JP3640840B2 - Nucleic acid fragment separation method and nucleic acid sequence pretreatment apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
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 nucleic acid fragment synthesized by adding a nucleotide to a primer and extending it. The present invention also relates to a nucleic acid sequence pretreatment method and apparatus using this purification method.
[0002]
[Prior art]
In recent years, in order to analyze the characteristics of a gene or the like, it has become indispensable to determine the base sequence of the gene. One of the methods for determining the base sequence of this gene or the like is the sanger method (also called the dideoxy method).
[0003]
In this Sangha method, four reaction solutions containing template DNA and a primer complementary to deoxyribonucleotide (dNTP; dATP, dCTP, dCTP, dGTP, dTTP) are prepared, and each of these four is separate from dNTP. A kind of dideoxyribonucleotide (ddNTP), that is, ddATP, ddCTP, ddGTP or ddTTP is added, and the extension reaction is carried out in the presence of a polymerase.
[0004]
In this extension reaction, bases complementary to the base sequence of the template DNA are successively added to the 3 ′ end of the primer to extend the strand, but when dideoxyribonucleotides are added during this extension process, the next base cannot be bound. Chain extension stops. Accordingly, single-stranded nucleic acid fragments of various lengths that are complementary to the template DNA are generated in each reaction solution from the difference in position where the dideoxyribonucleotide is added. 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]
This Sangha method is widely used from the viewpoint of easy operation and high analysis accuracy. In recent years, various sequence determiners based on this Sangha method have been developed.
[0006]
[Problems to be solved by the invention]
In the above sequence determination, it is required to be able to read a longer nucleic acid sequence, but in the Sangha method, unreacted nucleotides (deoxynucleotides, dideoxyribonucleotides) In some cases, it becomes an obstacle in reading the electrophoretic pattern, making it difficult to read, and limiting the length of reading. In particular, such a problem is remarkable in the internal labeling method and the terminator labeling method.
[0007]
That is, in the Sangha method, it is necessary to visualize each fragment in order to finally perform electrophoresis and read the electrophoresis pattern. Fragment labeling is performed to achieve this visualization. This fragment labeling method is roughly divided into three methods: an internal labeling method, a terminator labeling method, and a primer labeling method. In the primer labeling method, a primer used for synthesizing a sequence fragment is labeled with fluorescence or the like in advance, and the sequence fragment 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 those labeled with fluorescence or the like are used as nucleotides as substrates for synthesis. By synthesizing the fragments using the nucleotides thus labeled, the synthesized sequence fragments are labeled as a result. This internal labeling method and terminator labeling method are simple because there is no need to label the primer in advance as in the primer labeling method, and since any primer can be used, a primer suitable for the nucleic acid region to be determined is used. It is possible to freely determine the nucleic acid sequence by using it.
[0009]
However, the internal labeling method and the terminator labeling method are simple methods as described above, but unreacted labeled nucleotides that are present in a large amount together with the nucleic acid fragments when the nucleic acid fragments are finally fractionated and read by electrophoresis. Appeared in the background, and reading of the electrophoretic pattern (sequence ladder) of the extension fragments in the vicinity thereof was sometimes disturbed.
[0010]
Therefore, in order to solve such a problem and determine a base sequence with high accuracy over a wider range, removal of unreacted nucleotides is performed after the extension reaction. Methods for removing this nucleotide include ethanol precipitation, a method using a purification column, and the like.
[0011]
However, in ethanol precipitation, the amount of reaction solution is very small, and the amount of nucleic acid contained therein is also very small, so the recovery rate after ethanol precipitation is low and the recovery rate is likely to vary. There's a problem. In particular, since it is necessary to determine four bases (A, G, C, T) at the same time in the above sequence determination, any one of these bases significantly reduces the sample during the processing. When this occurs, the sequence cannot be determined as a whole.
[0012]
In addition, there is a method using a purification column instead of ethanol precipitation. In this method, the nucleic acid is diluted with an elution buffer when the nucleic acid adsorbed on the carrier is eluted. 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. Thus, in the conventional method for removing unreacted nucleotides, it has been difficult to improve the efficiency of the subsequent sequencing reaction.
[0013]
On the other hand, due to recent progress in human genome analysis, nucleic acid sequence analysis is expected to be used for diagnosis of diseases caused by genes. When sequence analysis is used for clinical diagnosis of such diseases, analysis with a simpler operation and higher accuracy is required.
