JP2914333B2 - Base sequence determination method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for determining the base sequence of DNA or RNA, and more particularly to a method and apparatus suitable for shortening the measurement time.

[0002]

2. Description of the Related Art Conventionally, base sequence determination of DNA and the like has been performed by DN.
The fragment A was labeled with a radioactive element, the pattern obtained by gel electrophoresis separation according to the length was transferred to a photograph by autoradiography, and the DNA band pattern was read. This had the disadvantage that it was troublesome and time-consuming.
Thus, a technique has been developed in which a DNA fragment is separated and detected in real time to determine a base sequence by using a fluorescent label instead of a radioactive label. About the real-time detection method using such a fluorescent label and the light detection type electrophoresis apparatus used for the method,
For example, Journal of Biochemical and
Bioactive Physical Methods 13 (1986) 315-
323 (Journal of Biochemical)
l and Biophysical Methods
13 (1986) 315-323), or Nature 321, December, June, 1986, pp. 674-679 (Nature Vol. 321 12 Ju).
ne 1986).

[0003]

In the above-mentioned prior art, there is a problem that it takes as long as 5 to 10 hours from the start of the electrophoresis to the end of the measurement. The reason why such long-time electrophoresis is required is that the details of the gel electrophoresis phenomenon are not known,
In the real-time detection method, the position resolution of light detection is lower than the position resolution when reading the band by looking at the autoradiogram, and a long electrophoresis path is required. This is due to the fact that the measurement is carried out using a migration separator having a high acrylamide concentration of usually about 8%. An object of the present invention is to provide a method and an apparatus for determining the base sequence of DNA or RNA, which can solve the above-mentioned difficulties and can complete the measurement in a short time.

[0004]

Accordingly, the object of the short-time measurement is achieved by optimizing various conditions of gel electrophoresis. Specifically, it is achieved by setting the polyacrylamide concentration of the gel used for the electrophoresis separator to 2% to 6%. More specifically, the gel concentration C is such that the DNA band is identified and the time t required for the electrophoresis is as small as possible under the electric field strength and temperature at which there is no problem in the measurement.
And the length L of the migration path.

[0005]

Using the migration speed V (N) of the DNA fragment length of base length N, the time t required to migrate the migration path length L can be expressed as (Equation 1).

[0006]

T = L / V (N) (Equation 1) V (N) is proportional to the electric field strength E in the gel, and a proportional coefficient V ₀
(N, C, T) is a function of the number of bases, that is, the base length N, the concentration C of polyacrylamide, and the temperature T of the DNA fragment.

[0007]

T = L / {E · V ₀ (N, C, T)} (Equation 2) When the electric field strength E is increased, the time t required for electrophoresis can be shortened. , The temperature of the DNA band increases, which hinders the separation of the DNA band. Under the electric field intensity E and temperature (T) at which there is no problem in the measurement, a short time measurement is realized by selecting the gel concentration C and the migration path length L so as to identify the DNA band and minimize the time t required for the electrophoresis as much as possible. it can.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to FIGS. FIG. 1 shows an example of a photodetection type electrophoresis apparatus according to the present invention. The excitation laser 1 obtained from the laser source 31 enters the electrophoresis gel 4 from the side through the lens 2.
The electrophoresis gel 4 is held on the quartz plate 3. The sample 32 such as a fluorescence-labeled DNA advances toward the lower end of the gel 4 while being subjected to electrophoretic separation with the upper end of the gel 4 as an electrophoresis starting point, for example. The laser 1 irradiates a certain distance from the electrophoresis start point, and the fluorescence from the fluorescently labeled DNA passing therethrough is collected by the lens 6 with the filter 5, amplified by the image amplifier 7, passed through the relay lens 8, Camera 9
To detect. The obtained signal is processed and output by the computer 10. Instead of inserting the laser from the side, it may scan and irradiate a fixed road from the front. As can be seen from (Equation 2), the migration time is determined by the distance L from the electrophoresis start point to the laser irradiation part, the concentration C of the polyacrylamide gel, the voltage v applied to the upper and lower ends of the gel plate (or the electric field intensity E in the gel). Dependent.

