JPS63124953A - Electrophoresis method - Google Patents

Abstract

PURPOSE:To separate component molecules having a specified mol.wt. difference on a gel at equally spaced distances without depending on the mol.wt. thereof by imparting the electric field intensity decreasing monotonously in accordance with a certain function to a migration direction. CONSTITUTION:The gel 1 for analyzing a nucleic acid sample is so molded that the front surface 3 thereof is curved and the rear surface 2 thereof is flat. The curved surface 3 is formed to kaT=k1Ta<2/3>, k1T=0.515, V0=17.5, a=100, L=50, lambda=0.2, in the equation I when the thickness of the section is designated as f(y). Said surface is so formed that the thickness f(y) of the section is a function of the distance y in the migration direction. The electric field intensity is A(B-Cy)<1->D exp{E(B-Cy)D} (where A-E are positive real constants) if a voltage is impressed across the gel. The component molecules having the specified mol.wt. difference are thereby separated on the gel at the equally spaced distance without depending on the mol.wt. thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for separating and analyzing proteins, nucleic acids, etc., and particularly to an electrophoresis method suitable for separating high molecular weight components of these substances.

[Conventional technology]

As is well known, the migration distance, y, of a charged molecule placed in an aqueous solution is expressed by the following equation (2), where Ex is the electric field strength applied to the solution, t is the migration time, and unit time is The migration speed per unit electric field strength is uo.

It is given by y==uoEzt-(2). In a gel-like medium, the procedure of
National Academy of Sciences (Proc N
at'l Acad, Sci, ) Volume 65, 970~
As discussed on page 977 (1970), the migration distance decreases from the value given by (2), y = uoEt t e−” ”(3
) is given by Here, k is a parameter that has a positive correlation with the size of the migrating molecules, and T is a parameter that is proportional to the gel concentration, both of which are positive real numbers. (3
) According to the equation, molecules can be separated because their migration distances differ depending on their size. In addition, denatured nucleic acids and
It is known that in protein molecules, uo does not depend on the molecular weight and remains a substantially constant value.

[Problem that the invention seeks to solve]

Based on the above principle, in the conventional technology, the electric field strength E□ is constant throughout the gel (if the gel length is L and the voltage difference between both ends of the gel is Eo, the thickness of the gel is constant, so E t = E
o/L), and there was a problem in that the difference in migration distance between two component molecules having a certain molecular weight difference rapidly decreases as the molecular weight of the components increases, making them impossible to separate.

An object of the present invention is to overcome this problem and provide a method for separating component molecules having a certain molecular weight difference at equal distances on a gel, independent of their molecular weights.

Furthermore, problems for explaining the second invention of the present application will be shown.

Based on the above principle, in the conventional technology, the electric field strength Ex is constant throughout the gel (the gel length is L, and the voltage difference between both ends of the gel is E).
If o, the thickness of the gel is constant, so E i = E o
/L), and the difference in migration distance, Δy, between two component molecules with a certain molecular weight difference rapidly decreases as the molecular weight of the components increases, leading to the problem that the separation bands overlap and become impossible to separate. there were. This is because the separated components diffuse in the gel and exist in a spread band shape.

The separation power of electrophoresis is maximum when Δy=λW (34), where W is the width of the separated band and λ is the minimum number (positive real number) necessary to confirm the separation of the bands. . λ is a parameter that depends on the performance of the device used to detect the band, the shape of the band, etc. When Δy is larger than λW, so to speak, unnecessary excessive separation occurs. In the conventional electrophoresis method, Δy is too large for low molecular weight components, and it can be said that gel sites that should be used for separating high molecular weight components are wasted. An object of the present invention is to overcome this problem and provide a separation method in which the relationship of formula (34) always holds true on a gel, regardless of the molecular weight of the separated components.

[Means for solving problems]

The above object is achieved by setting El to decrease in the direction of migration. This is clear from equation (3). In this case, as a method of changing the electrophoresis speed, or, if we will discuss it quantitatively below, the electric field strength depending on the position of the gel, for example, a method of increasing the thickness of the gel in the electrophoresis direction is used. The thickness of the gel at position my is f (y),
Let us consider a gel 1 whose bottom surface 2 is flat and whose top surface 3 is curved, as shown in FIG. 2, assuming the potential to be E.

Here, γ is the specific resistance of the gel, and Ro is the total resistance.

The total resistance is determined by the depth (thickness f (y) axis 4
(direction perpendicular to ) is defined as unit length.

In this case, let L be the length of the gel in the migration direction.

From equation (4), becomes. Solving this differential equation O ...(8) F(0) is the value of F(y) when 1=0.

