JP4320210B2 - Electrophoresis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrophoresis apparatus for measuring a fragment length of a gene (nucleic acid oligomer) such as DNA or RNA by an electrophoresis method under denaturing conditions, and analyzing a nucleic acid base sequence, a VNTR polymorphism, or the like.
[0002]
[Prior art]
Non-Patent Document 1 (Christoph Heller, Electrophoresis, Vol. 21, 593-602, 2000) describes a method for analyzing by denaturing electrophoresis using a fragment length of a nucleic acid oligomer as a clue. There is a method using capillary electrophoresis. In this non-patent document 1, a commercially available single capillary type capillary electrophoresis apparatus is used, a capillary having an inner diameter of 50 μm and an outer diameter of 375 μm, and a refillable polymer based on a commercially available polydimethylacrylamide as an electrophoresis medium, A 100 mM TAPS buffer (100 mM N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid, adjusted to pH 8.0 with NaOH, electric conductivity 1250 μS / cm) is used as the buffer. The electrophoresis temperature is 50 ° C., and the electrophoresis voltage is 150 V / cm in terms of electric field strength. Under the optimum conditions of Non-Patent Document 1, at least 10 cm in order to separate fragments having a length of one base different from each other with 400 base single-stranded DNA fragments with a resolution of 0.5 or more, at least 20 cm in the case of 600 bases In the case of 800 bases, it is concluded that an effective length of at least 37 cm is necessary. In these cases, about 30 minutes, 67 minutes, and 150 minutes are required for separating 400 bases with an effective length of 10 cm, 600 bases of 20 cm, and 800 bases of 37 cm (from FIG. 3).
[0003]
However, reflecting the increasing recognition of the usefulness of genetic information, there is a growing need for more rapid and rapid determination of nucleic acid base sequences. Therefore, attempts have been reported to increase the conventional analysis speed and shorten the analysis time. For example, Non-Patent Document 2 (Karel Kleparnik et al., Electrophoresis, Vol. 22, pp. 783-788, 2001) is an attempt to improve the migration speed by changing the composition of the electrophoresis buffer. In Non-Patent Document 2, 0.04M NaOH is used as a buffer, and 4 wt% LPA (linear polyacrylamide) is used as a polymer. The electrophoretic performance in Non-Patent Document 2 is reported to be about 3 times faster than the previous method (4% LPA, 7M urea, 0.1M Tris-0.1M TAPS, pH 8.3). However, the separation is poor unless the following special measures are taken, and for example, a fragment having a length of 180 bases is not separated at an effective length of 19.5 cm (FIG. 4). This problem was avoided by injecting PEG- immediately after sample injection. In the case of the effective length, a fragment of 163 bases can be separated in about 10 minutes, and a migration time of a fragment of 668 bases (although poor separation) is about 22 minutes (FIGS. 2 and 1), and an effective length of 7 cm In this case, the 163 base-length fragment (although poor separation) has a migration time of about 3.5 minutes (FIG. 3). In Non-Patent Document 2, in order to prevent hydrolysis of the polymer by alkali, the polymer is first dissolved in 0.03M NaCl and filled into the capillary, and then the capillary is placed in a pre-migration buffer composed of 0.04M NaOH. The buffer of the running medium is exchanged with 0.04 M NaOH by pre-running for about 5 minutes.
[0004]
[Non-Patent Document 1]
Christoph Heller, Electrophoresis, 21, 593-602, 2000
[Non-Patent Document 2]
Karel Kleparnik et al., Electrophoresis, Vol. 22, pp. 783-788, 2001
[0005]
[Problems to be solved by the invention]
The method of Non-Patent Document 1 requires a long migration time of about 30 minutes in order to perform one base separation of a 400 nt nucleic acid oligomer with a resolution of 0.5 or more. The method of Non-Patent Document 2 is 3 times faster than the conventional example. However, when the separation of the 163 nt nucleic acid oligomer is performed with an electrophoresis time of 3.5 minutes, the separation is poor, and additional injection of PEG- or preliminary electrophoresis is performed. Such a complicated operation is required. In particular, the pre-electrophoresis takes about 5 minutes and takes longer than the main electrophoretic migration. That is, in conventional nucleic acid electrophoresis, there is a trade-off between high separation and rapidity, and it takes a long time to obtain sufficient separation, and conversely, sufficient separation cannot be obtained with a short analysis time. there were.
[0006]
An object of the present invention is to perform electrophoresis of nucleic acids under denaturing conditions using an electrophoresis apparatus having one or a plurality of flow paths (capillaries, grooves formed on a substrate, etc.) and DNA sequencing or nucleic acid When performing fragment length analysis, it is intended to achieve rapid and high nucleic acid separation with a simple configuration and to enable analysis of even long nucleic acids.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, an electrophoresis apparatus according to the present invention includes one or a plurality of channels filled with an electrophoresis medium containing a polymer, a buffer, and a nucleic acid denaturant, and serves as a buffer. And a water-soluble primary amine and an amphoteric electrolyte. The electrophoresis medium contains urea as a denaturing agent, and as a polymer, an acrylamide polymer, particularly preferably an (N-alkyl) acrylamide polymer. The concentration of the polymer is preferably 2 to 8% by weight, particularly preferably 4 to 6% by weight, and the polymer is refilled for each measurement.
