JP4408290B2 - Sample analysis method, electrophoresis apparatus, and method for readjusting nucleic acid fragment concentration - Google Patents

Description

  The present invention relates to a method and an analysis apparatus for conducting analysis by electrically introducing a charged sample, for example, a method and apparatus for analyzing a sample by electrophoresis.

  Analysis of a charged biological sample such as nucleic acid is performed using gel electrophoresis, capillary electrophoresis, or the like.

  The method for adjusting the concentration of the sample was performed as follows.

  For example, if the charged sample is expected to be outside the measurement range due to its high concentration, the sample is diluted to a concentration recommended in the instruction manual of the analyzer. In general, there are the following two dilution methods.

  In one method, a stock solution of a sample is collected and diluted with a solvent used for dilution (hereinafter also referred to as “dilution solvent” or “dilution solution”). The diluted sample solution is collected and used for analysis (known example 1).

  The other is to dilute the sample stock solution directly by adding a dilution solvent (known example 2).

  In addition, when analyzing a sample by electrophoresis, a technique for injecting a chelating agent into a solution containing the sample has been proposed for the purpose of capturing (chelating) the metal ions contained in the sample. .

  For example, in JP-A-2-38966, a chelating agent is added to a terminal liquid when analyzing metal ions having hydrating properties contained in industrial wastewater by isotachophoresis. In this case, the chelating agent reacts with the metal ions of the sample to form a strong complex of reverse polarity ions (metal anions). That is, electrophoresis is made possible by converting metal ions into metal anions having no hydratability.

In JP-A-9-201200, a chelating agent is used in order to prevent excess Mg ⁺⁺ from binding to nucleic acid when nucleic acid such as DNA is analyzed by capillary electrophoresis. In this case, the chelating agent is added to the denaturing solvent and acts as a magnesium chelator that reacts with Mg ⁺⁺ contained in the solvent.

  In JP-A-2001-242139, the storage stability of the electrophoresis gel is improved by including a chelating agent in the buffer for electrophoresis used for electrophoresis of DNA.

  As typical chelating agents, ethylenediaminetetraacetic acid (EDTA), ethylene glycol bistetraacetic acid (EGTA) and the like are disclosed.

  In the conventional electrical sample introduction method, as described above, the sample concentration in the solution is adjusted with the diluent to control the amount of sample introduced into the analyzer, but this process takes time.

  For example, in the case of the known example 1 described above, it takes time to dispense and dilute the sample during the initial adjustment or readjustment of the sample.

  In the case of the known example 2, if the first analysis result is below the lower limit of the measurement range (the amount of sample introduced into the analyzer is small), a mixed solution (a mixture of the sample stock solution and the diluted solution) is used. Although it is necessary to readjust, since the stock solution itself was diluted from the beginning, it was necessary to procure the stock solution again and add it to the previously used mixed solution. When the measurement range is equal to or greater than the upper limit (when the amount of sample introduced into the analyzer is too large), it is necessary to dilute after discarding some of the mixture.

  Furthermore, when automating the analyzer including the sample preparation as described above, a mechanism for performing dilution and its space are required.

JP-A-2-38966

Japanese Patent Laid-Open No. 9-201200 JP 2001-242139 A

  The present invention provides a sample analysis method and apparatus that eliminates the need for a diluent when adjusting a sample in analysis of a biological sample and the like, and can simplify the adjustment of the amount of sample introduced into the analyzer. is there.

The present invention, in order to solve the above problems, the charged specimen (for example in the case of nucleic acids, negative ions are a) when performing electrical introduce and analyze the analyzer, sample solution containing the sample The amount of the sample introduced into the analyzer is adjusted by adding a substance (ionic substance) having the same charge as (1) and adjusting the amount of the substance having the charge.

For example, when the sample is electrically introduced by electrophoresis, a substance having the same charge as the sample in the stock solution (ex. PCR product if the sample is a nucleic acid) is added to the solution (solvent). .
The charged substance has a function of suppressing the amount of sample introduced into the analyzer. Although the mechanism of the suppression of the introduction amount has not been fully clarified theoretically, the addition of a charged substance (ionic substance) causes a change in the conductivity in the solution containing the sample, and the electrical introduction. It is presumed to be due to a phenomenon such as a so-called matrix effect in which the amount (for example, the amount of electrophoresis) decreases. Such a phenomenon was originally discussed as a phenomenon caused by undetectable ionic substances (coexisting substances such as sodium ions and chloride ions) originally contained in the solution, which reduces the measurement sensitivity of the analysis. It was recognized as a factor. Therefore, in the past, devoted to eliminating the effect, was not considered to use it, but in the present invention, paying attention to this phenomenon, positively charged substances are added The idea has been reached, thereby enabling an electrical concentration adjustment to replace the sample dilution.