[0014]
Therefore, 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 a nucleic acid fragment that can remove nucleotides mixed in by a simple operation, and further to use this separation method. It is an object of the present invention to provide a method and apparatus for pre-processing a sequence.
[0015]
[Means for Solving the Problems]
In order to achieve the above object, the inventors of the present application conducted intensive research, and as a result, added a phenol reagent to the reaction solution after nucleic acid synthesis, stirred, and then separated into an aqueous phase and a phenol layer, It has been found that unreacted nucleotides can be removed from the aqueous phase containing synthetic nucleic acids. Furthermore, it has been found that the aqueous phase and the phenol layer can be easily separated by heating the mixed solution of the reaction solution and the phenol reagent, and the present invention has been completed.
[0016]
That is, the method for separating a nucleic acid fragment of the present invention separates a nucleic acid fragment synthesized from an unreacted nucleotide 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, and a heating step of heating the mixed solution of the reaction solution and the phenol reagent, In the heating step, the mixture is heated to 40 to 80 degrees, In the heating step, the stirred mixture is separated into an aqueous phase and a phenol layer, and the aqueous phase is recovered to separate the nucleic acid fragments from unreacted nucleotides.
[0017]
According to the said invention, the liquid mixture containing the reaction liquid after completion | finish of chain | strand elongation reaction and a phenol reagent is stirred and heated, and a liquid mixture is isolate | separated into an aqueous phase and a phenol layer. And among these, a nucleic acid fragment can be isolate | separated from an unreacted nucleotide by collect | recovering an aqueous phase. As described above, in the present invention, the mixed liquid after stirring can be separated into the aqueous phase and the phenol layer only by applying heat without using an apparatus such as a centrifugal separator after the stirring, thereby simplifying the operation. It is possible to process a large amount of samples.
[0018]
The present invention This By heating in such a temperature range, the aqueous phase and the phenol layer can be effectively separated.
[0019]
The present invention is characterized in that the heating step is performed after completion of the stirring step. Thus, according to the present invention, by performing the heating step after the stirring step is completed, the reaction solution and the phenol reagent are sufficiently stirred in advance and then separated into the aqueous phase and the phenol layer by heating. It becomes possible to efficiently separate unreacted nucleotides from the nucleic acid fragment.
[0020]
Further, the present invention is characterized in that the nucleic acid fragment is a sequence fragment for nucleic acid sequencing, and the nucleotide is deoxyribonucleotide or dideoxyribonucleotide.
[0021]
That is, the separation method is used to separate unreacted dideoxynucleotides and deoxynucleotides from the sequence fragments, thereby separating the unreacted nucleotides that previously caused the sequence analysis from being hindered from the sequence fragments. It becomes possible. As a result, according to the present invention, the efficiency of sequence analysis can be improved.
[0022]
The nucleic acid sequence pretreatment apparatus of the present invention purifies a sequence fragment generated by adding a primer, deoxyribonucleotide and dideoxyribonucleotide 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 pretreatment apparatus for mixing nucleic acid sequence, wherein the reaction solution after completion of the chain extension reaction is mixed with a phenol reagent, a stirring means for stirring, and a mixed solution of the reaction solution and the phenol reagent 40 to 80 degrees And heating means for heating.
[0023]
According to the above invention, the mixture containing the reaction solution after completion of the chain extension reaction and the phenol reagent is stirred by the stirring means, the reaction solution stirred by the heating means is heated, and the mixture is mixed with the aqueous phase and phenol. Separated into layers. The aqueous phase separated here contains sequence fragments from which unreacted deoxynucleotides and the like have been removed.
[0024]
As described above, according to the present invention, unreacted nucleotides that have conventionally been an obstacle to sequence analysis can be separated from the sequence fragments. In addition, in the present invention, since the aqueous phase and the phenol layer can be separated by the heating means, the unreacted nucleotide is removed with a small apparatus even without a centrifuge that is easily increased in size. The objective can be achieved.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
Preferred embodiments of the present invention will be described below with reference to the drawings.
[0026]
[First embodiment]
In the nucleic acid separation method of this embodiment, a primer and a nucleotide are added to an aqueous solution containing a template nucleic acid, and a nucleic acid fragment produced by performing a chain extension reaction from the primer in the presence of a polymerase is separated from unreacted nucleotides. Used when.