FIG. 2 shows the migration times t of DNA fragments at various gel concentrations. The migration time t of the DNA fragment can be expressed as (Equation 2) from FIG. 2 that the migration speed V (N) is proportional to the electric field strength E. Note that V ₀ (N,
C, T) = f (C, T) N + g (T).

[0010]

T = (L / E) {f (C, T) N + g (T)} (Equation 3) where f (C, T) and g (T) are polyacrylamide concentration C, and It is a function of the temperature T. When the base length is asymptotically reduced to zero, the electrophoresis time t ₀ is L · g (T) / E in (Equation 3).
And does not depend on the concentration C. From (Equation 1) and (Equation 3), V (N) can be expressed as (Equation 4).

[0011]

V (N) = E / {f (C, T) N + g (T)} (Equation 4) The interval d between adjacent bands when electrophoresed for time t is (Equation 5).

[0012]

D = {V (N) -V (N + 1)} t = L / {N + (g (T) / f (C, T))} (Equation 5) From this (Equation 5), the band interval When the value of the base length N is large, d does not depend much on the concentration C of polyacrylamide.
/ N. For example, L = 220 mm
L / N = 2.2, 1.1, 0.73, 0.55 for N = 100, 200, 300, 400 bases
Becomes

FIG. 3 shows the concentration dependence of the measured band interval for various base lengths. Migration distance is 22
cm. When the base length is as short as about 100, increasing the gel concentration C increases the band interval d.
When the gel concentration is more than 00, especially 300 or 400, the band interval becomes almost constant at the gel concentration C and 6%.
It can be seen that the use of more than 10% gel merely leads to an increase in the migration time. On the other hand, it was confirmed from the experiment that the DNA bandwidth ω hardly depends on the gel concentration and is almost proportional to ΔL. Therefore, ω can be represented by (Equation 6).

[0014]

Ω = ω ₀ (N, T) √L (Equation 6) FIG. 4 shows the polyacrylamide gel concentration dependence of the migration time of various base lengths. It can be seen that the migration time increases in proportion to the square of the concentration C. When the temperature is kept constant, the migration time t is a function of the migration path length L and the gel concentration C. At least d to distinguish two adjacent bands
≧ ω is required. Then, with d = ω, the migration time t was made a function of the gel concentration C only, and the gel concentration C that minimized the migration time t was determined. L is obtained as in (Equation 7) from d = ω, (Equation 5) and (Equation 6), and substituted into (Equation 3) to obtain (Equation 8).

[0015]

L = ω ₀ ² (N, T) ｛N + (g (T) / f (C, T))｝ ² (Equation 7)

[0016]

T = ω ₀ ² (N, T) ｛N + (g (T) / f (C, T))｝ ³ / ｛Ef ² (C, T)｝ (Equation 8) (Equation 8) Is used, and when C is changed from (dt / dC) = 0, the C value that minimizes t is the gel concentration to be obtained.

[0017]

(Dt / dC) = {ω ₀ ² (N, T) / (Ef ⁴ (C, T))} × {3 (f (C, T) N + g (T)) ² f (C, T) N−2 (f (C, T) N + g (T)) ³ {f (C, T)} df (C, T) / dC} = 0 (Equation 9) (C, T) N = 2g (T) is obtained. That is, the gel concentration C that satisfies (t−t ₀ ) = (L / E) f (C, T) N = (L / E) 2g (T) = 2t _{0 in} FIG. 4 minimizes t. , Base length is 1
At the time of 00, 200, 300 and 400, 6.2 respectively
%, 4.3%, 3.2% and 2.6%. In actual analysis, analysis of 200 to 300 bases is often performed.
The gel path length L required for the separation is given by (Equation 7) as f (C, T)
It can be obtained by substituting N = 2g (T).

[0018]

L = (9/4) N ² ω ₀ ² (N, T) (Equation 10) ω ₀ (N, T) is the bandwidth of the migration path length L ₀ from (Equation 6). It can be obtained from the measured value ω _obs by ω _obs / L ₀ .

