to a separation detection system.

An eighth aspect of the present invention is to remove unreacted low molecular compounds in a DNA sequence sample,
A method for transferring an analyte in an NA sequence sample to an electrophoresis capillary for separation and detection, wherein one end of the electrophoresis capillary is immersed in a DNA sequence sample filled in a through hole of the plate of the present invention,
A voltage is applied between the membrane that seals the through-hole filled with the DNA sequence sample and the other end of the electrophoresis capillary, and the unreacted low-molecular compound in the DNA sequence sample passes through the membrane through the sample. Moving outside, and then applying a voltage between the membrane and the other end of the electrophoresis capillary to transfer the sample in the DNA sequence sample to the electrophoresis capillary,
And a method for transferring a DNA sequence sample to a separation and detection system.

A ninth aspect of the present invention is to remove unreacted low molecular compounds in a DNA sequence sample,
A method for transferring an analyte in an NA sequence sample to an electrophoresis capillary for separation and detection, wherein the DNA sequence sample is filled in the through hole of the plate of the present invention, and the opening of the through hole and the outside of the membrane are provided. A pressure difference is applied so that a negative pressure is applied to the outside of the membrane, so that unreacted low molecular compounds in the DNA sequence sample are transferred out of the sample through the membrane, and then the DNA sequence With one end of the electrophoresis capillary immersed in the sample, a voltage is applied between the other end of the electrophoresis capillary and the membrane that seals the through-hole filled with the DNA sequence sample, Transferring the sample to the electrophoresis capillary,
And a method for transferring a DNA sequence sample to a separation and detection system.

Container Precursor and Manufacturing Method Thereof (First and Second Embodiments) The container precursor of the present invention comprises a substrate having a plurality of through holes penetrating in the thickness direction. Further, this container precursor can be used for producing a container containing a reaction solution. A description will be given based on FIG. The container precursor 20 is
Substrate 22 having a plurality of through holes 21 penetrating in the thickness direction
Consists of The substrate 22 can be formed from, for example, synthetic resin, glass, or the like.
It can be appropriately determined in consideration of the size (inner diameter and depth) and the number of 1s. The dimensions (inner diameter and depth) and the number of the through holes 21 can be determined as appropriate according to the application.
05-10 mm, preferably 0.5-5 mm, the depth is
For example, it can be set to 0.2 to 200 mm. The number of through-holes 21 can be increased as many samples can be processed at one time, but is appropriately determined in consideration of the number of electrophoresis capillaries of the separation / detection device, the performance of the separation / detection device, and the like.

The container precursor 20 is immersed in a reaction solution 25 to fill the through-hole 21 with the reaction solution 25a. Then, one opening of the through-hole 21 is closed with a film 23 to close the through-hole. By using the reaction container 24, a container containing a reaction solution can be manufactured. The reaction solution is not particularly limited and can be appropriately determined depending on the application. For example, it can be a buffer containing various enzymes.

Plate for Transfer to Separation and Detection System (Third Embodiment) The plate for transfer to the separation and detection system of the present invention will be described with reference to FIG. The separation detection system transfer plate 1 is composed of a substrate 3 having a plurality of through holes 2 penetrating in the thickness direction, and a film 5 provided so as to block one opening 4 of the through holes 2. The substrate 3 can be formed from, for example, synthetic resin or glass, and its size and thickness can be appropriately determined in consideration of the dimensions (inner diameter and depth) and the number of the through holes 2. The size (inner diameter and depth) and the number of the through holes 2 can be appropriately determined according to the application.
-5 mm, and the depth can be, for example, 0.2-200 mm. The number of the through-holes 2 can be processed at a time as much as the number of the through holes 2 is large.

The membrane 5 can be appropriately selected according to the method of using the plate. For example, when an analyte in a DNA sequence sample is to be electrically transferred to an electrophoresis capillary, the membrane 5 may be made of a material that can be energized in contact with an electrolytic solution. For example, a molecular sieve such as an ultrafiltration membrane Can be used. When the sample in the DNA sequence sample is transferred to the electrophoresis capillary using the pressure difference, the membrane 5
May be a film made of a liquid-permeable material. For example, a film such as a cellophane film or a polyethersulfone film can be used.

