JP2001029070A - Separation of nucleic acid fragment and pre-treating device for nucleic acid sequence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating nucleic acid fragments, capable of recovering the nucleic acid fragments synthesized in the presence of a polymerase in a high efficiency, and also simply removing mingled nucleotides. SOLUTION: This method for separating nucleic acid fragments is a method for removing un-reacted nucleotides from the nucleic acid fragments obtained by adding a primer and a nucleotide to an aqueous solution containing a template nucleic acid and performing a chain extension reaction from the primer in the presence of a polymerase. A phenol reagent 3 is added to a container housing a reaction liquid 2 after completing the chain extension reaction and agitated. After the agitation, the agitated liquid is heated at 40-60 deg.C for separating it to an aqueous phase 5 and a phenol phase 6. In the aqueous phase 5, the objective nucleic acid fragments removed from the un-reacted nucleotides, is contained. By recovering the aqueous phase 5 and utilizing it for a nucleic acid analysis, it becomes possible to perform the nucleic acid analysis of a high accuracy without being inhibited by the un-reacted nucleotides.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a synthetic nucleic acid fragment, and more particularly to a method for removing unnecessary unreacted nucleotides from a synthesized nucleic acid fragment by adding nucleotides to a primer and extending the primer. In addition, the present invention relates to a nucleic acid sequence pretreatment method and apparatus using the purification method.

[0002]

2. Description of the Related Art In recent years, it has become essential to determine the base sequence of a gene in order to analyze the characteristics of the gene or the like. One of the methods for determining the nucleotide sequence of this gene or the like is the sanger method (also called the dideoxy method).

In this Sanga method, deoxyribonucleotides (dNTPs; dATP, dCTP, dGTP, dTT) are added to a reaction solution containing a template DNA and a primer complementary thereto.
4) to which P) was added, and dNTPs were added to these four.
Apart from each one of the dideoxyribonucleotides (ddNTPs), ddATP, ddCTP, dd
GTP or ddTTP is added, and an extension reaction is performed in the presence of a polymerase.

In this extension reaction, bases complementary to the base sequence of the template DNA are added one after another to the 3 ′ end of the primer to elongate the chain. When a dideoxyribonucleotide is added in the extension process, the next base is added. Unable to bind, chain elongation stops. Therefore, due to the difference in the position where the dideoxyribonucleotide was added, the template DNA was contained in each reaction solution.
A single-stranded nucleic acid fragment of various lengths complementary to is generated. Then, finally, each nucleic acid fragment in each reaction solution is fractionated by electrophoresis in parallel, whereby the base sequence of the template DNA is determined from this fractionation pattern (sequence ladder).

[0005] The Sanga method is widely used in terms of simplicity of operation and high analysis accuracy. In recent years, various sequence determiners and the like based on the Sanga method have been developed.

[0006]

In the above-mentioned sequence determination, it is required to be able to read a longer nucleic acid sequence. However, in the above-mentioned Sanga method, unreacted nucleotides (dexoxynucleotide, Deoxyribonucleotides) can interfere with reading the final electrophoresis pattern, making it difficult to read,
In some cases, the reading length is limited.
In particular, such a problem was remarkable in the internal labeling method and the terminator labeling method.

That is, in the Sanga method, it is necessary to visualize each fragment in order to finally perform electrophoresis and read the electrophoresis pattern. Fragment labeling is performed to perform this visualization. Labeling methods for this fragment include:
Broadly, there are three methods: internal labeling, terminator labeling, and primer labeling. In the primer labeling method, a primer used for synthesizing a fragment for sequencing is previously labeled with fluorescence or the like, and the fragment for sequencing is synthesized using the labeled primer.

[0008] On the other hand, in the internal labeling method and the terminator labeling method, the primer does not need to be particularly labeled, and a labeled nucleotide such as fluorescence is used as a nucleotide at the time of synthesis. By synthesizing a fragment using such labeled nucleotides, the synthesized sequencing fragments will be labeled as a result. In the internal labeling method and the terminator labeling method, unlike the primer labeling method, there is no need to label a primer or the like in advance, which is simple and any primer can be used. It is possible to freely determine a nucleic acid sequence using the method.

