JP2002506204A - Purification and detection process using reversible affinity electrophoresis - Google Patents

Abstract

  (57) [Summary] Simultaneously altering one or more properties of an electrophoretic medium between two states characterized by being suitable or not suitable for specific reversible binding of a sample analyte to an affinity ligand immobilized in the medium. While describing the affinity electrophoresis process, changing the direction of electrophoresis in a periodic manner. The resulting steps allow for the separation of specific analytes for detection or purification very efficiently and conveniently using simple materials and equipment.

Description

DETAILED DESCRIPTION OF THE INVENTION

Related Application This application is related to US Provisional Application No. 60 / 076,614 (filed March 3, 1998,
Name: purification and detection process using reversible affinity electrophoresis). The teachings of which are all incorporated herein by reference.

BACKGROUND OF THE INVENTION Zone electrophoresis, particularly gel electrophoresis, is the best known method for separating, purifying and characterizing charged molecules, especially macromolecules such as proteins or nucleic acids. (Freifelder, Physical Bi
Chemistry, 2nd edition (1982), pp. 276-310, Freem
an, San Francisco). Electrophoresis can be used to separate molecules based on their size, charge, conformation, and many combinations of these properties.

[0003] In most electrophoretic applications, charged molecules move through a supporting medium under the influence of an electric field. In most cases, electrophoresis is performed using a linear, constant voltage gradient with fixed orientation (two fixed electrodes, constant voltage). However, for very large DNA molecules (i.e., the size range is 30 kb-2000
kb), the polymer chains are oriented according to the electric field and meander in the gel. This makes the sieving action of the electrophoretic medium insufficient. To separate large DNA molecules, researchers have called "field inversion gel electrophoresis.
sis) "(Carle et al., Science (1986), 232: 65) or" pulse field "gel application (Schwartz and Cantor, Cel).
1 (1984), 37:67), an application was developed that periodically changes the direction of the electric field. Another technique is to apply a constant electric field in a periodic pulsed fashion. Finally, a method is described that combines the continuation of both alternating electric and pulse fields (Bio-Rad Life Sciences Product).
ence Research) catalog (1997), pp. 175-182).

[0004] In most electrophoretic applications, the support medium acts to prevent convection and diffusion and may be sieved or non-sieved. In affinity electrophoresis, the support medium also interacts specifically or non-specifically with one or more desired analytes, and thus affects their mobility and thereby the analyte and non-analyte during purification. The analyte is modified with a chemical group (ie, a ligand) to help achieve separation of the sample components.

[0005] Affinity electrophoresis has been used to measure the binding affinity of proteins (Horejsi and Kocourek, Biochim. Biop.
hys. Acta (1974), 336: 338-343 and Chu et al.
Med. Chemistry (1992), 35: 2915-2917). In addition, polyacrylamide media modified with vinyl-adenine have been used to enhance nucleic acid separation in capillary electrophoresis (Baba et al., Ana).
Lytical Chemistry (1992), 64: 1920-1924.
).

Although many improvements have been made in the resolution of electrophoresis, many biological macromolecules containing only slight structural differences (eg, point mutations in proteins or nucleic acids) are still satisfactory. Cannot be separated as much. In order for researchers to have the opportunity to further explore and understand biological systems, analytical techniques are needed to improve the separation of biological molecules.

SUMMARY OF THE INVENTION An affinity electrophoresis process is described that changes the direction of an electric field in a periodic manner while simultaneously changing one or more properties of an electrophoretic medium between two states. The property (s) being changed in the first state
Advantageously, the specific and reversible binding of the sample analyte to the affinity ligand immobilized in the medium. In the second state, the property (s) being altered favors binding of the sample analyte to the immobilized affinity ligand. This process provides a convenient way to obtain high resolution separations.