[0027]
That is, when DNA is artificially synthesized in a test tube or the like, excessively added nucleotides (dNTPs) remain after the synthesis of the nucleic acid fragments and are mixed with the generated nucleic acid fragments. Used to remove intermixed unreacted nucleotides. Therefore, as long as nucleic acid fragments and unreacted nucleotides are mixed after synthesis in this way, the method for synthesizing nucleic acid fragments to be separated and the purpose of use after nucleic acid fragment synthesis are not limited.
[0028]
Therefore, the above-mentioned “nucleic acid fragment” includes a nucleic acid fragment synthesized from a template DNA (hereinafter referred to as a sequence fragment) for determining a sequence by the Sangha method, or an amplified nucleic acid fragment generated by PCR (polymerase chain reaction). The nucleic acid fragment is single-stranded or double-stranded, as long as it is a nucleic acid fragment artificially synthesized in the presence of polymerase and nucleotides. In addition, the nucleic acid fragment is not limited to DNA but can also include RNA.
[0029]
The “nucleotide” to be removed is not limited to deoxyribonucleotide (dNTP) as long as it is added at the time of nucleic acid fragment synthesis, and also includes dideoxyribonucleotide (ddNTP) and the like. The “nucleotide” includes nucleotides labeled with radiation or fluorescence.
[0030]
Hereinafter, the separation method of this embodiment will be described with reference to FIG. FIG. 1 shows an outline of the nucleic acid separation operation.
[0031]
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. The phenol reagent 3 may be pure phenol, but preferably, neutral phenol, phenol-chloroform, or the like can be used.
[0032]
This neutral phenol is produced by injecting a buffer solution (for example, Tris EDTA buffer solution) into melted phenol to adjust the pH and saturate the phenol with the buffer solution. 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.
[0033]
Phenol-chloroform is prepared by mixing chloroform with the above neutral phenol. By adding chloroform in this way, the difference in specific gravity with the aqueous solution that is the reaction solution is increased, the separation ability between the aqueous phase and the phenol layer can be improved, and phenol remains in the aqueous phase. Can do.
[0034]
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. Therefore, instead of phenol, the use of a substituted phenol in which a substituent is inserted in phenol or a phenol resin processed as a resin makes it environmentally friendly and easy to handle. This is advantageous.
[0035]
After adding 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.
[0036]
After the 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 heating can be set to a temperature at which the stirring liquid can be separated into the aqueous phase and the phenol layer, for example, 40 degrees or more. However, if the temperature is too high, the ability to remove unreacted nucleotides tends to be reduced and mixed in the aqueous phase. Therefore, the temperature during heating is preferably 80 ° C., more preferably the upper limit. 60 degrees.
[0037]
Although the heating time depends on the heating temperature, if the heating temperature is 40 to 60 ° C., the stirring liquid is held at the above temperature for about 10 seconds or more, more reliably for about 30 seconds.
[0038]
The heating operation is not necessarily started after the stirring step, but may be started together with or before the stirring step, and at the end of the stirring step, the stirring liquid may reach a predetermined heating temperature. it can. Thus, by starting the heating operation simultaneously with or before the stirring process, efficiency is improved in terms of time compared to the case where the stirring process and the heating process are performed in two stages. It is also possible to plan.
[0039]
When the heating step is completed, the liquid in the container 1 is separated into the aqueous phase 5 and the phenol layer 6, and an intermediate layer 7 is formed between them, which may not be recognized with the naked eye. In other words, this heating step makes it possible to easily separate the aqueous phase and the phenol layer without using a centrifuge or the like.
[0040]
The aqueous phase 5 separated in the 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. Become. Therefore, finally, by recovering the aqueous phase 5, the nucleic acid fragments can be selectively recovered, and it is difficult to read the electrophoretic image when analyzing the synthetic DNA by electrophoresis or the like. The nucleotide of the reaction is removed. As a result, it is possible to improve the analysis accuracy by analyzing the sequence using the nucleic acid fragment in the aqueous phase 5.
[0041]
[Second Embodiment]
In the present embodiment, a case will be described in which the nucleic acid fragment separation method of the first embodiment is used for pretreatment of a nucleic acid sequence.
[0042]
First, synthesis of a sequence fragment to be separated in this embodiment will be briefly described with reference to FIG.