However, the internal labeling method and the terminator labeling method are simple methods as described above. However, when the nucleic acid fragments are finally fractionated and read by electrophoresis, unreacted amounts present together with the nucleic acid fragments are present in large amounts. May appear in the background, and reading of the electrophoresis pattern (sequence ladder) of the extended fragment around the labeled nucleotide may be hindered.

[0010] Therefore, in order to solve such a problem and to determine a base sequence with higher precision over a wider range, unreacted nucleotides have been removed after the extension reaction. Methods for removing the nucleotide include ethanol precipitation, a method using a purification column, and the like.

However, in ethanol precipitation, since the amount of the reaction solution is very small and the amount of nucleic acid contained therein is also very small, the recovery rate after ethanol precipitation is low and the recovery rate varies. There is a problem that it easily occurs. In particular, in the above sequence determination, it is necessary to determine four bases (A, G, C, T) at the same time. If this occurs, the sequence determination cannot be performed as a whole.

There is also a method using a purification column instead of ethanol precipitation. In this method, when eluting the nucleic acid adsorbed on the carrier, the nucleic acid is diluted by an elution buffer. . In addition, in order to concentrate the diluted nucleic acid solution, the ethanol precipitation must be performed, and the same problem as described above occurs. As described above, with the conventional method for removing unreacted nucleotides, it has been difficult to increase the efficiency of the subsequent sequencing reaction.

On the other hand, from the recent progress in human genome analysis,
In the future, it is expected that nucleic acid sequence analysis will be used for diagnosis of diseases caused by genes. When a sequence analysis is used for clinical diagnosis of such a disease, a simpler operation and high-precision analysis are required.

Accordingly, the present invention has been made in view of the above problems, and an object of the present invention is to provide a method for separating nucleic acid fragments that can remove mixed nucleotides by a simple operation. An object of the present invention is to provide a method and apparatus for pre-processing a sequence using the method.

[0015]

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted intensive studies, and as a result, after adding a phenol reagent to a reaction solution after nucleic acid synthesis and stirring,
By separating the aqueous phase and the phenol layer, it has been found that unreacted nucleotides can be removed from the aqueous phase containing the synthetic nucleic acid. Furthermore, they have found that by heating a mixed solution of a reaction solution and a phenol reagent, it is possible to easily separate an aqueous phase and a phenol layer, thereby completing the present invention.

That is, according to the method for separating nucleic acid fragments of the present invention, a nucleic acid fragment synthesized by adding primers and nucleotides to an aqueous solution containing a template nucleic acid and performing a chain extension reaction from the primers in the presence of a polymerase is used. A method for separating from a nucleotide, the reaction solution after the completion of the chain elongation reaction is mixed with a phenol reagent, a stirring step of stirring, and a heating step of heating a mixture of the reaction solution and the phenol reagent, Wherein the heating step separates the stirred mixture into an aqueous phase and a phenol layer, and the nucleic acid fragment is separated from unreacted nucleotides by collecting the aqueous phase. .

According to the invention, the mixed solution containing the phenol reagent and the reaction solution after the completion of the chain elongation reaction is stirred and heated to separate the mixed solution into an aqueous phase and a phenol layer. By recovering the aqueous phase, the nucleic acid fragment can be separated from unreacted nucleotides. As described above, in the present invention, the mixed liquid after the stirring can be separated into the aqueous phase and the phenol layer only by applying heat without using a device such as centrifugal separation after the stirring, thereby simplifying the operation. This makes it possible to process a large amount of samples.

According to the present invention, in the above-mentioned heating step,
The mixed solution is heated to 40 to 80 degrees. By heating in such a temperature range, the aqueous phase and the phenol layer can be effectively separated.

The present invention is characterized in that the heating step is performed after the completion of the stirring step. As described above, according to the present invention, by performing the heating step after the completion of the stirring step, the reaction liquid and the phenol reagent are sufficiently stirred in advance and then separated into an aqueous phase and a phenol layer by heating.
It is possible to efficiently separate unreacted nucleotides from nucleic acid fragments.

Further, the present invention is characterized in that the nucleic acid fragment is a sequencing fragment for determining a nucleic acid sequence, and the nucleotide is a deoxyribonucleotide or dideoxyribonucleotide.