In another aspect, an apparatus for separating a target analyte from a test sample is described. The device comprises an electrophoretic medium having immobilized affinity ligands, an electrode system having one or more electrodes capable of generating a reversible electric field, and one or more properties of the electrophoretic medium. It has the means to change between two states. In the first state, the property (s) being altered favors specific and reversible binding of the sample analyte to the affinity ligand immobilized in the medium. In the second state, the property (s) being altered favors binding of the sample analyte to the immobilized affinity ligand. The means for changing the properties of the electrophoretic medium may be a device for changing the temperature of the electrophoretic medium. Or,
The means for changing one or more properties of the electrophoresis medium may be to change the electrophoresis buffer manually or automatically.

DETAILED DESCRIPTION OF THE INVENTION The present invention disclosed herein relates to an electrophoresis process for separating sample components, and to an apparatus designed to perform this process, which has the following features: 1) using an electric field that changes direction at least once during the process; and 3) the analyte (s). A change in at least one other media property that affects the ability of the affinity ligand (s) to form a specific binding complex with the sample, which coincides with a change in the direction of the electric field, thereby causing Changes in media property (s) that allow for electrophoretic separation of analyte and non-analyte components.

[0010] The combination of these features provides a novel invention that is widely used for the separation, purification, and detection of molecules, including proteins, nucleic acids, and other charged species.

In one preferred embodiment of the invention, the process is a general method for performing a repetitive cycle of affinity separation to purify a particular analyte in a biological or test sample. In this embodiment, the analyte molecules are purified (eg, isolated or separated) from non-analyte sample components.
. Each cycle features two electrophoresis steps. The first step involves a first electric field orientation and a first media condition (also referred to herein as a "state").
This is done using This condition allows the formation of a specific binding complex between the sample analyte and the affinity ligand in the medium. Under such conditions, only non-analyte sample components migrate through the medium, allowing fractionation of the sample based on the affinity of the analyte for the ligand. The next step is performed under a second electric field orientation and a second medium condition obtained by changing one or more properties of the electrophoretic medium from the first medium condition. This second condition is designed to disrupt the formation of a specific binding complex between the sample analyte and the affinity ligand in the medium. In this step, all sample components move to new locations in the medium. When this step is partially or completely completed, the particular analyte has been separated from the other sample components, and both fractions have moved to new locations in the medium.

[0012] For a given sample / analyte / ligand combination, a single cycle can provide sufficient purification of the analyte for many applications. This purification cycle can be repeated if further purification is desired. For example, in the second cycle, purification proceeds as in the first cycle. However, the starting material of the second purification cycle is a partially fractionated product of the first purification cycle, which is now at a new location on the electrophoretic medium. The locations of the analyte and non-analyte fractions from the first cycle may or may not overlap, depending on the degree of purification achieved in the previous cycle. In each case, in the second and subsequent cycles, the two fractions are further separated. Thus, any number of affinity purification cycles can be performed in a single electrophoresis unit in an automated manner by successively removing non-bound sample components in each cycle. In systems where there is strong binding between the affinity ligand and the analyte immobilized on the gel and low non-specific binding of non-target molecules with respect to the affinity ligand, a small number of cycles (eg, 1-10 cycles) ) Allows the required purification to be obtained. In other cases, if the ligand-target binding is weak, or if there is significant binding of the non-target molecule to the affinity ligand, a number of cycles (eg, 10 to several thousand times) may cause the components to separate May be required.

[0013] The repetitive nature of this process allows for very efficient electrophoretic purification of analyte molecules. For example, when the purification efficiency of each cycle is 10 times (for example, 90 times)
(If% non-analyte sample components are removed each cycle) Four cycles provide 10,000 fold purification.

An important advantage of the present invention is that the periodic purification process can be performed in one device, such as an electrophoresis gel. This simplifies the purification process by eliminating preparation, loading and fraction collection from multiple columns.

In addition, for applications involving simple changes in state, such as temperature, to control ligand / analyte interactions, this process involves little (or no) moving parts.
It can be done in an automated fashion using equipment without.