  For example, in the first sample analysis, if the analysis result exceeds the upper limit of the measurement range (when the amount of sample introduced into the analyzer is too large), a substance for adjusting the sample introduction amount (has a charge) Add a larger amount of substance) (readjust the sample). Thus, in the second sample analysis, the amount of sample introduced into the analyzer is reduced, which is substantially the same as when the sample is diluted, and analysis within the measurement range is possible.

  Conversely, if the analysis result of the first sample analysis is below the lower limit of the measurement range (when the amount of sample introduced into the analyzer is too small), an appropriate amount of sample in the solution (mixture) containing the sample Add the stock solution.

  Examples of the charged substance used for adjusting the sample concentration (including the first adjustment and the second and subsequent readjustments) include, for example, ethylenediaminetetraacetic acid (EDTA), ethylene glycol when the sample is a nucleic acid. It has been found that bistetraacetic acid (EGTA) and the like are preferable. Nucleic acid, EDTA, and EGTA are ionic substances (anions) of the same polarity. EDTA, EGTA and the like are conventionally used as chelating agents for various purposes (for example, JP-A-2-38966, JP-A-9-201200, JP-A-2001-242139 described above), The technical idea used to electrically adjust the concentration of a sample as in the present invention is not known.

  In addition, as for the electrical introduction of the sample in the present invention, capillary electrophoresis is typically exemplified, and examples of the charged substance include those described above, but are not limited thereto.

According to the present invention, by adding a very small amount of liquid (a sample stock solution or a substance having a charge), the sample concentration in the sample solution and thus the amount of electrophoresis can be greatly adjusted. Further, the adjustment can be performed only by the procedure of adding the liquid to the micro container (without performing fractionation or dilution), and thereby the adjustment of the sample concentration and the amount of electrophoresis becomes simple.

FIG. 1 is a diagram showing a sample adjustment step of sample analysis in the present invention . FIG . 2 shows the analysis process . FIG . 3 is a diagram showing a reanalysis process . FIG . 4 is a plan view showing an apparatus configuration . FIG. 5 is a diagram showing the operation of the apparatus during sample adjustment, analysis, and reanalysis . FIG. 6 is a flowchart showing the operation of the automatic analyzer of FIG .

  Hereinafter, the best mode of the present invention will be described.

  As an apparatus for electrically introducing a sample, for example, there is an electrophoresis apparatus. It is used in a sample introduction part of a gene analyzer that determines the base sequence of a nucleic acid for gene analysis. Furthermore, in addition to nucleic acids, it is used for sample introduction of various substances such as metal ions and proteins. Examples of the electrophoresis system include a capillary electrophoresis system and a CE / MS system in which a capillary and a mass spectrometry system are combined. However, any other apparatus for introducing a sample may be used as long as it can embody the present invention, and is not limited to the electrophoresis apparatus as described above.

In the case of capillary electrophoresis, the capillary is filled with a sample separation medium (for example, electrophoresis solution, polymer, gel, etc.), and one end of the capillary is immersed in a sample solution for analysis filled in a micro container, A sample is introduced into the capillary by immersing the other end in an electrolyte solution (buffer solution) and applying a pulse voltage to both ends of the capillary. Specifically, when the sample has a positive charge, a pulse voltage is applied for a certain period of time with one end of the capillary (sample solution side) as a cathode and the other end (buffer solution) as an anod. Conversely, when the sample has a negative charge, the polarity at both ends of the capillary is reversed from the above and a pulse voltage is applied for a certain period of time. Thereby, the sample can be introduced into the capillary which is the migration path.
Further, the amount of sample introduction can be adjusted by the magnitude of the voltage applied to both ends of the capillary and the length of time. The larger the applied voltage and the longer the applied time, the greater the amount of sample introduced.

  However, if the application time is too long, there may be a problem that the separation performance deteriorates, and it is difficult to set the sample introduction conditions only by changing the applied voltage magnitude or application time for each sample. is there. The capillary electrophoresis apparatus achieves higher sensitivity than the gel plate electrophoresis apparatus, but has an upper limit in the measurement range. For this reason, in many cases of analyzing a sample, it is necessary to dilute the sample so that the amount of sample introduced (electrophoresis amount) is less than the upper limit of the measurement range. In actual analysis, it is necessary to set a reliable lower limit of the measurement range due to variations in measurement values caused by the magnitude of baseline noise.