When an unreacted low molecular compound in a DNA sequence sample is electrically removed outside the system via the membrane 5, the low molecular compound passes through the membrane 5, but the reaction product is Any material that does not transmit a certain DNA fragment may be used. For example, a membrane such as an ultrafiltration membrane can be used. Further, when unreacted low molecular compounds in the DNA sequence sample are electrically removed outside the system via the membrane 5, and then the specimen in the DNA sequence sample is electrically transferred to the electrophoresis capillary, The membrane 5 may be made of a material that transmits a low-molecular compound but does not transmit a DNA fragment that is a reaction product, and is a material that can be electrically connected to an electrolyte. As such a membrane, for example, a membrane such as an ultrafiltration membrane can be used. In the present invention, a polyethersulfone membrane can be used as the ultrafiltration membrane.

[0020]Removal method of unreacted low molecular compounds (pressure difference
Law)(Fourth Mode) A description will be given based on FIG. Of the plate 1 of the present invention
The DNA sequence sample 7 is filled in the through hole 2
Then, between the opening 2a of the through hole and the outside of the membrane 5,
A pressure difference is applied so that the outside has a negative pressure. In particular,
For example, the membrane 5 side of the plate 1 is set in the vacuum container 20 and
Pressure to reduce the pressure outside the membrane 5 to a negative pressure.
The difference can be added. In DNA sequence sample
The unreacted low-molecular-weight compound of
The compound, e.g., water, is transferred out of the sample.
Can be.

Method for removing unreacted low molecular compounds (electrical method
Method) (Fifth Aspect) The fifth aspect of the present invention includes the following two methods. The first method is to fill the D holes filled in the through holes of the plate.
One end of the electrophoresis capillary is immersed in the NA sequence sample, and the electrode immersed in the electrolyte that comes into contact with the other end of the electrophoresis capillary and the electrolyte that comes into contact with the membrane that seals the through hole filled with the DNA sequence sample. This is a method of applying a voltage between the immersed electrode. This method will be described with reference to FIG. The above-mentioned plate 1 of the present invention
DNA sequence sample 7 filled in the through hole 2 of FIG.
A voltage is applied between the membrane 5 and the other end 6b of the electrophoresis capillary 6 such that the other end 6b of the electrophoresis capillary 6 becomes a cathode while the one end 6a of the electrophoresis capillary 6 is immersed. The voltage is applied by bringing the membrane 5 into contact with the electrolyte 8 in which the electrode 9 is immersed, bringing the other end 6b of the electrophoresis capillary 6 into contact with the electrolyte 11 in which the electrode 10 is immersed, using the electrode 9 as an anode, and It can be performed by using a cathode. In this manner, the unreacted low molecular compound in the DNA sequence sample 7 can be transferred to the outside of the sample via the membrane 5. FIG. 4 shows one through hole 2
However, the transfer operation of the low-molecular compound can be simultaneously performed in a plurality or all of the through holes 2 in parallel.

In the second method, an electrode is immersed in a DNA sequence sample filled in a through hole of a plate, and the electrode is brought into contact with an electrolyte in which the membrane sealing the through hole filled with the DNA sequence sample comes into contact. This is a method of applying a voltage between the immersed electrode. FIG. 5 shows a case where an electrode is immersed in this DNA sequence sample. In a state where the electrode 12 is immersed in the DNA sequence sample 7 filled in the through hole 2 of the plate 1 of the present invention, a voltage is applied between the membrane 5 and the electrode 9 so that the electrode 12 becomes a cathode. Can be done with Even in this case, the unreacted low molecular compound in the DNA sequence sample 7 can be transferred to the outside of the sample via the membrane 5. FIG.
Although only one through hole 2 is shown in FIG.
The transfer operation of the low-molecular compound can be performed simultaneously and in parallel in a plurality or all of the through holes 2.

In the above method, the entire surface of the membrane 5 of the plate 1 is brought into contact with the electrolytic solution 8, the electrodes 12 or the electrophoresis capillaries 6 are immersed in the sample in each through-hole, and a voltage is applied to form a plurality or the like. DNA in all through holes 2
Unreacted low molecular compounds in the sequence sample can be simultaneously transferred out of the sample via the membrane 5 in parallel.