A description of the key components of the invention and a non-limiting extension are set forth below.

Test Sample A test sample can be any sample from any source in which analyte molecules are mixed with non-analyte molecules. An analyte molecule is any molecule of interest that can form a binding complex with an affinity ligand. Specifically encompassed by the present invention are samples derived from biological sources containing cells, using known techniques, for the preparation of bodily tissues (eg, skin, hair, internal organs) or A sample obtained from a body fluid (eg, blood, plasma, urine, semen, sweat). Other sources of samples suitable for analysis by the methods of the present invention are microbiological samples such as viruses, yeasts and bacteria: plasmids, isolated nucleic acids, and agricultural sources such as recombinant plants.

[0018] The test sample is processed in such a manner known to those skilled in the art so that the analyte molecules contained in the test sample are available for binding. For example,
Cell lysates can be prepared and such crude cell lysates (eg, containing the target analyte as well as other cellular components) can be analyzed. Alternatively, the target analyte can be partially isolated (so that the target analyte is substantially free of other cellular components) prior to analysis. Partial isolation can be performed using known laboratory techniques. For example, DNA, RNA and proteins can be obtained from various biological samples using TRI reagents (Sigma catalog, 154).
See page 5, catalog numbers T9424, T3809 and T3934;
Chomczynski et al., Biochem. (1987), 162: 156;
Chomczynski Biotechniques (1993), 15: 5.
32 (see also Southern Blotting (DNA), Northern Blotting (RNA) and Western Blotting (Protein) techniques. Antibodies can be isolated by binding to protein A immobilized on a solid support (Surolia et al., Tre.
nds Bioch. Sci. (1981), 7:74 and Sigma catalog, page 1462, catalog number PURE-1). Nucleic acid analytes can also be amplified (eg, by polymerase chain reaction or ligase chain reaction techniques) prior to analysis.

Affinity Ligand that Reversibly Binds to an Analyte An affinity ligand is any molecule that can form a specific binding complex with an analyte and can be immobilized in a suitable electrophoretic medium. It is.

[0020] Methods for determining the thermal stability of binding complexes, especially hybridization complexes, are well known in the literature. Wetmur, Critical
Reviews in Biochemistry and Molecul
ar Biology, 26: 227-259 (1991); Quartin and Wetmur, Biochemistry, 28: 1040-1047 (1).
989). Applying these methods to assess the stability of an analyte / affinity ligand complex involves the following reactions:

[0021]

(Equation 1)

Wherein D and D ′ are affinity ligands and analytes such as the first nucleic acid and a second nucleic acid containing a region complementary to the sequence of the first nucleic acid, and B is the analyte / affinity The ligand complex product, where k ₂ and k _r are
Kinetic rate constants for the formation and dissociation of the analyte / affinity ligand complex. In this equation, the reverse reaction is most related to the consideration of spontaneous dissociation of the analyte / affinity ligand complex, and the rate constant for dissociation, k _r , enhances the binding between the analyte and the affinity ligand. It is an important variable that needs to be minimized. For certain analyte / affinity ligand complexes,
Dissociation can be reduced by lowering the assay temperature; this reduces the dissociation constant.

When the magnitude of the dissociation constant is determined for one experimental temperature, the Arrhenius equation (2
) Can be modified as follows to calculate k _r for other temperatures:

[0024]

(Equation 2)

Where k _{r 1} and k _{r 2} are the dissociation rate constants of the analyte / affinity ligand complex at temperatures T ₁ and T ₂ , Ea is the activation energy for dissociation, and R is the universal gas Is a constant. In the case of a nucleic acid analyte / affinity ligand complex, the Ea term can be calculated from the base sequence of the nucleic acid sequence used to form the analyte / affinity ligand complex. Wetmur, Crit
Ial Reviews in Biochemistry and Mol
ecology Biology, 26: 227-259 (1991). Using the Arrhenius equation for this calculation is described in Tinocco et al. (Physical).
Chemistry: Principles and Applicatio
ns in Biological Sciences, Prentice H
all (issued), Englewood Cliffs, NJ, 290-294
P. (1978)).