That is, by using the above separation method to separate unreacted dideoxynucleotides and deoxynucleotides from a sequence fragment, unreacted nucleotides which have conventionally hindered sequence analysis can be used. Can be separated. As a result, according to the present invention, the efficiency of the sequence analysis is improved.

The nucleic acid sequence pretreatment device of the present invention comprises:
A nucleic acid sequence pretreatment device for adding a primer, deoxyribonucleotides, and dideoxyribonucleotides to an aqueous solution containing a template nucleic acid, and purifying a sequence fragment generated by performing a chain extension reaction from the primers in the presence of a polymerase, It is characterized by comprising a stirring means for mixing and stirring the reaction solution after the completion of the chain extension reaction with a phenol reagent, and a heating means for heating a mixture of the reaction solution and the phenol reagent.

According to the above invention, the mixture containing the phenol reagent and the reaction solution after the completion of the chain elongation reaction is stirred by the stirring means, and the stirred reaction liquid is heated by the heating means, and the mixed liquid is washed with water. Separate into phase and phenol layer. The aqueous phase separated here contains sequence fragments from which unreacted deoxynucleotides and the like have been removed.

As described above, according to the present invention, unreacted nucleotides, which have conventionally hindered sequence analysis, can be separated from sequencing fragments. In addition, in the present invention, since the aqueous phase and the phenol layer can be separated by the heating means, the unreacted nucleotides can be removed with a small device without a centrifuge that is easily increased in size. The purpose can be achieved.

[0025]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below with reference to the drawings.

[First Embodiment] The nucleic acid separation method of the present embodiment is produced by adding a primer and a nucleotide to an aqueous solution containing a template nucleic acid and performing a chain extension reaction from the primer in the presence of a polymerase. Used to separate nucleic acid fragments from unreacted nucleotides.

That is, DNA is artificially produced in a test tube or the like.
When performing the synthesis, the nucleotides (dN
Since TP) remains after the synthesis of the nucleic acid fragment and is mixed with the generated nucleic acid fragment, it is used to remove the mixed unreacted nucleotide. Therefore, the method for synthesizing the nucleic acid fragment to be separated and the purpose of use after the synthesis of the nucleic acid fragment are not limited as long as the nucleic acid fragment and the unreacted nucleotide are mixed after the synthesis.

Therefore, the above-mentioned “nucleic acid fragment” is a nucleic acid fragment synthesized from a template DNA for sequence determination by the Sanga method (hereinafter, referred to as a sequence fragment) or generated by PCR (polymerase chain reaction). Amplified nucleic acid fragments and the like are included, and as long as the nucleic acid fragments are artificially synthesized in the presence of polymerase and nucleotides in this way, the nucleic acid fragments may be single-stranded or double-stranded. The nucleic acid fragment may include not only DNA but also RNA.

The "nucleotide" to be removed is not limited to deoxyribonucleotide (dNTP) but may be dideoxyribonucleotide (ddNTP) as long as it is added at the time of nucleic acid fragment synthesis. The “nucleotide” also includes nucleotides and the like labeled with radiation or fluorescence.

Hereinafter, the separation method of the present embodiment will be described with reference to FIG. FIG. 1 shows an outline of the present nucleic acid separation operation.

First, a reaction solution 2 in which a synthetic nucleic acid synthesized in the presence of a polymerase and unreacted nucleotides are mixed is prepared in a container 1, and a phenol reagent 3 is added thereto. As the phenol reagent 3, pure phenol may be used, but preferably, neutral phenol, phenol-chloroform or the like can be used.

The neutral phenol is produced by injecting a buffer (eg, Tris EDTA buffer) into molten phenol to adjust the pH and saturate the phenol with the buffer. By using such a neutral phenol, nucleotides can be removed without changing the pH of the reaction solution after completion of synthesis and without changing the amount of the reaction solution.

Phenol-chloroform is prepared by mixing chloroform with the above neutral phenol. By adding chloroform in this manner, the difference in specific gravity between the aqueous solution as the reaction solution and the aqueous solution is increased, the separation ability between the aqueous phase and the phenol layer can be improved, and the phenol can be prevented from remaining in the aqueous phase. Can be.

