JPH0875703A - Method for determining length of DNA fragment using capillary gel electrophoresis - Google Patents

Abstract

(57) [Summary] [Objective] To determine the length of a DNA fragment of an unknown sample by using capillary gel electrophoresis. [Structure] By using capillary gel electrophoresis, DN
The mobility of a standard sample of the A fragment is measured, and the measurement result is fitted to a theoretical formula of the mobility of the DNA fragment in consideration of the effect of a high applied voltage to prepare a calibration curve. Next, the unknown sample is measured, and the DNA length of the unknown sample is determined from the prepared calibration curve. [Effect] DNA obtained by capillary gel electrophoresis
By using the theoretical formula of the mobility of the DNA fragment in consideration of the influence of the high applied voltage in creating the calibration curve of the standard sample of the fragment,
An accurate calibration curve of DNA fragments can be obtained, and DNA of unknown samples can be obtained.
The length can be easily determined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining a length of a DNA fragment by using a capillary gel electrophoresis method.

[0002]

2. Description of the Related Art Capillary gel electrophoresis is superior to high performance liquid chromatography in that it can be analyzed with a small amount of sample of 1/10000 or less and that it has several tens of times higher separation performance. Further, as compared with the conventional slab gel electrophoresis method and the like, a high voltage of one digit or more can be applied, so that the analysis time can be significantly shortened. Further, since one end of the capillary can be used as an injector, the sample injection can be easily automated. Therefore, in recent years, capillary gel electrophoresis has been actively used in many fields. In particular, it is being applied to DNA fragment analysis and DNA sequencer with the progress of researches on genome analysis. In the DNA fragment analysis and the DNA sequencer, the capillary gel electrophoresis method using acrylamide gel is usually used, and the sequencing of about 300 bases is performed in about 45 minutes. The theoretical consideration of the mobility of DNA fragments has been extensively studied since the reptation model was proposed. However, the current DN
As the theoretical formula of the A fragment mobility, an approximate formula assuming a low applied voltage is used. Regarding the application of capillary gel electrophoresis to DNA fragment analysis and DNA sequencer, Journal of Chromatography, Volume 516 (1990), pp. 33 to 48 (Jouranl.
of Chromatography, Vol. 51
6 (1900) pp. 33-48), and Analytical Chemistry, Vol. 62 (1990), pages 900 to 903 (Analytical Chemist).
ry, Vol. 62 (1900) pp. 900-90
3), regarding the reptation model and the theoretical formula of DNA fragment mobility, see Biopolymers, Vol. 25 (1986).
Year) 431 to 454 (Biompolyme
rs, Vol. 25 (1986) pp. 431-45
4), and Biopolymers, 27 (1988) pp. 509-524 (Biopolymers,
Vol. 27 (1988) pp. 509-524).

[0003]

The conventional theoretical formula for the mobility of DNA fragments is an approximate formula limited to the case of low applied voltage.

[0004]

[Equation 1]

Or

[0006]

[Equation 2]

Here, μ: mobility of DNA fragment μ ₀ : mobility of DNA fragment in solution at temperature T ° C. A: constant dependent on gel composition N: length of DNA fragment βi: constant a: gel Pore size E: Applied voltage intensity Q: Total charge of DNA fragment ξ: Friction coefficient of DNA fragment in gel network B, c: Constant was used. However, the capillary gel electrophoresis method uses an applied voltage that is higher by one digit than that of the conventional slab gel electrophoresis method. Therefore, the DNA fragment mobility that is approximated by assuming the conventional low applied voltage is used. In the theoretical formula of
The behavior of DNA fragments in capillary gel gel electrophoresis cannot be fully explained. Therefore, it is difficult to create an accurate calibration curve even if a standard sample of a DNA fragment of known length is measured using capillary gel electrophoresis, and the exact length of the DNA fragment of the unknown sample is determined. I couldn't. Therefore, DNA by capillary gel electrophoresis
A theoretical formula expressing the mobility of fragments was needed.