[0024]Transition method to separation detection system (electrical method)
(Sixth Aspect) The DNA chip filled in the through hole of the plate of the present invention described above.
Electrophoresis capillary for separation and detection of sequence samples
It is a method to shift to. A description will be given based on FIG. Electrical
One end 6a of the electrophoresis capillary was filled in the through hole 2.
While immersed in the DNA sequence sample 7, the membrane 5
Apply voltage between the other end 6b of the electrophoresis capillary 6
I do. As shown in FIG. 4, the application of a voltage
Is brought into contact with the electrolytic solution 8 immersed in the electrophoresis capillary.
6 is brought into contact with the electrolytic solution 11 in which the electrode 10 is immersed.
The electrode 9 as a cathode and the electrode 10 as an anode.
I can.

This electric injection can be used when the packing (separation system) of the electrophoresis capillary is a polymer solution or acrylamide. Electrophoresis is performed by applying a voltage of 1 to 10 kV between the electrode 9 and the electrode 10 for one to several tens of seconds. Thereby, DNA sequence sample 7
The sample inside can be transferred to the electrophoresis capillary 6 (near 6a). FIG. 4 shows one through hole 2
However, only the entire surface of the membrane 5 of the plate 1 is brought into contact with the electrolytic solution 8 and the other end 6b of each electrophoresis capillary 6 is set to the anode side, so that a plurality or all of the through holes 2 are simultaneously formed. In parallel, the sample in the DNA sequence sample can be transferred to each electrophoresis capillary.

Method for Transferring to Separation and Detection System (Pressure Difference Method) (Seventh Aspect) A method for transferring the DNA sequence sample filled in the through hole of the plate of the present invention to an electrophoresis capillary for separation and detection. is there. Explanation will be given based on FIG. With the DNA sequence sample 7 filled in the through hole 2 of the plate 1, one end 6a of the electrophoresis capillary 6 is
It is immersed in the DNA sequence sample 7 in the through hole 2 from the opening side of the through hole 2, and the filler (for example, a polymer solution) 6 c in the electrophoresis capillary 6 is sucked from the other end 6 b of the electrophoresis capillary 6. That can be done. Thus, the specimen in the DNA sequence sample 7 can be transferred into the electrophoresis capillary 6. By immersing and aspirating the electrophoresis capillaries 6 in each of the through-holes 2 of the plate 1, the samples in the DNA sequence samples in all the through-holes 2 are simultaneously transferred to the respective electrophoresis capillaries. Can be.

Method for Transition to Purification / Separation Detection System (Eighth Aspect) In this method, unreacted low molecular compounds in a DNA sequence sample are removed, and then a sample in the DNA sequence sample is separated and used for electrical detection. This is a method for transferring to an electrophoresis capillary, and is a method for sequentially performing the method for removing unreacted low-molecular compounds of the DNA sequence sample and the method for transferring to a separation detection system. The method according to the fifth aspect of the present invention is used for removing the low molecular weight compound. That is, as shown in FIG. 4, one end 6a of the electrophoresis capillary 6 is immersed in the DNA sequence sample 7 filled in the through hole 2 of the plate 1 of the present invention, and the membrane 5 and the electrophoresis capillary 6 are immersed. Electrophoresis capillary 6 between the other end 6b
Voltage is applied so that the other end 6b of the
Unreacted low molecular compounds in the sequence sample 7
Through the sample. Then, the method of the sixth aspect of the present invention is used for transferring the sample. That is, a voltage is applied between the membrane 5 and the other end b of the electrophoresis capillary 6 so that the other end 6b of the electrophoresis capillary 6 becomes an anode, and the sample in the DNA sequence sample 7 is moved to the electrophoresis capillary 6. Move to Removal of the low-molecular compound and transfer of the sample can be easily performed by switching between the positive and negative electrodes 9 and 10. However,
It is preferable that the electrolyte solution 8 in which the unreacted low-molecular compound is transferred via the membrane 5 is replaced before the transfer of the sample, from the viewpoint that the low-molecular compound is prevented from refluxing to the sample 7. .