In addition to these neutral phenols and phenol-chloroform, for example, substituted phenols and phenol resins may be used as long as they can remove nucleotides. Phenol is highly volatile and has an irritating odor, so using phenol resin instead of phenol instead of phenol with a substituted phenol or a phenol resin processed as a resin makes it environmentally friendly and easy to handle. This is advantageous in terms of.

After the addition of the phenol reagent, the reaction solution 2 and the phenol reagent 3 are stirred to form a suspension. This stirring operation may be performed using a mixer or the like, or may be performed by a pipetting operation in which suction and discharge are repeated with a pipette.

After the above stirring, the stirring liquid 4 is heated to separate the stirring liquid 4 into an aqueous phase and a phenol layer. The temperature at the time of this heating can be a temperature at which the stirring liquid can be separated into an aqueous phase and a phenol layer, for example, 40 ° C. or more. However, if the temperature is too high, the ability to remove unreacted nucleotides tends to decrease and tend to coexist in the aqueous phase. Therefore, the temperature at the time of heating is preferably set to an upper limit of 80 degrees, more preferably an upper limit. 60 degrees.

Although the heating time depends on the heating temperature, if the heating temperature is 40 to 60 ° C., the stirring liquid is maintained at the above temperature for about ten and several seconds, more certainly for about 30 seconds.

The heating operation is not necessarily started after the stirring step, but is started with or before the stirring step. When the stirring step is completed, the stirring liquid is brought into a state where the stirring liquid has reached a predetermined heating temperature. You can also. By starting the heating operation at the same time as or before the stirring step, the efficiency in terms of time is improved as compared to the case where the stirring step and the heating step are performed in two stages. It is also possible to aim.

When the above-mentioned heating step is completed, the liquid in the container 1 is separated into an aqueous phase 5 and a phenol layer 6, and an intermediate layer 7 is formed between the aqueous phase 5 and the phenol layer 6 in some cases. That is, the heating step makes it possible to easily separate the aqueous phase and the phenol layer without using a centrifuge or the like.

The aqueous phase 5 separated above contains the target nucleic acid fragment, and unreacted nucleotides to be removed are transferred to the intermediate layer 7 together with unnecessary proteins such as polymerase. Will do.
Therefore, finally, the nucleic acid fragment can be selectively recovered by recovering the aqueous phase 5, and the synthetic DNA
Unreacted nucleotides that make it difficult to read an electrophoretic image when analyzing by electrophoresis or the like are removed.
As a result, by performing analysis such as a sequence using the nucleic acid fragments in the aqueous phase 5, it is possible to improve analysis accuracy and the like.

[Second Embodiment] In this embodiment, a case will be described in which the method for separating nucleic acid fragments of the first embodiment is used for pretreatment of a nucleic acid sequence.

First, the synthesis of a sequence fragment to be separated in the present embodiment will be briefly described with reference to FIG.

As shown in FIG. 2A, a fragment for sequencing was prepared by using a template DNA,
Primer, dNTP (deoxynucleotide; a mixture of dATP, dCTP, dGTP, dTTP), a polymerase and a buffer are mixed, and this mixture is added to each of four wells 12 in a 96-well plate 10, for example. Is done. And these four holes 1
2, a different kind of dideoxynucleotide (any one of ddATP, ddCTP, ddGTP and ddTTP) is added.

The fragments for sequencing are labeled so that the fragments can be finally visualized. As the labeling method, either the internal labeling method or the end labeling method may be used. For example, in the internal labeling method, at least one type of base among the above-mentioned dNTPs is labeled with radiation or fluorescence. In the end labeling method, a primer or a terminator in which the above-mentioned primer or terminator is previously labeled with radiation or fluorescence is used.

After the reaction solution is prepared on the reaction plate 10 as described above, a DNA synthesis reaction is performed by a polymerase. In this DNA synthesis reaction, 3 ′ of the primer
Nucleotides complementary to the template DNA are successively incorporated into the ends, and the chain is extended. However, if a dideoxynucleotide without a hydroxyl group is incorporated at the 3 ′ end during the chain extension process, the next nucleotide cannot be bound and the chain extension is specifically stopped. Due to the difference in the position where the dideoxynucleotide was incorporated, as shown in FIG.
Within 2, nucleic acid fragments of various lengths are generated.