[0008]

In order to solve the above problems, in the present invention, DNA in consideration of the influence of high applied voltage is used.
An expression expressing the mobility (μ) of a fragment

[0009]

(Equation 3)

Here, μ ₀ (T): mobility of DNA fragment in solution at temperature T ° C. A: constant dependent on gel composition N: DNA fragment length E ′: specific electric field strength, E ′ = qEa / 2k _B T q: constant dependent on gel pore size E: applied voltage intensity a: gel pore size k _B : Boltzmann constant T: temperature was invented.

It is known that the mobility of DNA in solution depends on temperature. The temperature dependence of the mobility of DNA in this solution is mainly due to the change in the viscosity of the solution (eg, Biopolymers, Vol. 2 (19).
64) pp. 245-257, or Biochem
istry, Vol. 23 (1984) pp. 922-
927). Therefore, it is necessary to consider the temperature change due to the Joule heat generated in the capillary. The temperature inside the capillary has a temperature distribution as shown in FIG. The meaning of each symbol in FIG. 3 is as follows.

T: temperature at the center of the gel Ta: temperature outside the capillary ΔT: temperature difference between the gel center and the outside of the capillary ΔT ₁ : temperature difference inside the gel ΔT ₂ : temperature difference inside the fused silica ΔT ₃ : temperature inside the polyimide Difference ΔT ₄ : Temperature outside the polyimide and outside the capillary Therefore, the temperature distribution inside the capillary is calculated by the heat transfer equation.

[0013]

[Equation 4]

Here, T is the temperature at the center of the gel, G is the amount of heat generated in the gel, r _{1 is} the inner radius of the capillary, r _{2 is} the outer radius of the capillary (without polyimide), and r _{3 is} the outer radius of the capillary (with polyimide) k. ₁ : Thermal conductivity of gel k ₂ : Thermal conductivity of fused silica k ₃ : Thermal conductivity of polyimide h: Heat transfer coefficient Ta: Temperature outside the capillary was used for correction. Further, since the temperature dependence of the mobility of DNA is mainly due to the change of the viscosity of the solution, the mobility (μ ₀ (T)) of the DNA fragment in the solution can be calculated by using the corrected temperature (T).

[0015]

(Equation 5)

Where μ ₀ (T ₁ ) is the mobility of the DNA fragment in the solution at the temperature T _1, η (T ₁ ) is the viscosity of the solution at the temperature T _{1, and} η (T) is the viscosity of the solution at the temperature T. I asked.

[0017]

[Operation] D when using capillary gel electrophoresis
The theoretical formula for the NA fragment mobility does not approximate the high applied voltage in consideration of the high applied voltage, and therefore the behavior of the DNA fragment at the high applied voltage can be expressed. Therefore, it is possible to measure a known standard sample and create an accurate calibration curve, and to determine the correct DNA fragment length of the unknown sample.
Furthermore, since the mobility of the DNA fragments in the solution is determined in consideration of the temperature change in the capillary due to the Joule heat generated in the capillary, the temperature change due to the change in the Joule heat due to the change in the external temperature or the voltage change is also considered. Can handle.

[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the block diagram of FIG.

In the method for determining the length of a DNA fragment using the capillary gel electrophoresis according to the present invention, first, a standard sample of a known DNA fragment is measured. The mobility of the DNA fragment as the measurement result is curve-fit to (Equation 1), each parameter is determined, and a calibration curve is prepared. Next, an unknown sample for which the length of the DNA fragment is desired is measured. Finally, the DN of the unknown sample measured using the calibration curve prepared from the standard sample
Determine the length of the A fragment.

FIG. 2 schematically shows an apparatus for determining the length of a DNA fragment using the capillary gel electrophoresis according to the present invention.
Will be described.

An apparatus for determining the length of a DNA fragment is DN
It is composed of a capillary gel electrophoresis apparatus 1 for measuring the A fragment and a data processing apparatus 2 for analyzing the measurement result.