Now, considering the separation of macromolecules such as nucleic acids and proteins whose molecular weight is an integer (generally referred to as n) times the basic molecular weight (the molecular weight of mononucleotides is approximately 340, and the molecular weight of amino acids is approximately 100), one objective of the present invention is to is y=L−λ(n−a)
...The degree of polymerization n is set so as to satisfy the relationship (9).
The purpose is to determine the migration distance y of the component. Here, a is the degree of polymerization of the component that migrated only up to the end point of the gel, and λ is the difference in migration distance between components whose degree of polymerization differs by 1.0 Give the relationship between the molecular size parameter and n. For example, k (y) can be found as a function of y from equation (9).

Therefore, from equation (8), the shape of the gel we are looking for, ie, f (y), is dy Rod, y (10). From equation (10), it immediately becomes Rody. Therefore, the electric field strength E, is uot from equation (5).
dy Here, t is the time required for a component with a degree of polymerization a to migrate by L, and if this is specifically written as ta, then Vo is the volume of the gel (the unit is the distance on the upper plane of the gel in the direction perpendicular to the y-axis). ka is the molecular size parameter of the component with the degree of polymerization a. Therefore, from equation (11), it can be written as dy...(14). Also, from equation (12).

...(15) It can be written as

The gel shape shown in FIG. 2 is not the only one in which components having a certain molecular weight difference are arranged at equal intervals on the gel.

For example, a kind of conical gel (Fig. 3, 5) formed by rotating f (y) around the migration axis in Fig. 2 may be used. In this case, the thickness of the gel at the 1st position My (the rotating body (radius of gyration) as f (y), equation (5) is given by π as pi.

becomes.

Hereinafter, considering the curved gel in FIG. 2, instead of equation (14), we obtain...(17). Also, instead of equation (15), we obtain. It should be noted that equations (15) and (18) have the same functional type when y is used as a variable. Although there are various shapes of gels, the type of electric field strength distribution that provides an evenly spaced migration pattern is the same.

Furthermore, as expected from equation (3) which describes the means for solving the problem of the second invention of the present application, the above object can be achieved by setting Ex to change with respect to the electrophoresis direction. be done.

Exists and changes. That is, in the following quantitative discussion, as a method of changing the electric field strength depending on the position of the gel, for example, a method of changing the thickness of the gel with respect to the electrophoresis direction is used. Letting the thickness of the gel at position y be f(y) and the potential as E, consider a gel 11 whose bottom surface 2 is flat and whose top surface 3 is curved as shown in FIG. 4, where γ is the ratio of the gel. The resistance, Ro, is the total resistance.

The total resistance is determined by the depth (thickness f (y) axis 4
(direction perpendicular to ) is defined as unit length.

In this case, let L be the total length of the gel in the migration direction.

From equations (35) and (36), dt Rof(y) becomes. Solving this differential equation ...(39) F(0) is the value of F(y) when 1=0.

Now basic molecules like nucleic acids and proteins! (The molecular weight of mononucleotides is approximately 340, and the molecular weight of amino acids is approximately Zoo. Migration distance 11f of component with degree of polymerization n
The key is to determine ly. Here, a is the degree of polymerization of the component that migrated only up to the end point of the gel, and Wi is the width of the separation band of the component with the degree of polymerization i. If we give the relationship between the molecular size parameter and n, and the relationship between Wn and n, we get (4o
), k (y) can be found as a function of y. Therefore (
39) From the formula, we can find the shape of the gel we are looking for, that is, f
(y) becomes, and from equation (11), it immediately becomes...(32). Therefore, the electric field strength E1 is given by equation (36).

Here, t is the time required for a component with a degree of polymerization a to migrate by L, and if this is specifically written as ta, then V Ll is the volume of the gel (the distance in the direction perpendicular to the y-axis on the flat surface of the gel). From formula (39), γuoE.

ka is a molecular size parameter of a component with a degree of polymerization a.

Therefore, from equation (42), ...(45) becomes. Also, from equation (13).

... (46) It can be written as

The shape of a gel for arranging components having a certain molecular weight difference on a gel according to equation (40) is not limited to that shown in FIG. 4. For example, a kind of conical gel (FIG. 5) formed by rotating f (y) around the migration axis in FIG. 4 may be used. In this case, since the radius of one rotation is f (y),
Equation (36) becomes as follows, where π is the constant of pi. Below, considering the same way as the curved gel in Figure 4, (4
5) Instead of Eq., we obtain (48). Also, instead of equation (46).

... (49) is obtained. It should be noted that equations (46) and (49) have the same functional form with respect to y.