  In the present invention, when adjusting the amount of introduction of the sample as described above, basically, a charged substance is added without using a diluent. Specific examples thereof will be described later.

  Examples of the analysis method using an apparatus for electrically introducing a sample include a nucleobase sequencing method and a single-stranded DNA conformation polymorphism analysis method (SSCP electrophoresis method). It is not limited to the analysis method as described above. The nucleic acid base sequence determination method is a method for determining four types of bases constituting an analysis nucleic acid by electrophoresis. Single-stranded DNA higher-order structure polymorphism analysis method uses nucleic acid fragments that have different structures because they have partially different sequences as samples for analysis. This is a method of determination by electrophoresis.

  The sample introduction adjustment method according to the present invention is performed by the procedure shown in FIG. The procedure is roughly divided into an initial adjustment step (a specific example is shown in FIG. 2) of adding a charged substance as a sample introduction adjustment substance to the sample solution and adjusting the amount of introduction of the sample. An analysis step (a specific example is shown in FIG. 3) for introducing a sample into a capillary by applying a pulse voltage and measuring the amount introduced, and if the analyzed sample introduction amount is outside the measurement range, It includes a readjustment step (a specific example is shown in FIG. 4) for adjusting the sample introduction amount by adding a charged substance or sample stock solution based on the introduction amount.

  In the sample adjustment step, a charged substance and a solution containing the sample are placed in a container holding the electrophoresis solvent, and the amount of the sample introduced is adjusted to be within a detectable measurement range.

  Specifically, first, the approximate concentration of the target sample stock solution is measured. Examples of this measuring method include a UV measuring method and a fluorescence measuring method if the sample is a nucleic acid. However, the present invention is not limited to these examples, and any method may be used as long as the concentration of the sample (stock solution) can be measured. If it is expected from the result of this concentration measurement that the sample stock solution will exceed the measurement range of the analyzer, a substance having an appropriate amount of charge is added to the prepared mixed solution of the analytical solvent and the sample.

  As a specific example, when a single-stranded DNA conformation polymorphism analysis is performed on a PCR product using a 16-capillary electrophoresis apparatus, 5 μl of the PCR product stock solution is used on a 7.5% polyacrylamide gel. Electrophoresis is carried out at a voltage of 100 V and a time of 50 V, stained with ethylene bromide, and colored with UV (ultraviolet light), so that the approximate concentration of DNA is known from the luminance of the color. For samples that are expected to exceed the measurement range of the electrophoresis apparatus from the approximate concentration obtained from this, after preparing the sample solution for analysis using formamide as a solvent (specific examples are The amount of the sample introduced is adjusted by appropriately adding 100 mM EDTA, which will be described later with reference to FIG. The adjustment of the sample introduction amount is not limited to the above method, and any method may be used as long as a substance having the same charge as the sample can be added to the mixed solution of the sample.

  The charged substance in the present invention is a substance that increases the ionic strength of the sample solution for analysis, and preferably has the same charge as the sample and is not detected by the analyzer or does not enter the detection region of the sample. is there. For example, when the sample is a nucleic acid, the nucleic acid has a negative charge, and therefore negatively charged EDTA and EGTA are also suitable. Any substance having an electric charge may be used as long as it does not affect the analysis of the sample.

  The sample used for the analysis may be a sample having an electric charge that can be introduced by the electric sample introduction method, and the type thereof is not limited. Examples include nucleic acids extracted from cells and microorganisms, nucleic acid fragments amplified by PCR, synthetic nucleic acids, amino acids, peptides, proteins, and the like. More specifically, it is a substance having a positive or negative charge on the base, molecule, or amino acid structure. In the case of amphoteric electrolytes such as amino acids, peptides, and proteins, since they have an isoelectric point, it is possible to adjust the positive / negative ion concentration in the solvent by adjusting the pH (hydrogen ion concentration) of the solvent. However, since the charge of the amphoteric electrolyte is 0 at the isoelectric point, it is necessary to adjust the pH to a value other than the isoelectric point.

  The sample solution (solvent / solute) for electrophoresis to be prepared for analysis can be used with any solvent / solute as long as electrical sample introduction is possible. For example, when a single-stranded DNA conformation polymorphism analysis is performed on a PCR product using a 16-capillary electrophoresis apparatus, as shown in FIG. Formidid may use 34 μl per sample, and in some cases, 1 μl of TAMRA Size Standard (ABI) may be used as a size standard sample.