If the analyte / affinity ligand binding reaction occurs in discrete regions of a solid support matrix, such as an electrophoresis matrix, the temperature gradient method can be used to estimate the effective dissociation constant. it can. The melting behavior of the analyte / affinity ligand complex immobilized in the electrophoresis gel can be measured using a temperature gradient that increases in the lateral direction of the gel. 50% of the complex has an electrophoresis time t
The temperature Td dissociated during a can be used to estimate the dissociation constant.

If we consider dissociation as a first-order reaction with a kinetic rate constant of k _r , k _r follows in Td:

[0028]

(Equation 3)

Thus, by using a temperature gradient gel, k_rEffective values of Td and t
a can be measured. k_rOnce is evaluated at Td, use equation (3)
Thus, k at other lower temperatures that may be suitable for the first media condition_rCalculate
Can be In this case, the conditions are the sample analyte and the affinity ligand
Are selected to allow the formation of a specific binding complex between Then k _r Using these calculated values for the first order rate equation, the given assay temperature ta
And analyte / affinity ligand complex remaining at electrophoresis time ta
The fraction of the body can be calculated.

[0030]

(Equation 4)

Where B is the concentration of the remaining analyte / affinity ligand complex at time ta and Bo is the initial concentration of the complex. Equation (6) can be used to estimate the change in k _r required to increase B / Bo (reduce the dissociation of the analyte / affinity ligand complex) to any given amount. it can. If desired the value of k _r is once revealed, could be using equation (3), calculating the change in temperature required to achieve such k _r value.

The gradient gel method must noted that only provide an estimate of the Td and k _r of the actual analyte / affinity ligand complex. This is because the detached analyte can re-bound to the uncomplexed immobilized affinity ligand. In general, experimentally determined values overestimate the actual Td and underestimate the actual k _r for the reversible analyte / affinity ligand complex dissociation reaction. Nevertheless, equation (1)
The quantitative relationships shown in (6) design a reasonable and practical framework for predicting the stability of the analyte / affinity ligand complex, and media conditions for the separation protocol.

One particularly useful example of an affinity ligand is a single-stranded nucleic acid that can bind to an analyte containing a complementary nucleic acid sequence, for example, by hybridization. The affinity ligand of a single-stranded nucleic acid can be complementary to the entire analyte nucleic acid sequence or to a portion thereof. Single-stranded nucleic acids can also be used to isolate double-stranded nucleic acids by triplex formation (Hogan and Kessler, US Pat. No. 5,176,966 and Cantor).
Et al., US Pat. No. 5,482,836, the teachings of which are incorporated herein by reference). Double-stranded nucleic acids can also serve as useful affinity ligands for nucleic acid binding proteins or for nucleic acid analytes that bind to a ligand by forming a triplex or quadruplex.

The conditions under which a single-stranded nucleic acid binds to another nucleic acid immobilized in a gel use the techniques outlined above to estimate the stability of the analyte / affinity ligand complex. Can be estimated. Further, the melting temperatures (Tm) of two nucleic acids provide a reasonable framework for estimating the temperature at which a nucleic acid analyte will hybridize to a nucleic acid affinity ligand. Generally, Td is about 15 times greater than Tm.
The temperature at which the gel must be engineered to facilitate specific hybridization between the analyte and the affinity ligand must be between about 15 ° C. and 25 ° C. below the Tm. Must.