The thickness of the gel plate is set to 0.3 mm, and the electric field intensity is set to 5 mm.
The polyacrylamide concentration C and the migration path length L that minimize the migration time at 0 V / cm and the migration time t at that time are as follows: {base length N: polyacrylamide concentration C (%):
Migration path length L (cm): shown as migration time t (minutes)｝.

{N = 100: C = 6.2%: L = 6 cm: t = 22 minutes} {N = 200: C = 4.3%: L = 15 cm: t = 56 minutes} {N = 300: C = 3.2%: L = 28 cm: t = 106 minutes｝ {N = 400: C = 2.6%: L = 41 cm: t = 156 minutes} The values shown here are only a guide, and can be used for electrophoresis. It varies slightly depending on the voltage, the concentration of the electrophoretic gel, the thickness of the gel, and the like. However, it can be seen that the use of a gel having a polyacrylamide concentration of 2% to 6% is suitable for high-speed electrophoretic separation of a sample containing a DNA fragment having a long base length at a suitable band interval. This concentration range is much lower than the concentration conventionally used.

Under the conditions obtained here, measurement of up to 300 bases can be performed in about 1.5 hours. FIG. 5 shows the results of measurement using a 3% gel at a migration path length of 22 cm. Since the migration path length is shorter than 28 cm, separation is not sufficient, but it can be seen that bases near 300 bases can be identified.
The electrophoresis time of the DNA fragment having a length of 300 bases was 77 minutes.

[0022]

According to the present invention, the measurement of a DNA fragment, which conventionally required 5 to 10 hours, can be performed in less than 1 hour, and the time required for base sequence determination can be greatly reduced. . In the fluorescence measurement, the scattered light and the fluorescence from the gel serve as background light, and the lower limit of detection is determined. However, at a low gel concentration, the background light also decreases, so that there is an advantage that high sensitivity detection is possible.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram of one embodiment of a light detection type electrophoresis apparatus according to the present invention.

FIG. 2 is a diagram showing the relationship between base length and migration time at various polyacrylamide gel concentrations in Examples of the present invention.

FIG. 3 is a graph showing the gel concentration dependency of the band intervals of various base lengths in Examples of the present invention.

FIG. 4 is a graph showing the gel concentration dependence of the migration time of each base length in Examples of the present invention.

FIG. 5 is a view showing a DNA fragment spectrum obtained using a 3% polyacrylamide gel (migration path length: 22 cm) in an example of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Laser, 2 ... Lens, 3 ... Quartz plate, 4 ... Gel plate, 5
... a filter, 6 ... an imaging lens, 7 ... an image amplifier,
8: relay lens, 9: vidicon camera, 10: computer, 11 to 16: 2%, 3%, 4%, 5%, 6 respectively
%, Relationship between electrophoresis time and base length when using 8% polyacrylamide gel, 17 to 20 ... 100, 200, 300
And the change in the band interval or the migration time of the DNA fragment of 400 bases depending on the gel concentration, the spectrum of the group of fragments ending with guanine (G) at 16..., The spectrum of the group of fragments ending with thymine (T) at 17 ′. ... laser source, 32 ... sample.

Continuation of the front page (56) References JP-A-61-173158 (JP, A) Edited by Koichiro Aoki and Hiroshi Nagai, “Latest Electric Swing Method,” Hirokawa Shoten p. 378-386, p. 409 (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 27/447

Claims (1)

(57) [Claims] 1. A method according to claim 1, wherein the fluorescence-labeled fragment of the nucleic acid sample is electrophoresed using a migration medium, and a laser beam is irradiated to a light irradiation unit at a predetermined distance from a migration start point of the migration medium. A method for determining the nucleotide sequence of the nucleic acid sample by detecting the fluorescence generated from the fluorescent label of the fragment and obtaining the spectrum of the fragment, wherein the polyacrylamide concentration in the electrophoresis medium is 2% to 6%. %, And the polyacrylamide concentration and the predetermined distance are determined according to the base length for which the base sequence is to be determined, and the polyacrylamide concentration is low or high depending on the length or shortness of the base length. Wherein the predetermined distance is set to a long or short distance in accordance with the length or shortness of the base length.