[0043]
As shown in FIG. 2 (A), the sequence fragment comprises, as starting materials, template DNA, a primer complementary to the template DNA, dNTP (deoxynucleotide; mixture of dATP, dCTP, dGTP, dTTP), and further a polymerase and a buffer. Are mixed, and this mixed solution is added to each of the four holes 12 in the 96-hole plate 10, for example. Further, one different kind of dideoxynucleotide (any of ddATP, ddCTP, ddGTP, and ddTTP) is added to each of the four holes 12.
[0044]
The sequencing fragment is labeled so that the fragment 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 base of the dNTPs labeled with radiation or fluorescence is used. In the end labeling method, a primer or terminator previously labeled with radiation or fluorescence is used.
[0045]
In the above, after the reaction solution is prepared on the reaction plate 10, a DNA synthesis reaction by a polymerase is performed. In this DNA synthesis reaction, nucleotides complementary to the template DNA are successively incorporated at the 3 ′ end of the primer, and the chain is extended. However, if a dideoxynucleotide without a hydroxyl group is incorporated at the 3 ′ end during this chain extension process, the next nucleotide cannot be bound and chain extension specifically stops. Thus, due to the difference in position where the dideoxynucleotide is incorporated, nucleic acid fragments of various lengths are generated in each hole 12 as shown in FIG. 2 (B).
[0046]
Next, a pretreatment is performed in order to remove the mixed nucleotides from the nucleic acid fragment generated here.
[0047]
This pretreatment for removing nucleotides is performed based on the nucleic acid separation method using the phenol reagent shown in the first embodiment. Furthermore, here, a case where this pretreatment is performed automatically by a nucleic acid sequence pretreatment apparatus is shown. 3 and 4 respectively show a front view and a plan view of the nucleic acid sequence pretreatment apparatus.
[0048]
As shown in FIGS. 3 and 4, a preparation block 14 is provided on a substrate 13 in the apparatus, and a chip rack 16 and a reagent rack 18 containing a phenol reagent are arranged in the preparation block 14. As a phenol reagent accommodated in this reagent rack 18, neutral phenol, phenol-chloroform, etc. can be used similarly to 1st embodiment mentioned above.
[0049]
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 the reaction solution in the plate 10 by a dispenser 24 described later, and stirring is performed here. In addition, although stirring operation in this stirring block 20 can also be performed by providing a mixer etc. in this stirring block, it is supposed that stirring is implemented by the dispenser mentioned later here. The stirring operation will be described in detail later.
[0050]
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.
[0051]
The temperature in the heat block 22 is adjusted to a temperature at which the stirring liquid can be separated into the aqueous phase and the phenol layer, for example, 40 degrees to 80 degrees, preferably 40 degrees to 60 degrees. Moreover, a timer etc. are provided as needed, and heating time is controlled. In order to separate the stirring liquid into the aqueous phase and the phenol layer, it is necessary to hold at the heating temperature for at least ten seconds or more, more certainly about 30 seconds. Therefore, the heating operation can be reliably performed by installing this timer.
[0052]
On the other hand, a dispenser 24 is provided above the substrate 13. The dispenser 24 is held by the guide rail 26 so as to be movable up and down so that the tip thereof faces the substrate 13 and can be moved in the left-right direction. Further, both ends of the guide rail 26 itself are supported by the fixing portion 28 so as to be slidable in the front-rear direction. Therefore, the dispenser 24 is configured to be able to move back and forth and right and left freely within the apparatus, and is configured to be able to perform transfer of the reagent to the stirring rack 20, dispensing to the reaction liquid, and stirring operation described later.
[0053]
Hereinafter, the operation of the pre-processing apparatus will be described with reference to FIG.
[0054]
The plate 10 containing the reaction solution for which the sequence reaction has been completed is disposed in the stirring block 20. A tip rack 16 and a reagent rack 18 are placed on the preparation block 14. When these preparations are completed, the dispenser 24 transfers the phenol reagent from the reagent rack 18 to the plate 10 on the stirring block.
[0055]
As for the transfer of the reagent by the dispenser 24, first, the dispenser 24 is guided by the guide rail 26 and arranged on the tip rack 16 of the preparation block 14, and is lowered to attach the tip 16a and ascend. . Next, it is guided to the top of the reagent rack 18 and descends at that position to aspirate the phenol reagent. The dispenser 24 that has aspirated the reagent then moves to the top of the plate 10 where the stirring block is dispensed and descends to dispense the aspirated phenol reagent (S01).