[0035] The Tm of a pair of nucleic acids is typically the same as the UV absorption of a solution of two nucleic acids in an electrophoresis buffer while uniformly changing the properties of the solution that changes periodically during electrophoretic separation. Determined by monitoring the physical properties of For example,
The temperature can be lowered slowly while monitoring the UV absorption. At elevated temperatures, nucleic acids are single-stranded. As the temperature decreases, complementary bases pair and form hydrogen bonds. This hydrogen bond causes a change in UV absorption. If the nucleic acids are complementary, the transition between the hydrogen-bonded and non-hydrogen-bonded states occurs over a narrow temperature range. The midpoint of this temperature range is the Tm of the two nucleic acids. Similarly,
The ionic strength or pH of the buffer can be varied in a uniform manner while keeping the temperature constant and monitoring the UV absorption.

[0036] Nucleic acids form duplexes more easily at higher ionic strength and lower temperatures. “Stringency conditions” for hybridization are technical terms that refer to conditions of temperature and buffer concentration (ionic strength) at which a particular nucleic acid can hybridize to a second nucleic acid. In this case, the first nucleic acid can be completely complementary to the second nucleic acid, or the first and second nucleic acids can be
Nucleic acids may have some degree of complementarity that is less than perfect. For example, a number of high stringency conditions can be used to distinguish perfectly complementary nucleic acids from less complementary nucleic acids. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridization are described in Current Protocols in Molecular Bi.
pp. 2.10.1 to 2.10.16 (especially 2.10.8 to pp. 213) of Vol.
(See page 11) and pages 6.3.1-6. This teaching is incorporated herein by reference. The exact conditions that determine the stringency of hybridization include ionic strength, temperature, and concentration of destabilizing agents such as formamide, as well as the length of the nucleic acid sequence, base composition, mismatch ratio between the sequences to be hybridized, And within other non-identical sequences will also depend on factors such as the frequency of occurrence of subpopulations of such sequences. Thus, high or moderate stringency conditions can be determined empirically.

By changing the hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, the given sequence is compared to the most similar sequence in the sample (eg, (Selectively) hybridization conditions can be determined.
Binding conditions for triplex and quadruplex can be estimated in a similar manner.

Nucleic acid aptamers (Tuerk and Gold, Science (1990),
249: 5050; Joyce, Gene (1989), 82: 83-87; E
llington and Szostak, Nature (1990), 346:
818-822) can also be used as affinity ligands in the process of the invention. Aptamers can be selected for a wide variety of analytes including proteins, small organic molecules and carbohydrates (Klug and Fa).
Mulok, Molecular Biology Reports (1994)
), 20: 97-107). Thus, the choice of aptamer ligand provides a simple and flexible mechanism for obtaining affinity ligands for virtually any target molecule.

[0039] Other useful ligands include proteins or polypeptides that can bind to a particular analyte. A particularly useful class of protein ligands are antibody molecules that can be elicited by immunization methods against a wide range of analytes. Antibody ligands can be monoclonal or polyclonal. Further, the antibody fragment can be an affinity ligand. Similarly, receptor proteins may be useful as ligands for purifying and detecting analytes that bind to or are bound by the receptor.

[0040] Carbohydrates have been used without problems as affinity ligands for electrophoretic purification of lectins (Horejsi and Kocourek, Biochim. Bio).
phys. Acta (1974), 336: 338-343), and may be useful for the purification and detection of molecules that bind to particular carbohydrates or glycoproteins.

The binding or non-binding conditions of proteins, aptamers and lectins for a particular ligand may be estimated using the techniques outlined above for estimating the stability of an analyte / affinity ligand complex. Can be. Furthermore, equilibrium dialysis experiments can provide a rational way to predict the stability of an analyte / affinity ligand complex. For example, the dissociation constant of a protein for a particular ligand can be determined in an electrophoresis buffer at several different pHs, temperatures or ionic strengths. The larger the dissociation constant, the weaker the binding between the protein and the ligand (Segel, IH, Biochemical Cal).
uculations, 2nd edition (1976), John Wiley & Sons
, N .; Y. , Pp. 241-244). From this data, the binding and non-binding conditions can be estimated.