Method for Transition to Purification / Separation Detection System (Ninth Aspect) This method removes unreacted low-molecular-weight compounds in a DNA sequence sample, and then removes the analyte in the DNA sequence sample by an electric current for separation and detection. This is a method for transferring to an electrophoresis capillary, and is a method for sequentially performing the method for removing unreacted low-molecular compounds of the DNA sequence sample and the method for transferring to a separation detection system. The method according to the fourth aspect of the present invention is used for removing the low molecular weight compound. That is, as shown in FIG. 3, in a state where the DNA sequence sample 7 is filled in the through hole 2 of the plate 1 of the present invention, the opening 2a of the through hole is provided.
A pressure difference is applied between the outside of the membrane 5 and the outside of the membrane 5 so that the outside of the membrane 5 has a negative pressure. Specifically, for example, the membrane 5 is set by setting the membrane 5 side of the plate 1 in the decompression container 20 and reducing the pressure.
A pressure difference can be applied so that the outside of the space becomes a negative pressure. Unreacted low molecular compounds in the DNA sequence sample can be transferred to the outside of the sample via the membrane 5 together with other low molecular compounds such as water.

Next, the method of the sixth aspect of the present invention is used for transferring the sample. That is, as shown in FIG. 4, the one end 6a of the electrophoresis capillary 6 is immersed in the DNA sequence sample 7 from which the unreacted low molecular compounds in the through-hole 2 of the plate 1 of the present invention have been removed, and A voltage is applied between the other end b of the electrophoresis capillary 6 and the sample in the DNA sequence sample 7 is transferred to the electrophoresis capillary 6.

In the present invention, the DNA sequence sample to be treated is, for example, a large number of chemical or enzymatic reactions (96 reactions /
Plate / Trial) The type and number of reactions are not limited. For example,
The DNA sequence sample is a product of the DNA sequence reaction performed in the through-hole of the plate of the present invention,
Alternatively, it can be the product of a DNA sequencing reaction performed in the wells of another microtiter plate. Further, as a DNA sequencing reaction, for example,
Sanger reaction using dideoxynucleotide (dideoxynucleotide), PCR (polymerase chain reaction)
DNA cycle sequence performed using

When the DNA sequence reaction is performed in the space formed in the through hole of the plate of the present invention, it is preferable to use the following method. First, it is necessary to put a different kind in each hole (for example, mold DN
Injection of reagent enzymes such as reaction enzymes (such as thermostable polymerase) and reaction buffers other than A)) as described in the second embodiment of the present invention using the container precursor of the first embodiment of the present invention. Then, they are put together in each through hole of the container precursor plate. That is, a certain amount of the reagent enzyme solution can be held in each through-hole only by immersing the container precursor of the first aspect of the present invention in the reagent enzyme solution and pulling it up. By providing two or more different through-holes on the same plate, it is possible to form a reaction field having a different holding amount of the reagent enzyme solution. Next, a membrane is attached to one surface of the container precursor plate to form a reaction solution-containing plate in which the reagent enzyme solution is held in the through hole. This plate can be provided for a reaction such as a desired DNA sequencing reaction. As the reaction performed in the container, for example, a sequence reaction using DNA polymerase performed at 37 ° C. is advantageous in consideration of the retention of the reaction solution by the membrane. According to this method of the present invention, the dispensing process of initially adding the reagent enzyme solution can be extremely labor-saving. However, the reagent enzyme solution may be dispensed one by one without using such a method.

The sample transferred to the electrophoresis capillary according to the method of the present invention is separated and detected by a conventional method. As such a conventional method, a DNA base sequence determination method by DNA capillary electrophoresis can be exemplified. DN
As a DNA separation system used in the A capillary electrophoresis, for example, a capillary lane 31 in which capillaries as shown in FIG. 7 are arranged in a grid and three-dimensionally regularly arranged (for example, in a columnar shape) is used as an electrophoresis gel. be able to. The arrangement of the capillaries housed in a grid shape is determined by changing each of the through holes 3 of the multi through hole plate 32 of the present invention.
By making the arrangement coincide with the arrangement of 3, the capillary can be inserted exactly into the through-hole 33 of the multi-through-hole plate 32.