  The container used for analysis may have any material and size as long as the material of the container does not elute into the sample solution. A polypropylene tube or 96-well plate having a volume of about 200 μl is preferable.

  Next, an initial sample introduction amount adjustment procedure (adjustment procedure performed in FIG. 2) will be described. When a certain amount (for example, several μl) of a charged substance that is a sample introduction adjusting substance is added to the sample solution, the substance having that charge is adjusted in advance from high to low concentration according to rank. Have it ready. Next, a sample solution for electrophoresis is prepared by mixing 1 μl of a solvent for electrophoresis (Formamid including a size marker) and a stock solution of the sample. If the concentration of the sample solution is high (as described above, for example, it is estimated that the concentration of the sample solution for analysis is high or low from the approximate concentration measurement of the sample stock solution). Add a certain amount of substance (substance with charge). Conversely, when the concentration of the sample solution is not so high, a certain amount of a substance having a low concentration of charge is added. As a specific example, when the sample is a nucleic acid fragment amplified by PCR, 100 mM, 50 mM, or 25 mM EDTA is used as a substance having a charge with a concentration of multiple ranks. Add 1 μl of a stock solution of amplified nucleic acid by PCR to a solvent for electrophoresis. If this stock solution has a high concentration, a fixed amount of adjusted 100 mM EDTA is added (see FIG. 2). If the concentration is low but the measurement range is likely to be exceeded, a fixed amount of 25 mM EDTA may be added. In this case, if the rate of decrease in the amount of sample introduced by each adjusted concentration of EDTA is known, the amount of sample introduced can be adjusted by adding a certain amount and concentration of EDTA to the sample concentration. This adjustment method is not limited to the above-described method as long as a substance having a charge for sample introduction adjustment is mixed with the sample solution finally adjusted for analysis.

  For example, the substance added to adjust the sample introduction amount (substance with electric charge: here also referred to as the sample introduction adjustment substance) is made to have a constant concentration, and the constant concentration is added to the sample solution. Water may be added so that the necessary amount is variably added according to the sample concentration, and the total amount of the solution after adjustment is constant in consideration of the variable amount. In this way, it is not necessary to prepare the sample introduction adjusting substance in advance by dividing it into a plurality of concentration ranks.

  When adding a necessary amount of the sample introduction adjusting substance having a constant concentration, as a procedure, first, a sample introduction adjusting substance having a constant concentration is prepared in advance. Next, a sample solution is prepared by mixing the solvent for electrophoresis and the stock solution of the sample. Then, an appropriate amount of the sample introduction adjusting substance (substance having the same charge as that of the sample) is added to the solution of each sample in accordance with the concentration of the stock solution that has been roughly measured in advance. In this case, each sample subjected to analysis has a different volume in a strict sense. In such a sample introduction and adjustment method, as described above, it is not necessary to prepare a plurality of concentrations of the sample introduction and adjustment substance, so that it does not take time and effort. In this case, it is more preferable that the sample introduction adjusting substance has a high concentration in order to suppress fluctuations in the total amount.

  The analysis step shown in FIGS. 1 and 3 is a step of introducing a sample into an analyzer, performing electrophoresis, and measuring a waveform or the like obtained as a result. Hereinafter, this analysis process will be described in detail. In this step, the sample may be electrically introduced into the analyzer and is not limited to the specific examples described later.

  In this example, after setting the sample holding container adjusted for analysis in the analyzer, the sample introduction port of the analyzer is inserted into the sample holding container, and a pulse voltage is applied to the sample introduction part and the lead-out part. Samples are introduced by electrophoresis. More specifically, when a 16-capillary electrophoresis apparatus is used, a 200 μl capacity tube (container) dedicated to the electrophoresis apparatus holding a sample prepared for analysis is used as a sample plate on which 96 tubes can be set. Put it on. The application of the pulse voltage is, for example, 20 kV in voltage and 5 seconds in time through a program for controlling the analyzer. By this procedure, the sample is electrophoresed and introduced into the capillary.

  The introduced sample is preliminarily provided with a marker, and sample analysis is performed by measuring it with a detector. As a specific sample measurement method, a method for measuring loss of heterozygosity (hereinafter referred to as “LOH”) using a single-stranded DNA conformation polymorphism analysis method will be described. The measurement method varies depending on the purpose of analysis and is not limited to LOH. In this example, one or two peaks can be obtained as a result of electrophoresis of the sample. In the LOH measurement method, the heights of two peaks are measured, the ratio is calculated, and the reduction rate of one peak is calculated.