[0056]
When the dispensing of the reagent is completed by the dispenser 24, the dispenser 24 sucks and discharges the mixed solution in which the reaction solution in the hole 12 of the plate 10 and the phenol reagent are mixed as shown in FIG. The mixture is repeatedly stirred (S02).
[0057]
The stirred mixed liquid is then transferred to the heat block 22 (S03). The transfer to the heat block 22 can also be performed using a dispenser 24, for example. 3 and 4 can be transferred to the heat block 22 as it is with the plate 10 using arm means or the like not shown.
[0058]
The mixed liquid transferred to the heat block 22 is heated at the predetermined heating temperature and separated into an aqueous phase and a phenol layer (S04). Of these, the aqueous phase is recovered (S05) and used for subsequent sequence analysis. Thus, by separating into an aqueous phase and a phenol layer by heating operation, the said separation operation can be performed, without providing the apparatus which tends to enlarge like a centrifuge.
[0059]
As shown in FIG. 2D, the aqueous phase obtained by the series of pretreatment operations is subjected to electrophoresis, and the sequence is determined from the electrophoresis pattern. In particular, in this embodiment, unreacted nucleotides and proteins that inhibit reading of the electrophoresis result are removed by the pretreatment, so that it is possible to read a longer sequence length with high accuracy. .
[0060]
Here, the nucleic acid separation method of the first embodiment has been described as a pretreatment device that uses nucleic acid sequence pretreatment, but this device is used as a device that removes unreacted nucleotides in a nucleic acid amplification device such as a PCR. You can also
[0061]
Moreover, although the stirring block 20 and the heat block 22 were provided separately in this embodiment, these blocks can also be integrated. In this case, during the stirring operation of the reaction solution and the phenol reagent, the temperature is kept at room temperature or the like, the temperature of the block is increased after the stirring is completed, the mixed solution is heated, and the aqueous phase and the phenol layer are mixed. Can be separated.
[0062]
【Example】
The nucleic acid separation method of the present invention will be described in more detail using examples. Here, the case where this method is used for the nucleic acid sequence pretreatment method is shown.
[0063]
1. Sequence fragment synthesis
Four mixed solutions were prepared by mixing template DNA, primer, fluorescently labeled dNTP, polymerase, and buffer, and one different kind of ddNTP was added thereto. A plurality of sets were prepared, and each was subjected to a polymerase reaction to generate a sequence fragment.
[0064]
2. Preprocessing
The set of sequencing fragments generated above was pretreated to remove nucleotides. As the pretreatment method, pretreatment with a phenol reagent according to this example was performed.
[0065]
In the pretreatment with a phenol reagent, the same amount of neutral phenol was added to the reaction solution after the above synthesis reaction, and after stirring, the mixture was heated to 50 ° C. and separated into an aqueous phase and a phenol layer. Of these, the aqueous phase was recovered and used for the following sequence determination.
[0066]
At the same time, as a comparative control, a conventional ethanol treatment, a column purified treatment, and an untreated one were prepared. The pretreatment by ethanol precipitation, which is a comparative control, utilized a normal ethanol precipitation method. That is, 100% ethanol was added to the reaction solution, left on ice, then centrifuged, the sediment was collected, and the sediment was suspended in a buffer solution.
[0067]
Similarly, in the treatment using the other comparison column, the reaction solution is diluted to about 50 μl and added to the QIA quick column (QIAGEN Nucleotide Removal Kit), and the eluate after a certain period of time is recovered. In order to concentrate the recovered liquid, the ethanol precipitation treatment was performed.
[0068]
3. Sequence determination process
The sequence was determined using an automatic sequencer (LICOR Model 4200 DNA sequencer). The sample solutions pretreated by various methods in the above were injected into the wells of the electrophoresis gel of the sequencer, the electrophoresis was started, and the electrophoresis pattern (sequence ladder) was recorded. In addition, in FIG. 7, the electrophoresis pattern of 1-300 bp vicinity is shown from a primer.