The membrane mounted on the bottom of the multi-through-hole plate on which the capillaries are set is transferred to the cathodic electrophoresis tank 34.
And a voltage is applied using a power supply 36 between the cathodic electrophoresis tank 34 and the anodic electrophoresis tank 35 in which the other end of the capillary 31 is immersed. In this way, all the reaction samples on the multi-through hole plate are electrophoretically injected into the capillary at one time. Then
The fragments are separated by electrophoresis, and the separated fragments are detected by a detector 37. The detector 37 selects according to the type of the marker put on the fragment. For example,
In the case of a fluorescent marker, a fluorescence detector can be used, and in the case of an RI marker, an imaging analyzer or the like can be used.

[0034]

The present invention will be further described with reference to the following examples.
In the following example, DN was measured using the apparatus shown in FIG.
The unreacted low-molecular compounds in the A sequence sample were removed, and then the DNA sequence sample was transferred to the electrophoresis capillary using the capillary electrophoresis apparatus shown in FIG. Further, separation and detection were performed by a separation and detection apparatus based on the electrophoresis method shown in FIG.

(1) Pretreatment of inner wall of quartz capillary Quartz capillary having inner diameter of 0.1 mm and outer diameter of 0.22 mm
(OD 220 μm, ID 100 μm, SGE: Australia) was cut into about 30 cm, passed through a reagent having a functional group (such as 3-methacryloxypropyltrimethoxysilane), and allowed to stand for several hours.
Thereafter, the inside of the capillary was washed with methanol and water. (2) Filling of acrylamide Acrylamide solution [TBE buffer (100 mM Tris borat)
e, pH 8.0, 0.2 mM EDTA), 7 M urea, 6% acrylamide, 0-5% bisacrylamide]
Degas using a decompressor or the like, and remove ammonium persulfate (final concentration 0.05%), tetramethylethylenediamine (final concentration
0.01%), and the above-mentioned quartz capillary was filled with an acrylamide solution using a syringe or the like. The mixture was left at 4 ° C. for several hours until gel passage was completed.

(3) Preparation of template DNA In a sampling tube, 0.5 pmol of M13 primer, 0.5 pmol of template DNA (M13phage), Sequenase Ver.2.0 buffer (200 μM Tris.Cl. pH7.5, 100 mM MgCl ₂ , 250 mM NaC)
l) 2 μl was added, adjusted to 10 μl with sterile distilled water, mixed, heated at 65 ° C. for 10 minutes, and then returned to room temperature to anneal the template and the primer. Next, 1 μl of 0.1 M dithiothreitol and 10 μl of dNTPs mix (1 μM dATP, dGTP, dTT) were added to 10 μl of the annealed template DNA-primer solution.
P) 2 μl, [α- ³² P] dCTP (3000 Ci / mmol, 10 mCi / ml,
Amershame) 0.5μl, Sequenase Ver.2.0 (Amersham) 2
U was added. Then stop mixture 80 μM dATP, dGTP, dCTP, dTTP
TP, 8 μM ddTTP, 50 mM NaCl)
2.5 μl was added, mixed, and reacted at 37 ° C. for 10 minutes. Ten
μl of formamide dye [95% formamide, 10 mM
EDTA, 0.05% bromophenol blue (BPB),
0.05% xylene cyanol (XC)] at 80 ° C
For 5 minutes.

(4) Injection of Sample into Capillary and Electrophoresis A plate having a 384 (16 × 24) through-hole with an inner diameter of 3.5 mm is prepared, and its lower surface (surface to be brought into contact with the buffer solution)
Then, a polyether sulfone ultrafiltration membrane was welded using an adhesion method such as ultrasonic welding. The sample solution prepared by the above method was injected into each hole of this plate in contact with the ultrafiltration membrane. As shown in FIG. 8, the membrane-mounted plate containing the sample was connected to a suction device, and the unreacted low-molecular compound was removed under reduced pressure by applying a vacuum for 10 minutes from the membrane side.
Next, one of the capillaries filled with the acrylamide gel prepared above was immersed in a TBE buffer solution, and the other end was immersed in a sample solution as shown in FIG. The ultrafiltration membrane on the lower surface of the plate was immersed in a TBE buffer, and a voltage of 3 kV was applied between the cathodic electrophoresis tank 34 and the anodic electrophoresis tank 35 for about 30 seconds as shown in FIG. Then, one end of the capillary was transferred from the sample solution to the TBE buffer solution, and electrophoresis was performed at 3000 V for 1 hour.