Next, a pretreatment is performed to remove nucleotides mixed in from the nucleic acid fragments generated here.

The pretreatment for removing the nucleotide is performed based on the nucleic acid separation method using a phenol reagent described in the first embodiment. Further, here, a case is shown in which this pretreatment is performed automatically by a nucleic acid sequence pretreatment device. 3 and 4 show a front view and a plan view of the nucleic acid sequence pretreatment device, respectively.

As shown in FIGS. 3 and 4, a preparation block 14 is provided on a substrate 13 in the apparatus. In the preparation block 14, a chip rack 16 and a reagent rack 18 containing a phenol reagent are arranged. I have. This reagent rack 1
Neutral phenol, phenol-chloroform, and the like can be used as the phenol reagent contained in 8, as in the first embodiment described above.

On the substrate 13, a stirring block 20 is provided adjacent to the preparation rack 14, and the plate 10 containing the reaction solution after the sequence reaction is placed on the stirring block 20. In the stirring block 20, a phenol reagent is added to a reaction solution in the plate 10 by a dispenser 24 described later, and stirring is performed here.
The stirring operation in the stirring block 20 can be performed by providing the stirring block with a mixer or the like. Here, the stirring is performed by a dispenser described later. The stirring operation will be described later in detail.

A heat block 22 is provided on the substrate 13 adjacent to the stirring block 20. In the heat block 22, the stirring liquid stirred in the stirring block 20 is transferred, and the stirring liquid is heated in this place to separate the stirring liquid into an aqueous phase and a phenol layer.

The temperature in the heat block 22 is:
The temperature is adjusted so as to be a temperature at which the stirring liquid can be separated into an aqueous phase and a phenol layer, for example, 40 to 80 degrees, preferably 40 to 60 degrees. In addition, a timer or the like is provided as needed to control the heating time. In order to separate the stirring liquid into the aqueous phase and the phenol layer, it is necessary to maintain the heating temperature for at least ten and several seconds, more surely for about 30 seconds. Therefore, by installing this timer, the heating operation can be reliably performed.

On the other hand, a dispenser 24 is provided above the substrate 13. The dispenser 24 is held on a guide rail 26 such that the tip thereof can be moved in the left-right direction toward the substrate 13 and can be moved up and down. Also, this guide rail 26
The body itself is supported by the fixed portion 28 so as to be slidable in the front-rear direction. For this reason, the dispenser 24 can be freely moved back and forth and right and left inside the apparatus, and
It is configured to be able to execute transfer of a reagent to the reaction solution, dispensing to a reaction solution, and a stirring operation described later.

Hereinafter, the operation of the pre-processing apparatus will be described with reference to FIG.
This will be described with reference to FIG.

The plate 10 containing the reaction solution after the completion of the sequence reaction is placed in the stirring block 20. The preparation block 14 includes the chip rack 1
6. The reagent rack 18 is placed. When these preparations are completed, the dispenser 24 moves the plate 10 on the stirring block.
The phenol reagent is transferred from the reagent rack 18 to the phenol reagent.

For the transfer of the reagent by the dispenser 24, first, the dispenser 24 is guided by the guide rail 26, placed on the chip rack 16 of the preparation block 14, and lowered to attach the chip 16a. ,To rise. Next, the phenol reagent is guided to the upper part of the reagent rack 18 and descends at that position to suck the phenol reagent. Dispenser 24 that aspirated reagent
Then, the phenol reagent moves to the top of the plate 10 to be dispensed in the stirring block and descends to dispense the sucked phenol reagent (S01).

When the dispensing of the reagent by the dispenser 24 is completed, the dispenser 24 aspirates the mixed solution obtained by mixing the reaction solution in the hole 12 of the plate 10 and the phenol reagent, as shown in FIG. The mixture is stirred by repeating the discharge (S
02).

Next, the stirred liquid mixture is transferred to the heat block 22 (S03). The transfer to the heat block 22 can be performed by using, for example, a dispenser 24. Alternatively, the plate 10 can be transferred to the heat block 22 using the arm means or the like not shown in FIGS.