  When the measured value of the sample is out of the measurement range, a readjustment step for the sample solution is required. Hereinafter, an example of this readjustment process will be described in detail with reference to FIG. In the readjustment step, it is only necessary to adjust the amount introduced into the analyzer by adding the sample stock solution or the sample preparation substance (substance with charge) to the sample outside the measurement range, and is not limited to the specific examples described later.

  Outside the measurement range means that the sample concentration is higher or lower than the measurement range. Specifically, when the peak height ratio is measured using a 16-capillary electrophoresis apparatus, the measurement range is 4000 counts to 65000 counts. This value is specific to the device used, and is not limited to this value. If one of the two peaks exceeds the upper limit of the measurement range of 65000, the analysis cannot be performed because the exact height ratio of the two peaks cannot be calculated. On the other hand, when the height of both peaks is 4000 or less, the measurement error due to the influence of the baseline noise becomes large, so that reliable measurement becomes impossible.

  When the measurement result is optimal, that is, when the measurement value of the sample is within the measurement range, the measurement is completed for the sample. When measuring the peak height ratio or area ratio as described above, both or one of the two peaks does not exceed the upper limit of the measurement range, and both of the two peaks are within the measurement range. When the lower limit is not exceeded, the measurement is complete.

  When the amount of sample introduced into the analyzer is large, that is, when the sample concentration is higher than the measurement range, a very small amount of a substance having the same charge as the high concentration sample is added. For example, when the peak height ratio is measured using a 16-capillary electrophoresis apparatus, since the sample concentration is high, when the peak height exceeds the measurement range, the approximate peak height from the peak width at the upper limit of the measurement range. Guess. It is not necessary to dilute the sample based on the peak height predicted at this time, and an appropriate amount of EDTA having an appropriate concentration (for example, 100 mM) is added.

  Conversely, when the amount of sample introduced is small, that is, when the sample concentration is lower than the measurement range, a very small amount of the sample stock solution is added. For example, since the sample concentration is low, when the peak height falls below the lower limit of the measurement range, the peak height is measured, and an appropriate amount of sample stock solution is added.

  As described above, the description has been given based on the peak height, but the same can be applied to the case where the peak area is used.

  An automatic analyzer including the sample preparation of the present invention is shown in FIG. FIG. 5 is a plan view of the automatic analyzer. This apparatus performs the operation based on the flowchart shown in FIG. 6, that is, the sample adjustment process, the analysis process, and the reanalysis process fully automatically. The operation of this device will be described below. If this device includes the operation of adding a charged substance instead of the operation of injecting the diluent when the sample is prepared (initial adjustment, readjustment). Well, it is not limited to the following examples.

  As shown in FIG. 5, the sample adjustment unit 14 in the sample analyzer 10 includes a sample stock solution holding unit 11 for holding a sample stock solution, and reagents necessary for adjusting the sample for analysis (a high level for sample introduction adjustment). A reagent holding unit 12 that holds a solution of a substance having a charge of concentration), and a sample solution holding unit 13 that holds a solution (sample solution) in which the sample stock solution and the reagent are mixed.

  The sample solution is adjusted by controlling the operation of the sample dispensing arm 15 in accordance with the flowchart of FIG. This operation control is performed by controlling the arm drive mechanism 16 by the control unit 19. As the concentration measurement sensor 17, for example, an optical system is used (for example, a UV measurement device or a fluorescence measurement device), and is arranged on the side of the sample adjustment unit 14. With such a sensor arrangement, the concentrations of the sample stock solution set in the holding unit 11 and the sample solution for analysis set in the holding unit 13 can be easily detected at the side position of the sample adjusting unit.

  The flowchart of FIG. 6 is an automated analysis process from the adjustment process shown in FIGS. 1 to 4 described above.

  After adjusting the sample solution for the first time (steps S1 to S3 in FIG. 6), the control unit 19 controls the application of a pulse voltage to both ends of the capillary (not shown), the sample is introduced into the capillary, and electrophoresis is performed. (Steps S4 to S6).

  Steps S1 to S3 are steps for measuring the concentration of a sample stock solution (stock solution sample), adjusting a sample solution (such as a solvent and a sample stock solution) (sample adjustment), and adding a sample introduction amount adjusting substance.