[0008]
As the water-soluble primary amine contained in the buffer, a metanamine derivative can be used, and as the amphoteric electrolyte, aminocarboxylic acid can be used. Preferably, tris (hydroxymethyl) aminomethane is used as the water-soluble primary amine, and glycine is used as the amphoteric electrolyte. The buffering agent particularly preferably contains 0.01 to 0.05 [M] of tris (hydroxymethyl) aminomethane and 0.1 to 0.4 [M] of glycine.
[0009]
The average electric conductivity of the electrophoresis medium is preferably 1 [mS / cm] or less, particularly preferably 0.7 [mS / cm] or less. In the present invention, electrophoresis is performed under the condition that the electrophoresis current in the electrophoresis medium is preferably 8 μA or less per channel, particularly preferably 5 μA or less per channel. Further, the electrophoresis is performed under the condition that the power of the electrophoresis current per unit length of the flow channel is preferably 3 [mW / cm] or less, particularly preferably 2 [mW / cm] or less.
[0010]
Both ends of the flow path are connected to a high voltage power source via an electrophoresis buffer, and the electrophoresis buffer preferably contains a buffer having the same composition and concentration as in the electrophoresis medium. As the flow path, a capillary having an inner diameter of approximately 25 μm to 75 μm, particularly preferably an inner diameter of approximately 50 μm is used.
By adopting the above construction, nucleic acid electrophoresis was achieved by a simple method with high speed, high separation and good reproducibility.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
[Embodiment 1]
A first embodiment of the present invention will be described below. As the electrophoresis apparatus, a single capillary type capillary electrophoresis apparatus similar to that described in Non-Patent Document 1 was used. This device is equipped with an automatic filling mechanism for electrophoresis media, and can continuously measure multiple samples with an autosampler. A detector based on the principle of laser excitation fluorescence is provided. Specific experimental conditions are as follows.
[0012]
The preparation conditions of the sample solution are as follows.
0.5 μL of size standard (GeneScan® 500 ROX manufactured by Applied Biosystems) and 12 μL of formamide were mixed, heat-denatured at 94 ° C. for 2 minutes, and then ice-cooled.
[0013]
The electrophoresis conditions are as follows.
Capillary: A fused quartz capillary manufactured by Polymicro Technologies, Inc. having an inner diameter of 50 μm, an outer diameter of 363 μm, an effective length of 30 cm, and a total length of 41 cm.
[0014]
Electrophoresis buffer: A 100 mM TAPS buffer described in Non-Patent Document 1 was used as a conventional electrophoresis buffer (comparison target). Further, a buffer solution (hereinafter referred to as TG buffer) containing 25 mM Tris (hydroxymethyl) aminomethane (hereinafter referred to as Tris) and 192 mM glycine as a buffering agent was used as the electrophoresis buffer according to the present invention. The pH of the TG buffer was about 8.8, and was used as it was without adjusting the pH.
[0015]
Electrophoresis medium: An electrophoretic medium containing a commercially available polymer described in Non-Patent Document 1, a denaturant, and a TAPS buffer as a buffering agent was used as a conventional electrophoretic medium (comparative object). Hereinafter, this conventional electrophoresis medium is abbreviated as TAPS6. Moreover, as the electrophoresis medium according to the present invention, an electrophoresis medium containing the above-mentioned commercially available polymer, TG buffer, and 7M urea as a nucleic acid denaturant was prepared. Hereinafter, this electrophoresis medium is abbreviated as TG6. A specific method for preparing TG6 is to remove low molecular components (such as a buffer) in TAPS6 through an ultrafiltration membrane with an exclusion limit molecular weight of 30000, and then add a predetermined amount of the above TG buffer component, urea, and pure water to make it uniform. Mixed. According to Non-Patent Document 1, since TAPS6 contains polydimethylacrylamide as a polymer, TG6 also contains polydimethylacrylamide at the same concentration as a polymer.
[0016]
Filling time of electrophoresis medium into capillary: 3.5 minutes.
Sample electric field injection conditions: applied voltage 15 kV, time 5 seconds.
Electrophoresis voltage: 15 kV.
[0017]
Temperature setting of the thermostatic chamber: 50 ° C. unless otherwise specified, but other temperature conditions were also examined. The sample was denatured with formamide and heat as described above, and the electrophoresis medium contained urea as a denaturant and the electrophoresis temperature was as high as 50 ° C. That is, the electrophoresis was performed in a state where the nucleic acid was single-stranded, that is, under denaturing conditions.
[0018]
FIG. 1 (a) shows a typical electrophoresis pattern (electropherogram) in the first embodiment of the present invention, that is, when TG6 is used as the electrophoresis medium and TG buffer is used as the electrophoresis buffer. Further, FIG. 1B shows a typical electrophoresis pattern in the case where TAPS 6 is used as the electrophoresis medium and TAPS buffer is used as the electrophoresis buffer. The horizontal axis is a time axis for each unit, and the width of one count corresponds to 0.22 seconds. The vertical axis is the intensity axis (arbitrary scale) of the fluorescence signal at a specific fluorescence wavelength, and is proportional to the concentration of the DNA fragment labeled with a dye that emits fluorescence at that wavelength. The vertical axis | shaft of FIG. 1 shows the signal intensity of the fluorescence wavelength of the fluorescent pigment | dye (in this case ROX) which has labeled the GeneScan (R) 500 ROX sample. The meanings of the subsequent electropherograms are the same as in FIG.