The capillary gel electrophoresis apparatus 1 comprises a high-voltage power supply section 3, buffer tanks 4, 5, a sample injection device 6, a sample stage 7, a standard sample tank 8, an unknown sample tank 9, an electrophoresis capillary 10 and an optical detector 11. , A recorder 12. The high-voltage power supply unit 3 uses a high-voltage power supply with an output of 0 to 30 kV and uses a platinum electrode 13 in the buffer tank 4, a platinum electrode 16 in the buffer tank 5, and a platinum electrode 14 in the standard sample tank 8 or an unknown sample. A high voltage can be applied between the platinum electrode 15 in the tank 9 and the platinum electrode 16 in the buffer tank 5. First, a high voltage is applied to the platinum electrode 13 in the buffer tank 4 and the platinum electrode 16 in the buffer tank 5 using the high-voltage power supply unit 3 to perform pre-migration.

For the measurement of the standard sample, the tip 10a of the electrophoresis capillary 10 is inserted into the standard sample tank 8 on the sample stage 7 using the sample injection device 6, and the standard sample is electrophoresed by the electric transfer method. 10 is introduced and electrophoresis is performed. Capillary 10 for electrophoresis by electrophoresis
The components separated inside are detected by the optical detector 11, and the migration time and the concentration value of each detected component are sent to the recorder 12 and recorded. The recorded data in the recorder 12 is sequentially transmitted to the data processing device 2, and the mobility of the DNA fragment of the measurement result is curve-fitted to (Equation 1) in the data processing device 2 to determine each parameter. A calibration curve is created.

For the measurement of the unknown sample, the tip 10a of the electrophoresis capillary 10 is inserted into the unknown sample tank 9 on the sample stage 7 using the sample injection device 6, and the unknown sample is analyzed by the electric transfer method. 10 is introduced and electrophoresis is performed. Capillary 10 for electrophoresis by electrophoresis
The components separated inside are detected by the optical detector 11, and the migration time and the concentration value of each detected component are sent to the recorder 12 and recorded. The recorded data in the recorder 12 is sequentially transmitted to the data processing device 2. In the data processing device 2, the length of the DNA fragment of the unknown sample measured using the calibration curve prepared from the standard sample is obtained.

A detailed method for preparing a calibration curve for a DNA fragment according to an embodiment of the present invention will be described.

In the present invention, ethidium bromide (EB), which is an intercalator of DNA, is added to the migration buffer.
A calibration curve was prepared for the DNA fragments with and without addition of. N, N, N ', N'-tetramethylethylenediamine, and ammonium peroxodisulfate as polymerization agents were used for the preparation of the acrylamide gel.
The optical detector is an ultraviolet absorption detector for HPLC improved for on-column detection.
The absorbance at 60 nm was measured. The measured data from the optical detector was sequentially transmitted to the data processing unit, and the data processing unit recorded and processed the data. The detailed measurement conditions are shown in Table 1.

[0027]

[Table 1]

In order to obtain the temperature distribution inside the capillary, it is necessary to obtain the heat transfer coefficient (h). In the present invention, in order to keep the temperature constant, an air conditioner (wind speed, about 1.0 m
/ S) was used. When air cooling the capillary,
It can be considered as heat transfer by forced convection in the case of a cylinder. In that case, h can be obtained by the following formula, and it is known that h is in good agreement with the experimental value in the case of air (for example, Heat Transfer, SI Me.
tric Edition (1989) J. P. H
olman).

[0029]

(Equation 6)

Where d is the diameter of the cylinder, k _{f is} the thermal conductivity of the air, u is the wind velocity, _{f f is} the kinematic viscosity of the air, Pr is the Prandtl coefficient of the air, C and n are constants depending on the Reynolds number. The values of the parameters are shown below.

