JP2002272462A - Column and method for nucleic acid sample purification - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simplify a purification operation of nucleic acid sample. SOLUTION: (1) A gel filtration filler 3 is packed into a purification column 1. (2) Rinse water 9 is sucked into a syringe 5 and a needle 7 and then a Sanger' s reaction product 13 stored in a sample container 11 is sucked into the needle 7 and the cylinder 5. (4) One end 1a of the purification column 1 is attached to the tip of the needle 7 and then (5) a piston of the syringe 5 is advanced, the Sanger's reaction product 14 is introduced into the purification cylinder 1 and successively rinse water 9 is introduced into the purification cylinder 1. Since components of the nucleic acid-containing Sanger's reaction product 13 are transferred at a rate corresponding to a molecular weight in the gel filtration filler 3 to the other end 1b side of the purification cylinder 1 and discharged from the other end 1b of the purification cylinder 1 in the order from a nucleic acid having a larger molecular weight, a Sanger's reaction product 13a which is desalted, from which an unreacted product is removed and which is discharged at an early stage is recovered at the other end 1b side of the purification column 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for purifying a nucleic acid sample. Nucleic acid samples include nucleic acid samples prepared by the Sanger method and PCR (Polymerase chain Reactor).
Examples include nucleic acid samples in which nucleic acids are mixed with contaminants such as salts and unreacted primers, such as nucleic acid samples prepared by the ion) method. The purified nucleic acid sample is used for microanalysis using, for example, a microchip electrophoresis apparatus.

[0002]

2. Description of the Related Art As a conventional technique for purifying a nucleic acid sample in which a nucleic acid is mixed with contaminants such as salts and unreacted primers, such as a nucleic acid sample prepared by the Sanger method or a nucleic acid sample prepared by the PCR method, gels are used. Examples include filtration and ethanol precipitation. In these methods, centrifugation is performed in order to shorten the processing time and increase the nucleic acid recovery rate.

[0003]

In the transfer step from a reaction system for preparing a nucleic acid sample to an analysis system such as a microchip electrophoresis apparatus, it is necessary to perform a nucleic acid sample purification operation. Since the centrifugal operation is performed, the nucleic acid sample must be once stored in a centrifuge, and there has been a problem that there are many purification steps. Therefore, an object of the present invention is to simplify the operation of purifying a nucleic acid sample.

[0004]

The column for purifying a nucleic acid sample according to the present invention is a column in which both ends are open and a gel filtration filler is filled.

[0005] In the method for purifying a nucleic acid sample according to the present invention, a capillary containing a nucleic acid sample before purification is connected to one end of a purification column filled with a gel filtration filler, and the nucleic acid sample before purification is converted to one end of the purification column. From the other end of the purification column to discharge the nucleic acid sample.

[0006] A thin tube containing a nucleic acid sample before purification is connected to one end of a purification column in which a gel filtration packing material is filled in a tube whose both ends are open. When a nucleic acid sample before purification is introduced into the purification column and further moved to the other end of the purification column, the nucleic acid having a higher molecular weight is discharged from the other end of the purification column in the order of larger molecular weight by the action of the gel filtration filler. The nucleic acid sample can be purified by collecting the discharged nucleic acid sample before discharging impurities such as salts and primers having a low molecular weight from the other end of the purification column. Furthermore, by connecting the other end of the purification column to the analysis system, the purified nucleic acid sample can be introduced into the analysis system without separately collecting the purified nucleic acid sample in a container. In addition, since steps such as centrifugation are not required, automation is facilitated.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for purifying a nucleic acid sample according to the present invention, it is preferable that after introducing the nucleic acid sample before purification into the purification column, a solution for pushing out the nucleic acid is further introduced from one end of the purification column. . As a result, the nucleic acid adsorbed on the gel filtration filler can be further washed away, and the nucleic acid recovery rate can be increased. Furthermore, it has been difficult to purify a very small amount of nucleic acid sample, for example, a nucleic acid sample of 5 microliters or less by gel filtration or ethanol precipitation, which is a conventional technique. Therefore, a very small amount of nucleic acid sample can be purified.

[0008]

FIG. 1 is a schematic view showing the steps of one embodiment of a purification column and one embodiment of a purification method. This embodiment shows an example applied to a fluorescent DNA (deoxyribonucleic acid) sequencer. As a nucleic acid sample, a Sanger reaction product (solution) prepared by the Sanger method was used. In this example,
The dispensing nozzle of the fluorescent DNA sequencer is simulated with a syringe and a needle.

For example, a purification column 1 is formed by filling a gel filtration filler 3 adjusted to a predetermined concentration into a glass tube having an inner diameter of 1.0 to 1.5 mm and a length of 8.0 mm.
As the gel filtration filler 3, for example, a gel filtration filler such as Sephadex (a product of Amersham Pharmacia Biotech Co., Ltd.) can be used.