[0019]
The migration time of the last peak in FIGS. 1A and 1B (corresponding to a fragment having a base length of 500, hereinafter referred to as 500 nt) is 7382 counts and 9264 counts, that is, 27.0 minutes and 34.0 minutes, respectively. It is. Therefore, compared with the conventional TAPS6, the TG6 according to this embodiment has a migration time of about 20% shorter, in other words, a migration speed is about 25% faster. That is, in the first embodiment of the present invention, there is an effect that the migration speed of the nucleic acid is faster than the conventional example.
[0020]
Here, the quantitative treatment relating to the maximum length of a nucleic acid fragment that can be analyzed in sequence analysis of DNA or fragment length analysis, which is the main problem of the present invention, will be described in detail. Various methods are used as the DNA sequence analysis method. Here, the dye terminator method will be described as an example. This method uses a DNA fragment to be analyzed as a template, a primer complementary to a part of the sequence, a monomer of deoxyribonucleic acid (dNTP), and a dye labeled with different fluorescent dyes corresponding to four types of bases. Mix deoxyribonucleic acid monomer (ddNTP), DNA polymerase, and buffer solution. By performing a so-called cycle sequence reaction or the like using this mixed solution, deoxyribonucleic acid is added to the primer in various lengths (complementary to the template), and the terminal is a fluorescently labeled ddNTP complementary to the template. A mixture of nucleic acid fragments is obtained. This mixture of fragments is analyzed by electrophoresis under denaturing conditions, fragments having different base lengths are separated and detected from each other, the fluorescence color of each fragment is analyzed, and the DNA sequence as a template is determined from the corresponding base sequence. decide.
[0021]
When measuring cycle sequence reaction products using electrophoresis, it is important whether or not the peaks corresponding to nucleic acid fragments that differ in length by one base in the electrophoresis results (electropherogram) can be accurately separated and detected. is there. In general, when the fragment length is short, the peak interval (spacing) between fragments whose fragment lengths differ from each other by one base is wide and separation analysis is easy. However, when the fragment length is long, the spacing becomes narrow and separation is difficult. It becomes. If the spacing is too small compared to the half width (full width at half maximum) of the peak of the desired fragment length, two adjacent peaks overlap each other and cannot be separated. Plot the number of bases on the horizontal axis, and the half width of the spacing and peak on the vertical axis, and a guideline for the length of the nucleic acid fragment that can sequence the number of nucleic acid bases under boundary conditions where the spacing is less than or equal to the half width of the peak. did. This number of nucleobases is hereinafter referred to as an equal width. An index used for the same purpose as the equal width length is a read length. For example, as described in Non-Patent Document 1, the read length is often defined as the number of bases at which the resolution Rs of two peaks is 0.5. On the other hand, since Rs in the equal width length is about 0.59 from the definition, the equal width length according to the definition is a numerical value smaller than the reading length.
[0022]
The calculation process for obtaining the equal width for the electropherograms of FIGS. 1 (a) and 1 (b) is shown in FIGS. 2 (a) and 2 (b), respectively. The horizontal axis of FIG. 2 indicates the fragment length, and the vertical axis indicates the spacing and the half width of the peak. In the figure, squares are plots of spacing, and diamonds are plots of peak half-value widths.
[0023]
In FIG. 2 (a) when TG6 according to the present invention is used, the half width shown by the rhombus is generally less than 0.45 mm, and the spacing shown by the square is narrower than the half width. This is the case when it is longer than about 412 nt. That is, the equal width length in TG6 is 412. On the other hand, in the case of the conventional TAPS6, as shown in FIG. 2B, the half width tends to be about 0.05 to 0.1 mm wider than TG6, and the equal width is 325 and shorter than TG6. Therefore, the present invention has an advantage that DNA sequence analysis can be performed accurately up to a longer nucleic acid fragment as compared with the conventional example.
[0024]
Next, the electrophoresis current when performing electrophoresis according to the present invention will be described. When TG6 according to the present invention was used, the electrophoretic current I was initially 3 [μA], decreased as the electrophoresis continued, and the electrophoretic current 27 minutes after the 500 nt peak was detected was 1 [μA]. It was. The average value of the electrophoretic current within this electrophoretic time was about 2.3 [μA]. Assuming that the current is defined by the capillary portion, since the migration voltage E is 15 [kV], the resistance R of the migration medium in the capillary portion is 6.5 [GΩ] on average. The total length L of the capillary is 41 [cm], the inner diameter of the capillary is 50 [μm], and the cross-sectional area S is 0.0000196 [cm]. ² Therefore, the average electric conductivity of the electrophoresis medium is determined to be about 0.32 [mS / cm] on average from L / (RS). On the other hand, the electrophoretic current I of the conventional TAPS6 was 10 [μA] in the initial stage, the electrophoretic current 34 minutes after the 500 nt peak was detected was 9 [μA], and the average value of the electrophoretic current was about 9.5 [μA]. It was. Therefore, the electrical conductivity of TAPS6 is similarly determined to be about 1.33 [mS / cm] on average.