D = 3.75 × 10 ⁻⁴ m k _f = 0.02624 W / m · K u = 1.0 m / s v _f = 1.569 × 10 ⁻⁵ m ² / s Pr = 0. 708 Under the experimental conditions of the present invention, the Reynolds number is 23.9, and the constants of Equation 6 are C = 0.911 and n = 0.385 (Heat Transfer, SIMet).
ric Edition (1989) J. P. H
olman). As a result, from Equation 6, h = 193 W / m
I asked for K.

Using the h value thus obtained, the temperature distribution in the capillary was obtained using the heat transfer equation (Equation 4). In addition, the value of the experimental parameter in that case is shown below.

R ₁ = 3.75 × 10 ^-5 m r ₂ = 1.725 × 10 ^-4 m r ₃ = 1.875 × 10 ^-4 m k ₁ = 0.614 W / m · K k ₂ = 1.5 W / m · K k ₃ = 0.155 W / m · K h = 193 W / m · K Ta = 299 K Shows the temperature distribution in the capillary calculated from the heat transfer equation (Equation 4). 2 shows.

[0034]

[Table 2]

Then, the viscosity (η) of the solution and the mobility (μ ₀ ) of the DNA in the solution were calculated from the calculated temperature change using the _equation ( ₅ ). The reference μ ₀ (T) at this time is a value μ ₀ (23) = 3.5 × 10 ⁻⁵ cm experimentally obtained by extrapolation of% T → 0 and% C → 0 at a temperature of 23 ° C. ² / V · s) (for details, see Electrophoresis, V
ol. 12 (1991) pp. 612-649). Table 3 shows the temperature of the center of the capillary corresponding to each applied voltage, the viscosity of the solution, and the mobility (μ ₀ ) of the DNA fragment in the solution at that time obtained from the above results.

[0036]

[Table 3]

The temperature corresponding to each applied voltage and the value of the mobility (μ ₀ ) of the DNA fragment in the solution obtained by the calculation were introduced into the theoretical formula of the mobility (Equation 3), and the measurement result of the standard sample was further obtained. Was subjected to curve fitting by the method of least squares. The data used are the mobilities of 8 DNA fragments from 234 base pairs to 1353 base pairs. The measured values in the absence (Non) and presence (EB) of ethidium bromide and the curve fitting curve by the least squares method are shown in FIGS. Symbols (○, ●,
Δ, ×, □) are actual measured values at an applied voltage of 100 to 300 V / cm, and the dotted line represents a curve fitting curve.
Parameters obtained by curve fitting at that time,
Table 4 shows the correlation coefficient (R).

[0038]

[Table 4]

A detailed method for preparing a calibration curve for a DNA fragment under another measurement condition according to the embodiment of the present invention will be described.

In the present invention,% T (weight percentage of acrylamide monomer) is constant and% C (weight percentage of bisacrylamide in acrylamide) is 0.5 to 5.0%.
In this case, a calibration curve of the DNA fragment was prepared. For the preparation of acrylamide gel, N, N, N ', N'-as the polymerization agent was used.
Tetramethylethylenediamine and ammonium peroxodisulfate were used. As an optical detector, an ultraviolet absorption detector for HPLC improved for on-column detection was used, and DNA was detected.
The absorbance at a wavelength of 260 nm, which is the measurement wavelength of, was measured.
The measured data from the optical detector was sequentially transmitted to the data processing unit, and the data processing unit recorded and processed the data. Table 5 shows the detailed measurement conditions.

[0041]

[Table 5]

In the present invention, in addition to the air conditioner (wind speed, about 1.0 m / s), an air-cooled positive fan (O
RIX MU925S-11X, wind speed, 2.8 m / s)
It was used. At that time, h was obtained by using Equation 6. The parameter values at that time are shown below.

D = 3.75 × 10 ⁻⁴ m k _f = 0.02624 W / m · K u = 3.8 m / s v _f = 1.569 × 10 ⁻⁵ m ² / s Pr = 0. 708 Under the experimental conditions of the present invention, the Reynolds number is 90.6, and the constants of the equation 6 are calculated as C = 0.683 and n = 0.466 (Heat Transfer, SIMet).
ric Edition (1989) J. P. H
olman). As a result, from Equation 6, h = 348 W / m
I asked for K.