  Steps S4 to S6 are a step of moving the analysis sample (sample solution) after the initial adjustment from the adjustment unit 14 to the detection unit 18 via a transport mechanism (not shown), and then a pulse for introducing the sample into the capillary. It shows a sample introduction process for applying a voltage, and a process for performing electrophoresis of a sample.

  The sample separated by electrophoresis is detected by a measuring instrument (for example, a UV measuring instrument, a fluorescence measuring instrument, etc.) of the detecting unit 18, and the detection result is input to the control unit (analyzing unit) 19 for data analysis (analysis). (Step S9). The sample container subjected to the analysis is returned from the detection unit 18 to a predetermined position of the sample adjustment unit 10 after the sample is collected (step S7).

  In step 9, while the operator looks at the monitor or the analysis software of the control unit 19 automatically analyzes the sample, it is determined whether the analysis is completed or needs to be reanalyzed. Completion of analysis is executed when the detection (analysis) result of the sample is within the range. The need for reanalysis is executed when the sample detection (analysis result) is outside the measurement range.

  If it is determined that the analysis is to be reanalyzed, the addition amount of the sample introduction amount adjusting substance (substance with charge) or sample stock solution is calculated according to the analyzed sample introduction amount (sample measurement concentration) (step S10). ). When the sample introduction amount is excessive, an appropriate amount of a charged substance solution is added. When the sample introduction amount is too small, the sample is readjusted by adding an appropriate amount of the stock solution sample (step S11). ). After the readjustment, the process returns to step S4, and the analysis process similar to the above is performed.

  Next, examples of the present invention will be described. The present invention is not limited to these.

In Example 1, when preparing a sample introduction amount adjustment substance (substance with charge) divided into a plurality of concentration ranks and adjusting the sample introduction amount, select one of the adjustment substances. In this case, a certain amount is added to the sample solution.
1. Determination of measurement range

Since an analyzer has an upper limit and a lower limit as a measurement range, it is necessary to determine a reliable measurement range for each analyzer. For example, in the case of the 16-capillary electrophoresis apparatus used in the present invention, the sample concentration was about 0.2 μmol / l at a voltage of 20 kV and a time of 5 seconds as introduction conditions, and the upper limit was 65,000 counts in the measured value. The lower limit was 30 fmol / l for the analytical sample concentration and 10 counts for the measured value under the introduction conditions described above, and measurement below that was difficult. However, when analyzing the height and area of the peak obtained, it is necessary to set a reliable value for the lower limit in consideration of the influence of the noise of the baseline, and the measured value serving as the lower limit is set to 4000 counts or more.
2. Preparation of sample stock solution

The sample stock solution used in this experiment was generated in the following process. Specifically, 1.0 pM of each primer was added to 0.1 μg of genomic nucleic acid (template) extracted from a biological sample, 10 nM nucleotide triphosphate (dNTP), 10 μM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.001% (w / v) gelatin, and 1.25 unit Taq nucleic acid polymerase (Perkin Elmer) were added to make a total volume of 25 μl. Then, this solution was subjected to PCR under the following conditions. PCR amplification conditions were as follows: initial denaturation at 95 ° C for 12 minutes, followed by 35 cycles of 95 ° C for 30 seconds, 57 ° C for 80 seconds, and 72 ° C for 30 seconds, followed by extension at 72 ° C for 7 minutes The reaction conditions were set.
3. Preparation of sample solution for analysis

  In the preparation of the sample solution for analysis, the reagent and the sample are added in the following procedure, but the order of addition is not limited to this example. To a micro container for analysis, 1.0 μl of 4 nM TAMRA size standard (ABI) as a size standard marker was added to 34 μl of Formamid, a DNA denaturant, and 1.0 μl of a stock solution of DNA amplification product was added. Seven different concentrations of EDTA solutions (sample introduction preparation materials: materials having the same polarity of charge as the sample) shown in the results were prepared, and equal amounts (8 μl) were added to adjust the total amount to 44 μl.

  3. SSCP electrophoresis and sample analysis results

  The adjusted sample was subjected to SSCP electrophoresis at a voltage of 20 kV for 5 seconds as a sample introduction condition and at a voltage of 15 kV for a duration of 70 minutes as an electrophoresis condition. Table 1 shows the peak height of the sample and the peak reduction rate due to the EDTA concentration change.