[0025]
That is, the electrophoretic medium TG6 according to the present embodiment has a current and electrical conductivity that are about one-fourth lower on average than the conventional electrophoretic medium. The Joule heat associated with the current is calculated based on the current power (RI) assuming that the temperature is constant. ² ) And time. Regarding the above-described embodiment and the conventional example, when the power per unit length of capillary is obtained, the average is 0.84 [mW / cm] and 3.5 [mW / cm], respectively. That is, the amount of Joule heat generated per unit length and unit time is smaller in this embodiment, about an average of about one quarter of that in the conventional example.
[0026]
Joule heat is a typical instability factor in electrophoresis and is thought to adversely affect separation performance. As described above, the reason why the separation performance in the present embodiment is higher than that in the conventional example is considered to be due to less generation of Joule heat. In addition, when the Joule heat is high, the migration result may not be obtained at all due to thermal runaway, and the thermal runaway may cause damage to the capillary. The present invention has an effect that Joule heat is as low as about one-fourth that of the prior art, so that a high-resolution nucleic acid electrophoresis result can be stably obtained, thermal runaway does not occur, and the capillary is not damaged. When the electrophoresis medium and the electrophoresis buffer according to this embodiment are used, the migration speed of nucleic acid is essentially about 25% faster as described above, and the current and heat generation are small. Therefore, as in this embodiment, 366 [V / cm Good nucleic acid separation can be obtained with good reproducibility even if a slightly higher electric field strength is employed. Therefore, there is also an effect that the nucleic acid can be measured quickly by the synergistic effect of both factors.
[0027]
When the examination results of the running time and the running current are arranged from the viewpoint of the buffer composition, the TG buffer employed in the present invention has a shorter running time, lower running current, and higher separation than the conventional buffer. When changing physical conditions such as electrophoresis voltage or migration path length, the electrophoresis current increases and the separation decreases to shorten the migration time, and the migration time increases to reduce the electrophoresis current and increase the separation. The three characteristics of time, current, and separation are generally contradictory. Therefore, it is understood that the TG buffer that can improve the three characteristics at the same time has unexpectedly favorable characteristics as compared with the conventional buffer. Based on the above examination results, a TG buffer is employed in the present embodiment.
[0028]
In the present embodiment, a combination of Tris and glycine is used as the migration buffer and the buffer in the migration medium, but other buffers can be used as well. Examples of other buffering agents that exhibit the same performance as Tris include water-soluble primary amines such as various methanamin derivatives and a group of compounds collectively referred to as Good's Buffer. Examples of Good's Buffer include MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO, CAPS These can be suitably used as a buffering agent in the present invention. For glycine, so-called ampholytes having both positive and negative charges in the molecule can be used as well. For example, various aminocarboxylic acids such as alanine and valine, iminocarboxylic acids, aminosulfonic acids and the like. Moreover, what is an amphoteric electrolyte among said Good's Buffer can be used similarly to glycine in the pH range which sets each pKa corresponding to both charge sites as an upper limit and a minimum.
[0029]
In the present embodiment, a system containing 25 mM and 192 mM of Tris and glycine as the migration buffer and the buffer in the migration medium has been studied, but the concentration of both is not limited to this. As the concentration limit in the lower direction, good electrophoresis results were obtained even at about 10 mM and 100 mM, respectively. However, when the concentration was reduced by half, a good electrophoresis pattern could not be obtained. In the direction of higher concentration, an electrophoretic pattern can be obtained even when the concentration is twice or more. However, if the concentration is higher than that, the low current feature of the present invention is hardly exhibited. It is considered a practical upper limit.
[0030]
[Embodiment 2]
A second embodiment of the present invention will be described below. The second embodiment basically employs the same experimental conditions as the first embodiment, but is based on polydimethylacrylamide having an average molecular weight (MW) of about 1 million instead of TG6 as a nucleic acid migration medium. The point which used is different.
[0031]
As an object to be compared with the present embodiment (conventional example), an average molecular weight (MW) of about 100 synthesized by the method described in the literature (Christoph Heller, Electrophoresis, 20, 1962-1977, 1999). Ten thousand polydimethylacrylamide dissolved in a TAPS buffer containing 7 M urea so as to have a weight concentration of 5% was used as the electrophoresis medium. Hereinafter, this conventional electrophoresis medium is abbreviated as TAPS7. Moreover, the same TAPS buffer as the conventional example in Embodiment 1 was used as the electrophoresis buffer of the comparative example.
[0032]
On the other hand, as an electrophoresis medium according to the present embodiment, the polydimethylacrylamide is contained at a weight concentration of 5%, includes the same TG buffer as that of the first embodiment, and further includes 7M urea as a nucleic acid denaturant. The medium was prepared. Hereinafter, this electrophoresis medium is abbreviated as TG7. In the present embodiment, a TG buffer is used as the migration buffer as in the first embodiment.
[0033]
Examples of typical electrophoretic patterns (electropherograms) when TG7 according to the present invention and conventional TAPS7 are used are shown in FIGS. 3A and 3B, respectively. The migration time of the last peak (base length 500 nt) in FIGS. 3 (a) and 3 (b) is 2625 counts and 3777 counts, respectively 9.6 minutes and 14.6 minutes, respectively. Therefore, compared with the conventional TAPS7, the TG7 of this embodiment has a migration time of about 3.5% shorter, in other words, a migration speed is about 52% faster. That is, also in the second embodiment of the present invention, there is an effect that the migration speed of the nucleic acid is faster than the conventional example.