(5) Autoradiography When the BPB reaches the lower end of the capillary, the electrophoresis is terminated and the BAS2000 imaging analyzer (FUJI)
The electrophoresis pattern was analyzed by. The result is shown in FIG. As shown in FIG. 10, a sequence ladder was generated according to the above protocol.

[0039]

According to the present invention, it is possible to provide a means for easily filling a large number of reaction solutions into a large number of wells in a short time. Further, according to the present invention, it is possible to provide a method for removing a labeling substance or the like in a short time and easily from a plurality of DNA sequence samples containing an unreacted labeling substance or the like. Further, according to the present invention, it is possible to provide a means for transferring a large number of DNA sequence samples to an electrophoresis capillary in a short time and easily. In addition, according to the present invention, a plurality of DNAs containing unreacted labeling substances and the like
It is possible to provide a method for removing a labeling substance or the like from a sequence sample in a short time and easily, and for transferring a large number of DNA sequence samples to an electrophoresis capillary easily in a short time.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view of a method for manufacturing a container precursor of the present invention and a container incorporating the reaction solution of the present invention using the container precursor.

FIG. 2 is a schematic cross-sectional explanatory view of a plate for transferring a DNA sequence sample to a separation and detection system according to the present invention.

FIG. 3 is a schematic cross-sectional explanatory view of a method for removing unreacted low-molecular compounds (pressure difference method) of the present invention.

FIG. 4 shows a method for removing unreacted low-molecular compounds of the present invention (electric method) and a method for transferring to a separation detection system (electric method).
FIG.

FIG. 5 is a schematic cross-sectional view of a method (electric method) for removing unreacted low-molecular compounds according to the present invention.

FIG. 6 shows a method for transferring to a separation detection system of the present invention (pressure method).
FIG.

FIG. 7 is a schematic explanatory diagram of a separation detection method by an electrophoresis method.

FIG. 8: Method for removing unreacted low-molecular compounds (pressure difference method)
FIG.

FIG. 9 is a schematic explanatory view of a method for transferring to an electrophoresis capillary (electric method).

FIG. 10 shows a sequence ladder of an electrophoresis pattern obtained by an imaging analyzer.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 27/26 321Z (56) References JP-A-3-297379 (JP, A) JP-A-5-142198 (JP, A) JP-A-4-127049 (JP, A) JP-A-60-100054 (JP, A) WO 88/6723 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12M 1 / 00 G01N 27/447 WPI (DIALOG) EUROPAT (QUESTEL) JICST file (JOIS)

Claims (16)