Many other types of immobilized ligands are possible. This includes peptides, amino acids, nucleotides, small organic molecules, lipids, hormones, drugs, enzyme substrates, enzyme inhibitors, enzymes, coenzymes, inorganic molecules, chelating agents, polymer complexes, polysaccharides, monosaccharides, etc. included.

Electrophoretic Medium Containing At least One Immobilized Affinity Ligand Any medium suitable for electrophoresis can be used for the methods of the present invention.
Generally, suitable media fall into two classes. The first class includes media composed of gel-forming substances such as cross-linked polyacrylamide and agarose. The second class includes media composed of solutions of linear, non-crosslinked polymers such as polyacrylamide, poly (hydroxyethylcellulose) and poly (ethylene oxide). The latter category is widely used for capillary electrophoresis applications.

The immobilization of the ligand can be performed by directly binding to the polymer component of the medium. Such binding can be mediated by forming a covalent bond between the ligand and the polymer. Non-covalent linkages between the ligand and the polymer substituent can also be used. For example, the strong non-covalent bonds provided by the widely used biotin streptavidin system and the digoxigenin-antidigoxigenin system can be used to attach the ligand to a suitably modified polymeric medium. Covalent bonds are preferred.

A direct link between the polymer medium and the ligand is not strictly required.
For example, a ligand can be attached to a particulate support, such as microspheres, and the particulate support can be immobilized within a polymeric medium by physical encapsulation (Cantor et al., US Pat. 482,863, the teachings of which are incorporated herein by reference in their entirety). Such particles may be of macroscopic, microscopic or colloidal nature (Polycien
ces, Inc. 195-1996 Particle Catalog, Warrington,
See PA).

In a similar manner, ligands can be attached to highly branched, soluble polymers. Because of its branched shape, such ligand-polymer conjugates exhibit extremely large effective hydrodynamic radii and, therefore, do not move through electric fields in a wide variety of polymeric media of reasonably small pore sizes. Thus, such a composite can be encapsulated in a medium in the same manner as a particulate support.

[0047] Absolute immobilization of the ligand in the medium is not required for all aspects of the invention. For many applications, it is sufficient that the mobility of the analyte change when a binding complex with the ligand is formed. This condition can be satisfied by binding the ligand to a medium component having extremely low electrophoretic mobility. However, for efficient purification, it is desirable that the change in mobility be as large as possible. Accordingly, media that make use of the true immobilization of ligands in the media are preferred for use in the present invention.

[0048] Commonly used gel media useful in the present invention include acrylamide gels and agarose gels. However, other materials can be used. By way of example, modified acrylamide and acrylate esters (eg, Polysc
ices, Inc. Polymer & Monomer Catalog, 1996-1997, W
arrington, PA), starch (Smilies, Bi)
ochem. J. (1959), 71: 585; product number S5651, Sigma
a Chemical Co. , St. Louis, MO), Dextran (see, eg, Polysciences, Inc. Polymer & Monomer Catalog, 199
6-1997, Warrington, PA), and cellulosic polymers (eg, Quesada, Current Opinions i).
n Biotechnology (1997), 8: 82-93).
Any of these polymers can be chemically modified to allow specific binding of ligands (including nucleic acids, proteins, peptides, small organic molecules, etc.) for use in the present invention.

In some methods, it may be useful to use a composite medium that contains a mixture of two or more supporting materials. An example is an acrylamide-agarose composite gel. Such gels typically contain 2% -5% acrylamide and 0.5% -1% agarose. In these gels, acrylamide provides the primary sieving function, but in the absence of agarose, such low concentration acrylamide gels do not have the mechanical strength required for convenient handling. Agarose provides mechanical support without significantly changing the sieving properties of acrylamide. In such cases, the nucleic acids can be attached to components that provide the sieving function of the gel. This is because such components make the most intimate contact with the solution phase nucleic acid target.