[0034]
FIGS. 4A and 4B show calculation processes for obtaining the equal width for the electropherograms of FIGS. 3A and 3B, respectively. 4A and 4B, the horizontal axis represents the fragment length, and the vertical axis represents the half width of the spacing and peak. In the figure, squares are plots of spacing, and diamonds are plots of peak half-value widths.
[0035]
In the case of FIG. 4A using TG7 according to the present invention, the half width of the peak is approximately 0.3 to 0.4 mm, and the equal width is approximately 439 nt. On the other hand, in the case of the conventional TAPS7, as shown in FIG. 4B, the peak half-value width tends to be 0.5 to 0.6 mm wider than 300 nt and about 0.1 to 0.2 mm wider than TG7, etc. The width was 326 nt and 100 bases shorter than TG7. Therefore, the second embodiment of the present invention also has a feature that DNA sequence analysis can be accurately performed up to a longer fragment length as compared with the conventional example.
[0036]
Next, the electrophoresis current will be described. When TG7 according to the present invention was used, the electrophoretic current I was 5 [μA] in the initial stage, the electrophoretic current at the final time point was 4 [μA], and the average value of the electrophoretic current was about 4.4 [μA]. According to the same calculation as that in the first embodiment, the resistance R of the electrophoresis medium is 3.4 [GΩ] on average and the electric conductivity is about 0.61 [mS / cm]. On the other hand, the migration current of the conventional TAPS7 was 11 [μA] at the initial stage, the migration current at the final time point was 10 [μA], and the average value of the migration current was about 10.0 [μA]. Therefore, the electrical conductivity of TAPS7 is required to be about 1.4 [mS / cm]. That is, the electrophoretic medium TG7 according to the present embodiment has a current and electrical conductivity that are as low as about 1 / 2.2 compared with the corresponding conventional electrophoretic medium.
[0037]
For the present embodiment and the conventional example, the power per unit length of the capillary is found to be 1.6 [mW / cm] and 3.7 [mW / cm] on average, respectively. That is, the amount of Joule heat generation per unit length and per unit time is smaller in this embodiment, about 1/2 of the average of the conventional example. Therefore, also in this embodiment, there is an effect that the electrophoresis result of the nucleic acid can be obtained with high separation and stability.
[0038]
When the electrophoresis medium and the electrophoresis buffer according to the present embodiment are used, the migration speed is originally about 52% faster as described above, and since current and heat generation are small, 366 [V / cm] as in the present embodiment. Good nucleic acid separation can be obtained with good reproducibility even if a slightly higher electric field strength is employed. Therefore, there is an effect that rapid nucleic acid measurement is possible due to the synergistic effect of both.
[0039]
[Embodiment 3]
A third embodiment of the present invention will be described below. The third embodiment is a capillary array type apparatus using a plurality of capillaries based on the same principle as the apparatus described in Anazawa et al., 68, 2699, 1996 as an electrophoresis apparatus. However, the other points are the same as those in the first and second embodiments.
[0040]
FIG. 5 is a schematic configuration diagram of the capillary array electrophoresis apparatus used in the present embodiment. A feature of this apparatus is that a capillary array having a plurality of capillaries for electrophoresis is employed, and a lateral incidence on-column detection method (multifocus method) is used as a detection method.
[0041]
Hereinafter, the configuration of the present apparatus will be described. One end of the capillary array 4 and a platinum cathode 5 are set in the electrophoresis buffer 3, and the other end of the capillary array 4 is bundled and then connected to the flow path 7 of the pump block 6. Syringes 8 and 8 ', a solenoid valve 9, a check valve 10, and a flow path 11 are connected to or built in the pump block 6, and a platinum anode 5' is installed in the migration buffer 3 '. That is, an electrophoresis buffer 3, a capillary array 4, a flow path 7 of a pump block 6, a solenoid valve 9, a flow path 11, and a migration buffer 3 'are provided between the cathode 5 and the anode 5', and these form an electrophoresis path. To do. The cathode 5 and the anode 5 ′ are connected to a high voltage power source 12. The syringes 8 and 8 ′ are respectively connected to a syringe drive mechanism (not shown), and the syringes 8 and 8 ′ are filled with the electrophoresis media 13 and 13 ′. Most of the capillary array 4 except for both ends is contained in the temperature controller 14, and in particular, a part close to the polymer block is in contact with the detector 15. The detector 15 includes light sources 16 and 16 ′ (laser) and a light receiver (including a spectroscope and a CCD camera, not shown). The electrophoresis buffer 3 is held by the autosampler 17, and the washing buffer 18, the sample solution 19, and the like are held on the autosampler 17 in addition to the electrophoresis buffer 3. The sample solution 19 contains anionic substances to be measured such as DNA fragments, and these are labeled with a fluorescent dye. The entire apparatus is connected to a measurement control apparatus (not shown).