The mixed solution transferred to the heat block 22 is heated at the above-mentioned predetermined heating temperature and separated into an aqueous phase and a phenol layer (S04). Then, the aqueous phase is collected (S05), and used for the subsequent sequence analysis. By separating the aqueous phase and the phenol layer by the heating operation in this way, the above-described separation operation can be performed without providing a device such as a centrifuge that tends to be large.

The aqueous phase obtained by the above series of pretreatment operations is subjected to electrophoresis as shown in FIG. 2D, and the sequence is determined from the migration pattern. In particular, in the present embodiment, since unreacted nucleotides that inhibit reading of the electrophoresis result by the pretreatment and further proteins are removed, the accuracy is high, and reading of a longer sequence length can be performed. .

Although the nucleic acid separation method of the first embodiment has been described as a pretreatment device used for pretreatment of a nucleic acid sequence, this device removes unreacted nucleotides of a nucleic acid amplification device such as PCR. It can also be used as a device.

In this embodiment, the stirring block 20
Although the heat block 22 and the heat block 22 are provided separately, these blocks may be integrated. In this case, while performing the stirring operation of the reaction solution and the phenol reagent, the temperature is maintained at room temperature or the like, and after the stirring, the temperature of the block is increased.
The mixture can be heated and separated into an aqueous phase and a phenol layer.

[0062]

EXAMPLES The nucleic acid separation method of the present invention will be described in more detail with reference to Examples. Here, a case where the present method is used for a nucleic acid sequence pretreatment method is shown.

1. Synthesis of Fragment for Sequencing Four mixed solutions in which a template DNA, a primer, a fluorescently labeled dNTP, a polymerase, and a buffer were mixed were prepared, and one different ddNTP was added thereto. A plurality of this set was prepared, and a polymerase reaction was performed on each of the sets to generate a fragment for sequencing.

2. Pretreatment Pretreatment was performed on the set of sequencing fragments generated above to remove nucleotides. As the pretreatment method, the pretreatment with the phenol reagent according to the present example was performed.

In the pretreatment with the phenol reagent, the same amount of neutral phenol is added to the reaction solution after the above synthesis reaction,
After stirring, the mixture was heated to 50 ° C. and separated into an aqueous phase and a phenol layer. The aqueous phase was collected and used for the following sequence determination.

At the same time, a sample which had been subjected to conventional ethanol treatment and a column purification treatment and a sample which had not been treated were prepared as comparative controls. The pretreatment by ethanol precipitation as a comparative control utilized a normal ethanol precipitation method. That is, 100% ethanol was added to the above reaction solution, left on ice, and then centrifuged to collect the sediment, and the sediment was suspended in a buffer.

Similarly, in the treatment using a column as another comparative control, the above reaction solution was diluted to about 50 μl, and the QIA qu
The eluate was added to an ick column (QIAGEN Nucleotide Removal Kit), and after a certain period of time, the eluate was collected. The ethanol precipitation was performed to concentrate the eluate.

3. Sequence determination process The sequence determination is performed using an automatic sequencer (LICOR Mode
l 4200 DNA sequencer). The sample solutions pretreated by the various methods described above were respectively injected into wells of an electrophoresis gel of a sequencer, electrophoresis was started, and an electrophoresis pattern (sequence ladder) was recorded. In addition, in FIG.
The migration pattern around 300 bp is shown.

In FIG. 7, the electrophoresis pattern (sample 1) of the untreated reaction solution shown at the left end of the drawing is 1
Around ３００300 bp, a plurality of dumpling-like signals due to unreacted nucleotides appeared, indicating that it was extremely difficult to read surrounding bands. Although not shown in FIG. 7, in the portion of 300 bp or more, although no signal due to untreated nucleotides does not appear, each band becomes an image with a tail, and in the portion where the bands are close to each other, it is difficult to read due to overlapping. there were.

In the pretreatment by the ethanol treatment (samples 4 and 5), the signal due to unreacted nucleotides decreased, but did not completely disappear. In addition, the signal intensity of the ladder-like band as a whole also decreased, and the signal intensity was uneven in each nucleotide (lane), resulting in a narrower range in which the sequence could be determined. In addition, pretreatment using a column (sample 6,
In 7), the recovery rate of the fragment after the treatment was reduced, and the signal intensity of each band was significantly reduced as compared with the pretreatment by the ethanol treatment. In addition, it is difficult to determine the sequence because the signal intensity is uneven in each nucleotide (lane).