[Operation] By applying an electric field strength as shown in the above equation (15) or (18) to the gel, components having a certain difference in molecular weight can be separated in the gel with a certain difference in migration distance. That is, according to the present invention, the difference in migration distance between the two formulas is proportional to the difference in molecular weight. As a result, high molecular weight components can be separated based on the same molecular weight differences as low molecular weight components.

Furthermore, the second invention will be described. (16) above or (
By applying an electric field strength as shown in the formula (19) to the gel, components having a certain molecular weight difference are arranged tightly on the gel, so to speak, with the separation bands in contact with each other. That is, according to the present invention, a maximum number of components can be separated.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples.

The sample to be separated used is a mixture of multiple components (sample for base sequencing based on the well-known M13 method) with deoxyribonucleic acid polymerization degrees differing by 1, and each component is labeled with radioisotope 8Zp. First, as a preliminary experiment, the above nucleic acid sample whose base sequence was known was analyzed by conventional electrophoresis.

That is, a gel with a polyacrylamide concentration of 8%, a distance of 50 cm in the migration direction, and a constant thickness (0.35 m+a) (the gel was made of 7 M urea and 90 mM Tris-borate buffer PH).
8, 3 and 1mM ethylenediaminetetraacetic acid)
The above sample was electrophoresed using a 1.2 kV voltage applied to both ends of the gel. After electrophoresis, an X-ray film was placed on top of the gel, and an autoradiogram was prepared using amp beta rays in a conventional manner. Analyzing the relationship between the migration distance y of each nucleic acid component appearing on the autoradiogram and the degree of polymerization n, we found that if 1 degree of polymerization n is kti-, then knT=kzTn
” - (19) was found to hold, where T is the length of gel fiber per unit gel volume.

It was found that kzT=0.0515. Applying this result to both equation (9) and equation (14).

We obtain λ f(y)=Voe. Further, the formula (15) becomes the formula %, kaT=kzTa'', where the formula (21) is placed in the formula (1).

In this example, a nucleic acid sample was analyzed using the curved gel 1 shown in FIG. In formula (2o), kz
T=0.0515. Vo=17.5cc.

a=100. Assuming L=50cm and λ=0.2cm, f
A specific functional form of (y) (Fig. 2, 3) was determined, and a gel was molded using a stainless steel mold. Using this gel,
When nucleic acids were analyzed for the same sample under the same electrophoresis conditions as previously performed using a uniform thickness gel, only components in the polymerization degree range of 100 to 165 were observed to be separated using the uniform thickness gel. In the curved gel, separation of components with a degree of polymerization ranging from 100 to 350 was observed. According to this embodiment, about 3.8
It is now possible to analyze nucleic acids over twice the molecular weight range. In addition, FIG. 1 shows the electric field strength Ez (formula (2)) used in this example.
The distribution pattern [9] was shown. El decreases monotonically with y. As in this example, monotonically decreasing Ei is a necessary condition for increasing the separation ability of high molecular weight components. For this purpose, the fact that D<1 is a necessary condition in equation (1) can be easily proven by calculating the differential of E with respect to y. If D>1, any glue.

y Example 2 The sample to be separated used is a mixture of multiple components with deoxyribonucleic acid polymerization degrees differing by 1 (sample for base sequencing based on the well-known M13 method), and each component is labeled with radioisotope 8Zp. has been done. First, as a preliminary experiment, the above nucleic acid sample whose base sequence was known was analyzed by conventional electrophoresis. That is, the polyacrylamide concentration is 8%, the distance in the migration direction is 50 cm, and the thickness is constant (0.35 cm).
The above samples were analyzed using a gel containing 6.33 M urea and 90 mM Tris-borate buffer pH 8.3 and 1 mM ethylenediaminetetraacetic acid.
A voltage of 1.2 kV was applied to both ends of the gel and the gel was run at 50°C. After electrophoresis, an X-ray film was placed on top of the gel, and an autoradiogram was created using beta rays of δzP in a conventional manner. By analyzing the relationship between the migration distance y of each nucleic acid component appearing on the autoradiogram and the degree of polymerization n, we found that k of the degree of polymerization n
If we set it to 7, then ! It was found that kI,T=ktTn8-(50) holds, and kzT=0.0515. Furthermore, as a result of measuring the separated gel using beta ray detection, the shape of the separated band is expressed by a normal distribution, and the width of the separated band defined as twice the standard deviation is... (51) I understand. Here, Dn is the diffusion coefficient of a single-stranded molecule of a nucleic acid with a degree of polymerization n. In this experiment, DI
= 7.00 X I 0-6cm"/S.

t is the migration time.

The results of equations (50) and (51) are combined with equation (40) into equation (4).
Applying to 5)...(52) is obtained. Here, the differential of h (n) with respect to n, h
'(n), h'(n)=-λWn.