[0042]
Next, an outline of the operation of this apparatus will be described with reference to FIG. A part of the replenishment electrophoresis medium 13 in the syringe 8 is filled in the flow path 7 and the flow path 11 of the pump block 6. The temperature of the capillary array 4 is kept constant by the thermostatic chamber 14. After closing the solenoid valve 9, by operating the syringes 8 and 8 ′, a part of the electrophoresis medium 13 in the syringe 8 passes through the flow path 7 and the check valve 10 to inject the electrophoresis medium 13 in the syringe 8 ′. Move to '. The autosampler 17 is operated and the tip of the capillary array is immersed in the cleaning liquid 18. The electrophoresis medium 13 ′ is injected into the capillary array 4 via the flow path 7 inside the pump block 6 by driving the syringe 8 ′ at a constant volume with a constant pressure. Open the solenoid valve 9. The autosampler 17 is operated and the tip of the capillary array is immersed in the sample solution 19. The high voltage power supply 12 is operated to inject an electric charge component in the sample into the capillary array. The autosampler 17 is operated and the tip of the capillary array is immersed in the electrophoresis buffer 3. The high voltage power supply is operated, and the components in the sample in the capillary array 4 are electrophoresed in the direction of the detector 15.
[0043]
The light sources 16 and 16 ′ are continuously driven to irradiate from both side surfaces of the capillary array 4. Due to the lens effect of each capillary, laser light sequentially passes through the capillary from the outside toward the center capillary to excite the sample (lateral incidence on-column detection method, also called multi-focus method). The fluorescence spectrum of the sample component is spectroscopically measured by the detector 15 to acquire the electrophoresis spectrum of the fluorescence-labeled sample component. Even when a plurality of samples are labeled with different fluorescent dyes and run simultaneously, the mutual spectral interference is corrected and each sample is measured simultaneously and independently. Measure the size standard at the same time and normalize the run time. When measuring another sample set after the measurement of one sample set is completed, the procedure is repeated from the place where the electrophoresis medium 13 is transferred to 13 '. All the above operations are automatically performed by the measurement control device based on an instruction from the operator.
[0044]
Next, the outline of the use conditions of this apparatus will be described. In the present embodiment, the above apparatus is used for DNA fragment length analysis. The conditions for this were basically the same as those in the single capillary device described in the first embodiment. The main changes are as follows. As the capillary array, a capillary array 4 having 96 capillaries having an inner diameter of 50 μm, an outer diameter of 365 μm, an effective length of 22 cm, and a total length of 33 cm was used. The electric field injection conditions of the sample are an applied voltage of 10 kV, a time of 8 seconds, and an electrophoresis voltage of 15 kV. As a migration medium and a migration buffer, a combination of TG6 and TG buffer was used as in the first embodiment.
[0045]
In the process of examining the experimental conditions of the present embodiment, when the combination of the conventional electrophoresis medium and the electrophoresis buffer described in the first embodiment is used, a normal electropherogram may not be obtained, and generally the separation is poor. In some cases, the pump block portion is damaged by heat generation. Under the above electrophoresis conditions (electric field strength of 455 V / cm), the electrophoresis current was as high as about 1130 μA (about 11.8 μA per capillary). In order to obtain a stable migration result using a combination of a conventional migration medium and a migration buffer, the electric field strength must be about 313 V / cm or less.
[0046]
On the other hand, in the present embodiment, as in the first embodiment, the TG buffer and the migration medium based thereon are employed. As a result, there is no problem as in the conventional example, and normal and high separation equivalent to the case of a single capillary is achieved. Nucleic acid electrophoresis patterns were obtained for all capillaries. In this embodiment, the electrophoretic current was as low as about 270 μA (about 2.8 μA per capillary). For the present embodiment and the conventional example, the power per unit length of capillary is found to be 1.3 [mW / cm] and 5.4 [mW / cm] on average, respectively. That is, the amount of Joule heat generation per unit length and per unit time per capillary is smaller in this embodiment, about an average of about one quarter of that in the conventional example.
[0047]
In the conventional example, Joule heat is large, and in the part where many capillaries are arranged densely like in the vicinity of the detector like this device, temperature rise, separation decline, reproducibility decline, thermal runaway, capillary damage, While problems such as damage to the capillary holder are likely to occur, in this embodiment, since Joule heat is as low as about one-fourth that of the conventional example, these adverse effects can be prevented and good results can be obtained. It is thought that it was done. Therefore, the TG buffer employed in the present invention and the electrophoresis medium based on the TG buffer have high reproducibility of nucleic acid electrophoresis results and high reliability, particularly in an apparatus configuration having a portion where a large number of capillaries are concentrated as in this embodiment. effective. Further, since a high electric field strength of 455 [V / cm] can be adopted, there is an effect that the nucleic acid migration time is short.
[0048]
In this embodiment, a capillary array having 96 capillaries is used. However, the present invention is not limited to this configuration, and an apparatus configuration having a plurality of capillaries with more than 96 or fewer than 96 capillaries is also used. The same applies. When more capillaries are used, in the conventional method, the problem due to the heat generation becomes more prominent, so the effect of the present invention becomes more prominent. In this embodiment, the case where the capillary array is used for holding the electrophoresis medium has been described. However, even when a so-called chip electrophoresis apparatus that holds the electrophoresis medium in a groove provided on the substrate is used, the present invention The present invention can be applied in the same manner, and particularly in a high-throughput application in which nucleic acid electrophoresis is performed simultaneously using a large number of migration paths, the present invention exhibits extremely excellent effects.
[0049]
The effect peculiar to the present embodiment is that parallel processing is possible by using a plurality of capillaries at the same time, the throughput is high, the nucleic acid migration speed is about 25% faster than conventional, and there is less current and heat generation. The multi-capillary device can adopt 455V / cm, which is 45% higher than the conventional electric field strength, and the high-resolution nucleic acid electrophoresis results can be obtained stably with good reproducibility. It is possible to measure nucleic acid accurately.