[0069]
In FIG. 7, in the migration pattern of the untreated reaction solution (sample 1) shown at the left end of the drawing, a plurality of unreacted nucleotide-like signals appear in the vicinity of 1 to 300 bp, and reading of the surrounding bands Has been shown to be extremely difficult. In addition, although not shown in FIG. 7, a signal due to unprocessed nucleotides does not appear at a portion of 300 bp or more, but each band becomes an image with a tail and overlapped at a portion where the bands are close to each other, making it difficult to read. there were.
[0070]
In addition, in the pretreatment with 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 not uniform in each nucleotide (lane), resulting in a narrower range where the sequence could be determined. In addition, in the pretreatment using the column (samples 6 and 7), the recovery rate of the fragments after the treatment was lowered and the signal intensity of each band was significantly reduced as compared with the pretreatment by ethanol treatment. In addition, it is difficult to determine the sequence because the signal intensity is not uniform in each nucleotide (lane).
[0071]
In contrast, 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 disappeared completely, and the resolution of each band was improved and clear. A rudder band was obtained. In addition, the signal intensity of each band was uniform, and the rate of decrease was within a range that did not hinder the reading of the band, indicating that the pretreatment was very good.
[0072]
In the untreated migration pattern, normally unreacted nucleotides appear at the tip of the migration, but in the results shown in FIG. 7, they appeared in the form of dumplings around 200 bp. This is expected to be a cluster of unreacted nucleotides and proteins such as polymerase. Therefore, the phenol treatment of this example also shows that nucleotides and proteins can be removed simultaneously by one operation.
[0073]
【The invention's effect】
As described above, according to the present invention, it is possible to remove unreacted nucleotides without losing the synthesized nucleic acid fragment, thereby enabling highly accurate nucleic acid analysis such as sequence analysis. 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 amount of samples.
[Brief description of the drawings]
FIG. 1 is a diagram showing an outline of a method for separating nucleic acid fragments of a first embodiment.
FIG. 2 is a diagram showing a series of steps of a sequence determination operation including the nucleic acid sequence pretreatment method of the second embodiment.
FIG. 3 is a front view of a nucleic acid sequence pretreatment apparatus according to a second embodiment.
FIG. 4 is a plan view of a nucleic acid sequence pretreatment apparatus according to a second embodiment.
FIG. 5 is a diagram showing an agitation operation in the nucleic acid sequence pretreatment apparatus according to the second embodiment.
FIG. 6 is a process diagram showing the operation of the nucleic acid sequence pretreatment apparatus according to the second embodiment.
FIG. 7 is a diagram showing a ladder band near 1 to 300 bp when a sample after performing each pretreatment operation in an example is subjected to electrophoresis.
[Explanation of symbols]
2 reaction liquid, 3 phenol reagent, 5 aqueous phase, 6 phenol layer, 10 reaction plate, 14 preparation block, 16 chip rack, 18 reagent rack, 20 stirring block, 22 heat block, 24 dispenser.

Claims (5)

A method of separating a nucleic acid fragment synthesized from an unreacted nucleotide by adding a primer and a nucleotide to an aqueous solution containing a template nucleic acid and causing 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 a phenol reagent;
Heating a mixed solution of the reaction solution and the phenol reagent,
In the heating step, the mixed solution is heated to 40 to 80 degrees, the mixed solution of the reaction solution and the phenol reagent is separated into an aqueous phase and a phenol layer by the heating step, and the aqueous phase is A method for separating nucleic acid fragments, wherein the nucleic acid fragments are separated from unreacted nucleotides by recovery. The method for separating nucleic acid fragments according to claim 1, wherein the heating step is performed after the stirring step. The method for separating nucleic acid fragments according to claim 1 or 2 , wherein the nucleic acid fragment is a sequence fragment for nucleic acid sequencing, and the nucleotide is deoxyribonucleotide or dideoxyribonucleotide. The method for separating nucleic acid fragments according to claim 3 , wherein the deoxyribonucleotide or dideoxyribonucleotide is labeled. A nucleic acid sequence pretreatment apparatus for purifying a sequence fragment generated by adding a primer, deoxyribonucleotide and dideoxyribonucleotide 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 means for mixing and stirring the reaction solution after completion of the chain extension reaction with a phenol reagent;
A pretreatment apparatus for nucleic acid sequence, comprising: a heating means for heating the mixed solution of the reaction solution and the phenol reagent to 40 to 80 degrees .

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