Furthermore, RoVokIT can be obtained from equation (36). Note that equation (23) is placed in equation (1).

Now, in this example, six equations (52) and (40) were used to analyze a nucleic acid sample using the curved gel 1 shown in FIG.
Niite, kzT=0.0515°Vo=1,88
cc, λ=2. L=25cm, DI=7, OOXlo
-Bcm”/S, ta=4hrs, a=30, f
A specific functional form of (y) (Fig. 4, 3) was determined, a stainless steel mold was made, and a gel was created.

Using this gel, we analyzed nucleic acids on the same sample under the same electrophoresis conditions as when using the uniform thickness gel above, and compared the results. Only components in positions 30 to 70 are separated 2
However, in the thickness gradient gel, the polymerization degree range was 30.
~200 components were analyzed 1 Comparing the degree of polymerization that can be analyzed, the degree of polymerization in the uniform thickness gel was 40, but the degree of polymerization in the thickness gradient gel was 170, and the sequence was determined for more than 4 times the number of nucleic acid bases. did it.

As can be seen by differentiating the right side of equation (42) with respect to n (or y), f (y) has a maximum at n = (2ktT). For kxT = 0.0515, n
=30. Care must be taken when analyzing components where n is 30 or less.

〔Effect of the invention〕

According to the present invention, biopolymers such as nucleic acids and proteins can be separated and analyzed with higher resolution for higher molecular weight components than conventional techniques, making it suitable for a wide range of research and production in biology, medicine, biotechnology, etc. It is valid. In particular, in the case of determining the base sequence of a nucleic acid, since the analysis sample is a mixture of many components with a molecular weight difference of about 340, the present invention allows us to calculate the number of bases that can be determined in one electrophoretic separation (the number of bases that can be separated). If it increases, the effect of sequencing will increase.6 Also, in sequencing, determination errors become a problem. According to the present invention, since the intervals at which the bases (nucleotides) migrate are equal, the migration positions can be accurately predicted, and the accuracy of reading and analyzing migration patterns is also increased.

Next, the second invention of the present invention will be described.

According to the present invention, biopolymers such as nucleic acids and proteins can be separated and analyzed with higher resolution9 for high-molecular-weight components than before, making it effective for a wide range of research and production in biology, medicine, biotechnology, etc. be. Particularly in the case of determining the base sequence of nucleic acids, an analysis sample is a mixture of many components with a molecular weight difference of about 340, so the present invention increases the number of bases that can be determined in one electrophoretic separation (the number of bases that can be separated). This increases the efficiency of sequencing.

Further, according to the present invention, even if a smaller gel (gel with a shorter migration distance) than before is used, it is possible to separate the same or higher amount of polymer components than before, so the entire apparatus can be miniaturized.

[Brief explanation of the drawing]

Fig. 1 is a distribution diagram of electric field strength in an embodiment of the present invention, Fig. 2 is a cross-sectional view of a gel whose upper surface is curved and whose lower surface is flat, and Fig. 3 is a cross-sectional view of a conical gel whose upper and lower edges are curved. 4 is a sectional view of a gel with a curved top surface and a flat bottom surface; FIG. FIG. 2 is a cross-sectional view of a conical (rotating body) shaped gel with a curved line at the bottom. 1... Curved gel, 2... Bottom surface of curved gel (migration axis)
, 3...Top surface of curved gel, 4...Gel axis, 5...
・Conical gel, 6... Center line of conical gel (migration axis), 7
...Side curved surface of conical gel, 8...Gel axis, 9...
Electric field strength as a function of migration distance.

Claims (1)

[Claims] 1. In an electrophoresis method using a gel-like substance as a separation medium, an electric field strength that monotonically decreases in accordance with a function type as shown in the following equation (1) with respect to the migration direction. An electrophoresis method characterized by imparting. Electric field strength = A(B-Cy)^1^-^De^E^(^B
^-^C^y^)^^D... (1) (Here, y is the distance in the migration direction, and A, B, C, D, and E are positive real constants.) 2. Gel-like In an electrophoresis method that uses a substance as a separation medium to separate a mixture of components having molecular weights that are an integer number, n, times a certain molecular weight, the following equation (
31) An electrophoresis method characterized by applying an electric field strength shown in 31) to a gel. Electric field strength = An＾Be＾C＾n＾F...(31) (However, y=L−λΣ＾n_i_=_a_+_1Wi Here, A, C, F, and λ are positive real numbers, and B is a negative real number. , L
is the total length of the gel and the migration distance of the component with the degree of polymerization a,
Wn indicates the width of the separated band of the na-th separated component counted from the gel edge. )