[0010] In order to increase the recovery rate of nucleic acids in the purification of a small amount of nucleic acid sample, for example, 5 microliters of the Sanger reaction product during the filtration through the purification column 1, the Sanger reaction product is introduced into the purification column 1. In order to further push out the nucleic acid adsorbed on the gel filtration packing material 3, a needle (capillary tube) 7 connected to the inside of the syringe 5 and to the tip of the syringe 5
For example, 5 microliters of the clean water 9 is suctioned in advance.

After the tip of the needle 7 enters the Sanger reaction product 13 accommodated in the sample container 11, the piston of the syringe 5 is retracted, and the Sanger reaction product 13 is sucked into the needle 7 and the syringe 5. I do. One end 1 a of the purification column 1 is attached to the tip of the needle 7.

By moving the piston of the syringe 5 forward, the Sanger reaction product 13 is introduced into the purification column 1 and subsequently the clean water 9 is introduced into the purification column 1. The components of the Sanger reaction product 13 containing nucleic acids move inside the gel filtration packing material 3 toward the other end 1 b of the production column 1 at a speed according to the molecular weight. The other end 1 of the purification column 1 in order of nucleic acid having a large molecular weight.
b, the Sanger reaction product 13a discharged at the initial stage after desalting and removal of unreacted substances is collected at the other end 1b of the purification column 1. Sanger reaction product 13a
Is directly introduced into the sample injection portion of the fluorescent DNA sequencer, the analysis can be performed while suppressing the decrease in the amount of the Sanger reaction product 13a and the contamination.

In this embodiment, the present invention is applied to a fluorescent DNA sequencer. However, the present invention is not limited to this, and the present invention can be applied to other automatic analyzers and purification by manual operation. can do. Further, the material of the purification column 1 is not limited to glass, and may be another material. Further, in this embodiment, clean water is used as a solution for pushing out the nucleic acid adsorbed on the gel filtration packing material. However, the present invention is not limited to this, and another solution may be used. Further, the numerical values shown in the embodiments are merely examples, and the present invention is not limited to the numerical values, and various changes can be made within the scope of the present invention described in the claims.

[0014]

According to the purification column for nucleic acid samples of the present invention, a gel filtration filler is filled in a tube having both ends opened, so that the nucleic acid sample introduced into the purification column from one end of the purification column is purified by the purification column. And discharge from the other end.

In the method for purifying a nucleic acid sample according to the present invention,
A capillary containing a nucleic acid sample before purification is connected to one end of the purification column filled with the gel filtration packing material, and the nucleic acid sample before purification is introduced into the purification column from one end of the purification column, and the other end of the purification column is introduced from the other end of the purification column. Since the nucleic acid sample is purified by discharging, the nucleic acid sample can be purified by collecting the discharged nucleic acid sample before discharging impurities such as salts and primers having a low molecular weight from the other end of the purification column. ,
The purification operation can be simplified. Furthermore, by connecting the other end of the purification column to the analysis system, the purified nucleic acid sample can be introduced into the analysis system without separately collecting the purified nucleic acid sample in a container. Further, since steps such as centrifugation are not required, automation becomes easy.

In the method for purifying a nucleic acid sample according to the present invention, after introducing the nucleic acid sample before purification into the purification column, a solution for pushing out the nucleic acid is further introduced from one end of the purification column. The nucleic acid adsorbed on the filler can be further washed away, and the recovery rate of the nucleic acid can be increased. Further, by increasing the recovery rate of nucleic acids, it is possible to purify a small amount of nucleic acid samples.

[Brief description of the drawings]

FIG. 1 is a schematic view showing the steps of one embodiment of a purification column and one embodiment of a purification method.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Purification column 1a One end of purification column 1b The other end of purification column 3 Gel filtration packing material 5 Syringe 7 Needle 9 Clean water 11 Sample container 13 Sanger reaction product (nucleic acid sample) 13a Sanger reaction product after purification

 ────────────────────────────────────────────────── ─── Continued on the front page F term (reference) 2G052 AA24 AA28 AB20 AB27 AD06 AD26 AD46 BA02 CA04 CA20 EA01 ED07 GA11 GA22 4B024 AA11 AA20 CA01 HA19 4B029 AA23 4B063 QA01 QA13 QQ42 QQ52 QR55 QS12

Claims (3)

[Claims] 1. A purification column for a nucleic acid sample, wherein a gel filtration packing material is filled in a tube having both ends opened. 2. A capillary tube containing a nucleic acid sample before purification is connected to one end of a purification column filled with a gel filtration packing material, and the nucleic acid sample before purification is introduced into the purification column from one end of the purification column. Purifying the nucleic acid sample by discharging the nucleic acid sample from the other end of the purification column. 3. The purification method according to claim 1, wherein after introducing the nucleic acid sample before purification into the purification column, a solution for pushing out the nucleic acid is further introduced from one end of the purification column.