[0050]
Next, the effect of the present invention will be described.
Conventional examples compared with the present invention are as described in each embodiment. That is, the migration medium and the migration buffer, which are the characteristics of each embodiment of the present invention, are replaced with those according to the prior art, and other conditions are aligned with each embodiment of the present invention. Specifically, the first conventional example is a method of performing DNA fragment length analysis using a commercially available electrophoresis apparatus, electrophoresis medium, and electrophoresis buffer described in Non-Patent Document 1, and specifically, electrophoresis. A single-capillary capillary electrophoresis apparatus was used as the apparatus, a capillary with an effective length of 30 cm as the capillary, TAPS 6 as the electrophoresis medium, and 100 mM TAPS buffer as the electrophoresis buffer. In the second conventional example, nucleic acid fragment length analysis is performed using a commercially available electrophoresis apparatus described in Non-Patent Document 1, a migration medium based on a polymer synthesized by the method described in Non-Patent Document 2, and an electrophoresis buffer. Specifically, a single capillary type capillary electrophoresis apparatus was used as the electrophoresis apparatus, a capillary having an effective length of 30 cm as the capillary, TAPS7 as the electrophoresis medium, and a TAPS buffer as the electrophoresis buffer. The third conventional example is a method of performing DNA fragment length analysis using a multicapillary electrophoresis apparatus, a commercially available electrophoresis medium described in Non-Patent Document 1, and an electrophoresis buffer. Specifically, electrophoresis is performed. As the apparatus, the multicapillary electrophoresis apparatus described in the third embodiment, a capillary array including 96 capillaries having an effective length of 22 cm as capillaries, TAPS6 as an electrophoresis medium, and 100 mM TAPS buffer as an electrophoresis buffer were used.
That is, the present invention is different from the conventional example using TAPS in that Tris-glycine is mainly used as a buffering agent in the electrophoresis medium and the electrophoresis buffer.
[0051]
An example of the comparison result between the conventional example and the present invention is shown in FIG. FIG. 6 shows the results of performing electrophoresis for each embodiment of the present invention and corresponding conventional examples, and comparing the representative characteristics of the results. The properties studied are separation properties (equal width), migration time, and migration current. Note that the conventional example corresponding to the third embodiment often fails to obtain good results when compared under the same conditions as the third embodiment. Characteristic values when good results are obtained () ^* This represents the best result under this condition, and in many cases, a lower result or nothing was obtained. Therefore, as a modified example of the third conventional example, the conditions under which the migration results were obtained reliably, that is, the results when the migration voltage was reduced to 10 kV (from 15 kV adopted as the standard condition) are also shown.
[0052]
As is clear from FIG. 6, each embodiment of the present invention has the features that the equal width is longer, the migration current is smaller, and the nucleic acid migration time is shorter than the conventional example under the same conditions. In addition, the third embodiment has a feature that nucleic acid electrophoresis can be performed with higher reproducibility than the conventional example under the same conditions. Further, as compared with the modified example of the conventional example 3 having different conditions, the electrophoretic current is small and the nucleic acid electrophoretic time is short. Further, the equal width per unit time is 13.7 [nt / min] in the third embodiment, and 8.7 [nt / min] in the modified example of the conventional example 3, and the analysis base length per unit time is long. It has the feature.
[0053]
Comparing in detail the average electrophoretic current per capillary, as described above, the first, second, and third embodiments are about 2.3 [μA], 4.4 [μA], and 2.8 [μA], respectively. On the other hand, the conventional examples 1, 2, and 3 are about 9.5 [μA], 10 [μA], and 11.8 [μA], respectively. That is, there is a clear difference between each embodiment of the present invention and the conventional example, each embodiment of the present invention is 5 [μA] or less, and the conventional example exceeds 8 [μA]. In terms of heat generation, separation, migration stability, etc., the present invention has obtained better characteristics than the conventional example because the average migration current per capillary (ie, one flow path) is compared with the conventional example. Therefore, it is thought that it is a big factor that it is extremely low. In other words, the conventional example employs electrophoresis conditions in which the average migration current per capillary is larger than 8 [μA], and thus there are problems such as high heat generation, low separation, and reduced migration stability. On the other hand, the present invention adopts electrophoresis conditions in which the average migration current per capillary is 8 [μA] or less, particularly preferably 5 [μA] or less, thereby reducing heat generation, high separation, and electrophoresis compared to the conventional example. Excellent effects such as high stability were obtained.
[0054]
Similarly, when compared in terms of electrical conductivity, the average of TG6 used in the first and third embodiments is about 0.32 [mS / cm] and the average of TG7 used in the second embodiment is about 0 as described above. The TAPS6 used in Conventional Examples 1 and 3 has an average of about 1.33 [mS / cm] and TAPS7 has an average of about 1.4 [mS / cm]. That is, there is a clear difference between each embodiment of the present invention and the conventional example, and each embodiment of the present invention is 0.7 [mS / cm] or less, whereas the conventional example is 1 [mS]. / Cm]. It is considered that the reason why the present invention obtained favorable characteristics compared to the conventional example in terms of heat generation, separation, migration stability, etc. is that the electrical conductivity is remarkably lower than that of the conventional example. . In other words, the conventional example employs electrophoresis conditions in which the electric conductivity is greater than 1 [mS / cm], and thus has problems such as high heat generation, low separation, and reduced stability of electrophoresis. On the other hand, according to the present invention, by adopting the electrophoresis conditions in which the electric conductivity is 1 [mS / cm] or less, particularly preferably 0.7 [μA] or less, the heat generation is low, the separation is high, and the electrophoresis is low. Excellent effects such as high stability were obtained.