From these results, it can be seen that the rate of peak reduction increases in proportion to the added EDTA concentration. That is, by preparing sample introduction preparation substances having different concentrations and adding a solution of the substance having an appropriate concentration among them, the sample introduction amount can be adjusted. For example, when the concentration of the sample stock solution is predicted to be about twice the measurement range (about 120,000 counts) by examining the concentration of the sample, the effective measurement range is 4000 to 65000 in this SSCP electrophoresis analysis. Therefore, the sample can be measured within the measurement range by adding 8 μl of EDTA of 12.5 mM to 100 mM.
5). Re-analysis of samples outside the measurement range

  In this SSCP electrophoretic analysis, an effective measurement range is 4000 to 65000. Therefore, in this table, No. 5 and No. Since the peak height of 6 is lower than the lower limit of the measurement range, reanalysis is required. At this time, the former (No. 5) should be added 2 times or more of the sample stock solution, and the latter (No. 6) should be added 4 times or more of the sample stock solution. In addition, reanalysis was performed under the same conditions. The results are shown in Table 2.

  From the results in this table, this method does not dilute the sample stock solution, and therefore, if the peak is low, it can be reanalyzed by adding a small amount of the sample stock solution. For this reason, it is possible to shorten the time, labor, and cost of readjusting the stock solution compared to the case of using it after diluting directly without holding the stock solution.

In Example 2, when adjusting the sample introduction amount, the concentration of the adjustment substance (substance with charge) is made constant, and a necessary amount of the adjustment substance is added by changing the amount according to the measured concentration of the sample.
1. Preparation of sample solution for analysis

Here, the reagent and the sample are added in the following order, but the order of addition is not limited to this example. To a micro container for analysis, 34 μl of Formid, a DNA denaturant, 1.0 μl of 4 nM TAMRA size standard (ABI) as a size standard marker, 1.0 μl of a stock solution of DNA amplification product, and 100 mM A solution of the sample preparation material (a material having the same polarity as the sample) was added according to the sample concentration.
2. Sample introduction and analysis by electrophoresis

  The prepared sample solution was subjected to SSCP electrophoresis at a voltage of 20 kV for 5 seconds as a sample introduction condition and at a voltage of 15 kV for a duration of 70 minutes as an electrophoresis condition. Table 3 shows the peak height of the sample at this time.

From the results in this table, it can be seen that the peak height can be kept within the measurement range by increasing the amount of high concentration EDTA added. That is, the amount of introduction can be adjusted by preparing a solution of a substance having a high concentration of charge and adding an appropriate amount. For example, when the concentration of the sample stock solution is predicted to be about twice the measurement range (about 120,000 counts) by examining the concentration of the sample, the effective measurement range is 4000 to 65000 in this SSCP electrophoresis analysis. Therefore, the sample can be measured within the measurement range only by adding 2 μl of 100 mM EDTA.
3. Re-analysis of samples outside the measurement range

  In this SSCP electrophoretic analysis, an effective measurement range is 4000 to 65000. Therefore, in this table, No. The peak height of No. 1 is lower than the measurement range. Since the peak height of 7 is higher than the measurement range, reanalysis is required. At this time, since the former only needs to be added twice or more of the sample stock solution, 2 μl of the retained sample stock solution is added, and in the latter, 4 μl of 100 mM EDTA is added to reduce the peak height to ¼ or less. Analysis was performed. The results are shown in Table 4.

  From the results in this table, this method retains the sample stock solution, so if the peak is low, it can be reanalyzed by adding a small amount of sample stock solution, and if the peak is high, a substance with the same charge as the sample can be added. Reanalysis can be performed by adding a small amount. For this reason, it became possible to shorten the dilution process in reanalysis.

The effect of the present embodiment is as follows.
(1) By adding a very small amount of liquid (sample stock solution or substance having electric charge), the amount introduced can be greatly adjusted.
(2) The adjustment can be made only by the procedure of adding the liquid to the micro container (without performing the fractionation and dilution), and the adjustment of the introduction amount becomes simple.
(3) Even when many samples are prepared at the same time, the dilution process is shortened, so that it can be processed in a short time.
(4) In addition, when this adjustment method is applied to an automatic analyzer including sample adjustment, it is possible to omit operations and spaces necessary for the dilution process.
(5) In addition, when reanalysis is performed because the measurement result is out of the measurement range, only the operation of adding the sample stock solution or the charged substance solution is performed without performing the operation of dilution or concentration of the sample. It is possible to adjust the sample introduction amount within the measurement range.

10: Sample analyzer, 14: Sample adjustment unit.