(57) [Claims] 1. A plurality of through holes penetrating in a thickness direction.
A reaction vessel precursor comprising a substrate is immersed in a reaction solution to fill the through-hole with the reaction solution, and then one opening of the through-hole is closed with a film to make the through-hole a reaction vessel. A method for producing a container containing a reaction solution. Wherein the inner diameter of the through hole 0.01~10mm
The production method according to claim 1, wherein 3. A process according to claim 1 or 2 is a buffer containing the reaction solution enzyme. 4. A method of removing low-molecular compounds unreacted in the DNA sequence samples, transmural in the thickness direction
Plate precursor comprising a substrate having a plurality of through holes through
Dip the body into the DNA sequence sample and place it in the through hole.
Fill the DNA sequence sample and then
Sequester one of the open mouth of the hole in the membrane, in this state, between the outside of the membrane and the open port of the through hole, by applying a pressure difference so that the outside of the membrane is negative, the A method comprising transferring unreacted low molecular compounds in a DNA sequence sample to outside the sample through the membrane. 5. A method for removing unreacted low molecular weight compounds in a DNA sequence sample, the method comprising penetrating in the thickness direction.
A substrate having a plurality of through holes, and one of the through holes
DNA membrane consisting of a membrane provided to block the outlet
A DNA sequence sample filled in a through-hole of a plate for transfer of a separation sample to a detection system;
A voltage is applied between the outside of the membrane that blocks the through hole filled with A, and the unreacted low-molecular compound in the DNA sequence sample is transferred outside the sample through the membrane. Method. 6. An end of an electrophoresis capillary is immersed in a DNA sequence sample filled in the through hole,
A voltage is applied between an electrode immersed in an electrolyte that contacts the other end of the electrophoresis capillary and an electrode immersed in the electrolyte that contacts a membrane that seals the through hole filled with the DNA sequence sample. Item 6. The method according to Item 5 . 7. immersing the electrode in the DNA sequence sample filled in the through hole, and the electrode, the DNA
The method according to claim 5 , wherein a voltage is applied between the membrane closing the through hole filled with the sequence sample and the electrode immersed in the electrolytic solution in contact with the membrane. 8. The method according to claim 4 , wherein the unreacted low molecular weight compound is an unreacted fluorescent labeling substance. 9. DNA sequence sample The method according to any one of claims 4-8 wherein the product that are made DNA sequencing reaction in the through-hole. 10. The method according to any one of claims 4 to 8 , wherein the DNA sequence sample is a product of a DNA sequencing reaction performed in a microtiter plate. 11. A plurality of through holes penetrating in a thickness direction.
A plate precursor consisting of
Immersed in the DNA sequence sample
Sample, and then open one of the through holes
In blocked, the DNA sequence sample filled in the through hole to a method of transition to electrophoresis capillary for separation and detection, the end of the electrophoretic capillary DNA sequence sample filled in the through-hole In the immersed state, a voltage is applied between the membrane that seals the through hole filled with the DNA sequence sample and the other end of the electrophoresis capillary, and the sample in the DNA sequence sample is transferred to the electrophoresis capillary. A method for transferring a DNA sequence sample to a separation / detection system. 12. DNA sequence sample The method of claim 11 wherein a transmembrane <br/> hole in products of DNA sequencing reaction was carried out at. 13. DNA sequence sample The method of claim 11, the product of DNA sequence reactions were performed in a microtiter plate. 14. A plurality of through holes penetrating in a thickness direction.
A plate precursor consisting of
Immersed in the DNA sequence sample
Sample, and then open one of the through holes
In this state, one end of the electrophoresis capillary is immersed in the DNA sequence sample in the through-hole from the open side of the through-hole, and the filler is sucked into the electrophoresis capillary from the other end of the electrophoresis capillary. A method for transferring a DNA sequence sample to a separation / detection system, wherein the sample in the DNA sequence sample is transferred into the electrophoresis capillary. 15. A method for removing low molecular compounds unreacted in the DNA sequence sample, then migrating analyte in DNA sequence samples to electrophoresis capillaries for separation and detection, a plurality of penetrating in the thickness direction Through-hole
To close the substrate with one of these through holes
DNA sequence sample consisting of a membrane provided
Filled in the through hole of the separation detection system transfer plate
In a state where one end of the electrophoresis capillary is immersed in the NA sequence sample, a voltage is applied between the other end of the electrophoresis capillary and a membrane that seals the through hole filled with the DNA sequence sample, The unreacted low-molecular compound in the sample is transferred to the outside of the sample through the membrane, and then a voltage is applied between the membrane and the other end of the electrophoresis capillary to cause the DN.
A method for transferring a DNA sequence sample to a purification and separation / detection system, wherein the sample in the A sequence sample is transferred to the electrophoresis capillary. 16. A method for removing unreacted low molecular compounds in a DNA sequence sample, and then transferring an analyte in the DNA sequence sample to an electrophoresis capillary for separation and detection, wherein the plurality of molecules penetrate in a thickness direction. Through-hole
Plate precursor comprising a substrate having
DNA sequence in the through hole
Fill the sample, then open one of the through holes
Was sealed in film, in this state, between an outer mouth opening and the membrane of the through hole, the outer membrane by applying a pressure difference such that the negative pressure, the unreacted in the DNA sequence sample The low-molecular compound is transferred to the outside of the sample through the membrane, and then, with the one end of the electrophoresis capillary immersed in the DNA sequence sample, the membrane that seals the through hole filled with the DNA sequence sample and the electrophoresis A method for transferring a sample in the DNA sequence sample to the electrophoresis capillary by applying a voltage between the other end of the capillary and the DNA sequence sample, and transferring the sample to a system for purifying and separating a DNA sequence sample.