Using the h value thus obtained, the temperature distribution in the capillary was obtained using the heat transfer equation (Equation 4). In addition, the value of the experimental parameter in that case is shown below.

R ₁ = 3.75 × 10 ^-5 m r ₂ = 1.725 × 10 ^-4 m r ₃ = 1.875 × 10 ^-4 m k ₁ = 0.614 W / mK k ₂ = 1.5 W / m · K k ₃ = 0.155 W / m · K h = 348 W / m · K Ta = 299 K The temperature distribution in the capillary calculated by the heat transfer equation (Equation 4) is shown. 6 shows.

[0046]

[Table 6]

Next, the viscosity (η) of the solution and the mobility (μ ₀ ) of the DNA in the solution were calculated from the calculated temperature change using the _equation ( ₅ ). The reference μ ₀ (T) at this time is a value μ ₀ (23) = 3.5 × 10 ⁻⁵ cm experimentally obtained by extrapolation of% T → 0 and% C → 0 at a temperature of 23 ° C. ² / V · s) (for details, see Electrophoresis, V
ol. 12 (1991) pp. 612-649). Table 7 shows the temperature of the central portion of the capillary, the viscosity of the solution, and the mobility (μ ₀ ) of the DNA fragment in the solution at each time, which are obtained from the above results and correspond to each applied voltage.

[0048]

[Table 7]

The temperature corresponding to each applied voltage obtained by the calculation and the value of the mobility (μ ₀ ) of the DNA fragment in the solution were introduced into the theoretical equation of the mobility (Equation 3), and the measurement result of the standard sample was obtained. Was subjected to curve fitting by the method of least squares. The data used are the mobilities of 8 DNA fragments from 234 base pairs to 1353 base pairs. 0.5 ~
The actual measurement value at 5.0% C and the curve fitting curve by the least squares method are shown in FIGS. 5 to 10, respectively. Symbols (○, ●, △, ×, □) are applied voltage 100-300V /
The measured value in cm, the dotted line represents the curve fitting curve. Table 8 shows the parameters obtained by curve fitting and the correlation coefficient (R).

[0050]

[Table 8]

The result of determining the length (N) of the DNA fragment of the unknown sample obtained by the example of the present invention will be described.

In the present invention, restriction enzyme Hind III degradation product of λ-DNA and DQA1 region of human leukocyte antibody gene are used as unknown samples for polymerase chain reaction (Po).
lymase chain reaction, P
The DNA fragment amplified by the CR) method was used. Table 9 shows the detailed measurement conditions.

[0053]

[Table 9]

In the present invention, FIG. 4 (a) was used as the calibration curve. The results are shown in Table 10. As a result, the measured value (value obtained from the calibration curve) was in good agreement with the actual value.

[0055]

[Table 10]

[0056]

According to the example of the present invention, an accurate calibration curve of a DNA fragment can be prepared by using the theoretical formula of the mobility of the DNA fragment in consideration of the influence of the high applied voltage.
Further, the accuracy is improved by considering the influence of temperature at that time. As a result, the length (N) of the DNA fragment of the unknown sample can be easily determined by using capillary gel electrophoresis.

[Brief description of drawings]

FIG. 1 is a block diagram showing the constitution of a method for determining the length of a DNA fragment using the capillary gel electrophoresis according to the present invention.

FIG. 2 is a diagram showing a configuration of a DNA fragment length determination device using a capillary gel electrophoresis method according to the present invention.

FIG. 3 is a diagram showing a temperature distribution in a capillary.

FIG. 4 is a diagram showing a calibration curve of a standard sample obtained in the present invention.

FIG. 5 is a diagram showing a calibration curve of a standard sample obtained in the present invention.