[0055]
Similarly, when comparing from the viewpoint of the power per unit length per capillary, the first, second, and third embodiments are about 0.32 [mW / cm] and 0.61 [respectively] as described above. mW / cm] and 1.3 [mW / cm], whereas the conventional examples 1, 2 and 3 are about 3.5 [mW / cm], 3.7 [mW / cm] and 5.4, respectively. [MW / cm]. That is, there is a clear difference between each embodiment of the present invention and the conventional example, each embodiment of the present invention is 2 [mW / cm] or less, and the conventional example is greater than 3 [mW / cm]. . In the items such as heat generation, separation, and migration stability, the present invention has obtained better characteristics compared to the conventional example because the work rate per unit length per capillary is lower than that of the conventional example. This is considered to be a major factor. In other words, the conventional example employs the electrophoresis conditions in which the power per unit length per capillary is larger than 3 [mW / cm], and thus there are problems such as high heat generation, low separation, and low stability of electrophoresis. It was. On the other hand, the present invention is compared with the conventional example by adopting electrophoresis conditions in which the power per unit length per capillary is 3 [mW / cm] or less, particularly preferably 2 [mW / cm] or less. As a result, excellent effects such as low heat generation, high separation, and high migration stability were obtained.
[0056]
Although a direct comparison experiment between the method described in Non-Patent Document 2 and each embodiment of the present invention was not performed, according to the description in Non-Patent Document 2, 163 at an effective length of 19.5 cm at which sufficient separation can be obtained. The migration time of the base length fragment is about 10 minutes. On the other hand, in the second embodiment of the present invention, the migration time of a nucleic acid fragment having a length of 500 bases at an effective length of 30 cm can be separated in 9.5 minutes, so that the migration is faster than in Non-Patent Document 2. In Non-Patent Document 2, in order to prevent hydrolysis of the polymer by alkali, the polymer is first dissolved in 0.03M NaCl and filled into the capillary, and then the capillary is placed in a pre-migration buffer made of 0.04M NaOH. The polymer buffer was exchanged with 0.04M NaOH by soaking and pre-running for about 5 minutes. That is, since this pre-electrophoresis time is required in addition to the sample electrophoretic time, it is clear that the total electrophoretic time is shorter in the present invention. Therefore, the present invention has a feature that it is a simple and quick method as compared with the method of Non-Patent Document 2.
[0057]
As described above, the present invention can provide a nucleic acid electrophoresis apparatus that is faster, more accurate, and more separated than conventional techniques, and has a feature that the number of nucleobases that can be analyzed is long.
[0058]
【The invention's effect】
According to the present invention, in a capillary electrophoresis apparatus, rapid and high nucleic acid separation can be achieved with a simple configuration, and even long nucleic acids can be analyzed.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of a result (electropherogram) of measuring a size standard using a capillary electrophoresis apparatus, (a) is an electropherogram of the first embodiment of the present invention based on TG6. (B) is an electropherogram of a conventional example based on TAPS6.
FIGS. 2A and 2B are diagrams showing a calculation process for obtaining a uniform width for the electropherogram of FIG. 1, wherein FIG. 2A corresponds to the first embodiment of the present invention, and FIG. To do.
FIG. 3 is a diagram showing an example (electropherogram) of a result of measuring a size standard using a capillary electrophoresis apparatus, where (a) is an electropherogram of a second embodiment of the present invention based on TG7. (B) is an electropherogram of a conventional example based on TAPS7.
FIGS. 4A and 4B are diagrams showing a calculation process for obtaining a uniform width for the electropherogram of FIG. 3, wherein FIG. 4A corresponds to the second embodiment of the present invention, and FIG. To do.
FIG. 5 is a schematic configuration diagram of a capillary array electrophoresis apparatus according to the present invention.
FIG. 6 is a diagram showing a comparison of electrophoretic characteristics between the present invention and a conventional example.
[Explanation of symbols]
3, 3 '... migration buffer, 4 ... capillary array, 5 ... cathode, 5' ... anode, 6 ... pump block, 7 ... flow path, 8, 8 '... syringe, 9 ... solenoid valve, 10 ... check valve, DESCRIPTION OF SYMBOLS 11 ... Flow path, 12 ... High voltage power supply, 13, 13 '... Electrophoresis polymer, 14 ... Temperature controller, 15 ... Detector, 16, 16' ... Light source, 17 ... Autosampler, 18 ... Cleaning solution, 19 ... Sample solution

Claims (2)

In an electrophoresis apparatus comprising a plurality of capillary channels filled with an electrophoresis medium containing a polymer, a buffer, and urea as a nucleic acid denaturant,
The electrophoretic device, wherein the buffer comprises 0.01 to 0.05 [M] tris (hydroxymethyl) aminomethane and 0.1 to 0.4 [M] glycine .   The electrophoresis apparatus according to claim 1, wherein an average electric conductivity of the electrophoresis medium is 1 [mS / cm] or less.

Images