Claims (18)

A method of conducting an analysis by electrically introducing a charged sample into an electrophoresis apparatus, wherein a substance having the same charge as the sample is added to a solution containing the sample, and the amount of the substance having the charge added the by adjusting to adjust the electrical levels of the sample analysis method of the sample and adjusts the electrophoresis of the sample. According to claim 1, when the electrophoretic amount of the sample is large, re-adjust the electrical levels of the sample by increasing the material having the same charge as the sample, the this readjustment Sample analysis method that reduces the amount of electrophoresis . According to claim 1, when the electrophoretic amount of the sample is large, the solution of the sample was re-adjusted by extending the material having the same charge as the sample, if the electrophoretic small amount Is a sample analysis method for adjusting the amount of electrophoresis by re-adjusting the sample solution by increasing the amount of sample stock solution. According to claim 1, when analysis by the analysis of the first sample is a concentration higher than the upper limit of the measurement range, by adding the substance having the same charge as the sample solution of the sample , A sample analysis method for performing reanalysis by reducing the amount of electrophoresis . In claim 1, a solution of a substance having a pre-Symbol conductive load is more prepared by concentration, when the measured value of the sample exceeds the upper limit of the measurement range, the sample of the preferred concentration depending on the measured value A method for analyzing a sample, wherein a solution of the substance having the same charge as that of the sample is selected and added to the sample solution. In claim 1, a solution of a substance having a pre-Symbol conductive load is of a fixed concentration are prepared, when the measured value of the sample exceeds the upper limit of the measurement range, and the sample according to the measurement value method for analyzing a sample added to the sample solution by changing the amount of solution of the substance having the same charge. 2. The electrophoresis amount according to claim 1, wherein an approximate concentration of the sample stock solution is measured at the time of adjusting the sample solution for mixing the solvent and the sample stock solution, and the concentration of the adjusted sample solution is estimated from the concentration of the stock solution. determination and if it is determined in large, an adjustment step of adding the substance having the same charge as the sample to the sample solution, the result of the analysis of the adjusted sample solution, and the electrophoretic amount is still large method for analyzing a sample and a step of the material to readjust in addition to the sample solution having the same charge as the sample when. The sample analysis method according to claim 1, wherein the electrophoresis apparatus is a capillary electrophoresis apparatus.   2. The method for analyzing a sample according to claim 1, wherein the sample is a biological sample such as a nucleic acid. 2. The method for analyzing a sample according to claim 1, wherein the substance having the same charge as the sample is ethylenediaminetetraacetic acid or ethylene glycol bistetraacetic acid. A sample introduction unit for introducing the charged sample electrophoretic the analysis section, the electrophoretic amount by the addition of substances having the same electric charge as the sample solution of the sample to adjust the concentration of the sample electrophoresis apparatus characterized by the adjustment portion for adjusting includes. 12. The electrophoresis apparatus according to claim 11 , further comprising a calculation unit that measures a sample concentration and calculates an addition amount of the substance having the same charge as the sample based on the measurement result. In claim 11, the sample concentration was measured and an arithmetic unit for the addition of the substance having the same charge as the sample based on the measurement result to calculate the amount and the determination of whether or not it is necessary, before Symbol tone-save, based the results of the calculation, the electrophoresis apparatus having a function of adding the optimum amount of the substance having the same charge as the sample solution of the sample. According to claim 11, when the concentration of the sample exceeds the upper limit of the measurement range, if the material having the same charge as the sample was added to a solution of the sample, the lower limit of the measurement range, the sample An electrophoresis apparatus for adding a stock solution to the sample solution. From a sample solution adjusted nuclear acid fragment concentration, the migration path of the electrophoresis apparatus, the nucleic acid fragments is introduced by electrophoresis, nucleic acid fragments to electrophoresis analysis,
A method of re-adjusting the nucleic acid fragment concentration by adding a negatively charged nucleic acid fragment concentration adjusting substance to the sample solution when the electrophoretic analysis value exceeds the upper limit of the measurement range . In claim 15,
A method of readjusting a nucleic acid fragment concentration in which the negatively charged sample preparation substance contains EDTA and / or EGTA. In claim 15,
How electrophoresis analysis of the nucleic acid fragments when exceeding the upper limit of the measurement range of the electrophoresis apparatus, to readjust the nucleic acid fragment concentration the sample adjusting material of the negative charges in addition to the sample solution. In claim 15,
A method of readjusting the concentration of a nucleic acid fragment in which the nucleic acid fragment is a nucleic acid extracted from a cell or a microorganism, a nucleic acid fragment amplified by PCR, or a synthetic nucleic acid.

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