FIG. 6 is a diagram showing a calibration curve of a standard sample obtained in the present invention.

FIG. 7 is a diagram showing a calibration curve of a standard sample obtained in the present invention.

FIG. 8 is a diagram showing a calibration curve of a standard sample obtained in the present invention.

FIG. 9 is a diagram showing a calibration curve of a standard sample obtained in the present invention.

FIG. 10 is a diagram showing a calibration curve of a standard sample obtained in the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Capillary gel electrophoresis apparatus, 2 ... Data processing apparatus, 3 ... High-voltage power supply part, 4, 5 ... Buffer tank, 6 ... Sample injection apparatus, 7 ... Sample stage, 8 ... Standard sample tank, 9
... unknown sample tank, 10 ... migration capillary, 10a ... migration capillary tip, 11 ... optical detector, 12 ... recorder, 13-16 ... platinum electrode, 31 ... gel, 32 ... fused silica, 33 ... polyimide .

Claims (6)

[Claims] 1. In a capillary gel electrophoresis method for analyzing a DNA fragment, a calibration curve prepared using the theoretical formula of mobility (μ) shown below is used to measure the length (N ) Determining the length of a DNA fragment. μ = μ ₀ (T) [A / N {1 / E ′ ² −1 / sinh
² (E ′)} + {coth (E ′) − 1 / E ′} ² ] where μ ₀ (T): mobility of DNA fragment in solution at temperature T ° C. A: depends on gel composition constant N: DNA fragment length E ': specific electric field _{strength, E' = qEa / 2k B} T q: constants dependent on Gerupoasaizu E: applied voltage intensity a: Gerupoasaizu k _B: Boltzmann constant T: temperature 2. A step of measuring the mobility of a standard sample of a DNA fragment in a capillary gel electrophoresis method for analyzing a DNA fragment, and the measurement result of the mobility of the standard sample is used as the DN.
The method comprises the steps of creating a calibration curve by substituting it into the theoretical equation of A fragment mobility, measuring an unknown sample, and determining the DNA length (N) of the unknown sample from the calibration curve. The method for determining the length of a DNA fragment according to claim 1. 3. A device for determining the length of a DNA fragment, wherein a capillary gel electrophoresis device for analyzing a standard sample of a DNA fragment and an unknown sample and the mobility measurement result of the standard sample are used to determine the mobility of the DNA fragment. 2. The data processing device, which is configured to perform the operation of creating a calibration curve by substituting it into a theoretical formula and determining the DNA length (N) of the unknown sample from the calibration curve. DNA fragment length determination device. 4. The method for determining the length of a DNA fragment according to claim 1, wherein the temperature (T) in the capillary gel is determined using the formula shown below. ^{_{T = Gr 1 2 / 4k 1}} + Gr 1 2/2 {1 / k 2 ln (r 2 /
r ₁ ) + 1 / k ₃ ln (r ₃ / r ₂ ) + 1 / r ₃ h} + Ta Here, G: calorific value per unit length k ₁ : heat transfer coefficient of gel k ₂ : heat of fused silica transfer rate k _3: polyimide heat transfer coefficient r _1: inside diameter of the capillary r _2: the outer diameter of the capillary without the polyimide r _3: the outer diameter of the capillary containing polyimide h: heat transfer coefficient Ta: capillary external temperature 5. The length of the DNA fragment according to claim 1, wherein the mobility (μ ₀ (T)) of the DNA fragment in the solution at the temperature T ° C. is determined by using the formula shown below. Decision method. μ ₀ (T) = {η (T ₁ ) / η (T)} μ ₀ (T ₁ ) where η (T ₁ ) is the viscosity of the solution at the temperature T ₁ ° C. η (T) is the temperature T ° C. Viscosity of solution at temperature μ ₀ (T ₁ ): Mobility of DNA fragment in solution at temperature T ₁ ° 6. The DNA according to claim 1, wherein the length (N) of the DNA fragment is 200 base pairs or more.
A method for determining the length of fragments.