On the other hand, in the treatment using the phenol reagent of this example (samples 2 and 3), the signal due to unreacted nucleotides in the vicinity of 1 to 300 bp completely disappeared, and the resolution of each band was reduced. An improved and clear ladder band was obtained. In addition, the signal intensity of each band was uniform, and the rate of decrease was within a range that would not interfere with band reading, indicating that the pretreatment was very good.

In the untreated electrophoresis pattern, unreacted nucleotides usually appear at the leading end of electrophoresis, but in the results shown in FIG. 7, they appeared in a cluster around 200 bp. This suggests that unreacted nucleotides and proteins such as polymerases are clustered. Therefore, it is also shown that the phenol treatment of this example can remove nucleotides and proteins simultaneously by one operation.

[0073]

As described above, according to the present invention, unreacted nucleotides can be removed without losing the synthesized nucleic acid fragments, so that nucleic acid analysis such as high-precision sequence analysis can be performed. And In particular, in the present invention,
The target nucleic acid fragment can be recovered by a simple operation without performing a cumbersome operation such as centrifugation, which is advantageous for processing a large number of samples.

[Brief description of the drawings]

FIG. 1 is a diagram schematically illustrating a nucleic acid fragment separation method according to a first embodiment.

FIG. 2 is a diagram showing a series of steps of a sequence determination operation including a nucleic acid sequence pretreatment method according to a second embodiment.

FIG. 3 is a front view of a nucleic acid sequence pretreatment device according to a second embodiment.

FIG. 4 is a plan view of a nucleic acid sequence pretreatment device according to a second embodiment.

FIG. 5 is a diagram showing a stirring operation in a nucleic acid sequence pretreatment device according to a second embodiment.

FIG. 6 is a process chart showing the operation of the nucleic acid sequence pretreatment device according to the second embodiment.

FIG. 7 is a view showing a ladder band around 1 to 300 bp when a sample after performing each pretreatment operation in the example is subjected to electrophoresis.

[Explanation of symbols]

2 reaction solution, 3 phenol reagent, 5 aqueous phase, 6 phenol layer, 10 reaction plate, 14 preparation block,
16 chip racks, 18 reagent racks, 20 stirring blocks, 22 heat blocks, 24 dispensers.

Claims (6)

[Claims] 1. A method for separating a synthesized nucleic acid fragment from unreacted nucleotides by adding a primer and a nucleotide to an aqueous solution containing a template nucleic acid and performing a chain extension reaction from the primer in the presence of a polymerase, A stirring step of mixing and stirring the reaction solution after completion of the chain extension reaction with the phenol reagent, and a heating step of heating a mixture of the reaction solution and the phenol reagent; A method for separating nucleic acid fragments, comprising separating a mixture of a liquid and a phenol reagent into an aqueous phase and a phenol layer, and collecting the aqueous phase to separate the nucleic acid fragments from unreacted nucleotides. 2. The method according to claim 1, wherein the heating is performed in the heating step.
The method for isolating a nucleic acid fragment according to claim 1, wherein the method is heated to 0 to 80 degrees. 3. The method for separating nucleic acid fragments according to claim 1, wherein the heating step is performed after the stirring step. 4. The nucleic acid fragment according to claim 1, wherein the nucleic acid fragment is a sequencing fragment for determining a nucleic acid sequence, and the nucleotide is a deoxyribonucleotide or a dideoxyribonucleotide. Separation method. 5. The method for separating nucleic acid fragments according to claim 4, wherein the deoxyribonucleotide or dideoxyribonucleotide is labeled. 6. A nucleic acid sequence pretreatment in which a primer, deoxyribonucleotide and dideoxyribonucleotide are added to an aqueous solution containing a template nucleic acid, and a chain extension reaction is performed from the primer in the presence of a polymerase to purify a fragment for sequencing generated. An apparatus, comprising: a stirring means for mixing and stirring the reaction solution after the completion of the chain extension reaction with a phenol reagent; and a heating means for heating a mixed solution of the reaction solution and the phenol reagent. Characteristic nucleic acid sequence pretreatment device.