For capillary electrophoresis (CE) applications, it is convenient to use a medium containing a soluble polymer. Examples of soluble polymers that have been shown to be useful for CE analysis include polyacrylamide, poly (N, N-dimethylacrylamide), poly (hydroxyethylcellulose), poly (ethylene oxide) and poly (vinyl alcohol). Linear polymers, which are Que.
Sada, Current Opinion in Biotechnology
y (1997), 8: 82-93. Solutions of these polymers can also be used to practice the method of the present invention.

[0051] Methods of conjugating various ligands to create affinity electrophoretic media are well known to those skilled in the art. Many ligands can be attached to agarose, dextran, cellulose and starch polymers using activation with cyanogen bromide or cyanuric chloride. Polymers containing carboxyl groups can be attached to ligands with primary amine groups using carbodiimide coupling. Polymers with primary amines can be attached to amine-containing ligands using glutaraldehyde or cyanuric chloride.
Many polymers that can be attached to thiol-containing ligands can be modified with thiol-reactive groups. Many other suitable methods are known in the literature. See, eg, Wong, "Conjugation and cross-linking chemistry of proteins", C
See RC Press, Boca Raton, FL, 1993.

Methods have also been developed for covalently attaching a ligand by copolymerization of an electrophoretic medium with a polymeric material. In this method, the ligand is chemically modified with a copolymerizable group. When such modified ligands are copolymerized with a suitable mixture of polymerizable monomers, a polymeric medium containing a high concentration of immobilized ligand can be obtained. Examples of methods for covalently attaching nucleic acids to polymerizable chemical groups are described in U.S. patent application Ser. No. 08 / 812,105 (named: polymerizable complexes containing nucleic acids) and U.S. patent application Ser. 845 (name: electrophoretic analysis of molecules using immobilized probes). These teachings are incorporated herein by reference in their entirety. (Rehman et al., Nucleic Ac
ids Research (1999), 27: 649). Other useful methods that have been used to immobilize proteins and small organic molecules inside polymer layers and gels are described in Bille et al., Eur. J. Bioche
m. (1989), 180: 41-47; Wang et al., Nature Biot.
technology (1997), 15: 789-793; and Holtz.
And Asher, Nature (1997), 389: 829-832.

Other methods for attaching nucleic acid probes to preformed polyacrylamide polymers, including gels or linear soluble polymers, are described by Ghosh and Fa.
Hy, US Patent No. 5,478,893, the teachings of which are incorporated herein by reference in their entirety, and Timofev et al., Nucleic.
Acids Res. (1996), 24: 3142-3148.

Processes and Means for Changing the Orientation of an Electrophoretic Field During Separation An electrode system is a system that, together with a power supply, produces an electric field gradient.
The electric field gradient is the voltage drop across the gel caused by the electrode system (Gid
dings, Unified Separations Science (19
91), John Wiley & Sons, New York, pp. 155-17.
See page 0). The orientation of the electric field gradient used for electrophoresis determines the form of separation between analyte and non-analyte sample components. Many electric field configurations can be used. For example, using a conventional two-electrode device, one-dimensional separation is performed by field-reversal gel electrophoresis (Carle et al., Science (1986), 23).
2:65) can be achieved by simply switching the polarity of the two electrodes. In this type of separation, the analyte only migrates under conditions that favor binding to the ligand, whereas non-analyte sample components migrate under both conditions. For illustrative purposes, the direction of net analyte movement during the purification process will be referred to as "forward." If the purification cycle is designed such that the duration of the reverse electric field orientation is longer than the duration of the forward electric field orientation, the analyte molecules will move forward during each purification cycle, but not The sample components of the analyte will only temporarily enter the gel in order to be efficiently removed from the gel by the long duration of the reverse electric field orientation.

Pulse field applications (Schwartz and Cantor, Cell (1
984), 37:67) and CHEF application (CHEF gel, US Pat.
No. 49,769; Bio-Rad Life Science Research Product Catalog (1997)
175-182), the separation process of the present invention can be performed in two dimensions. In principle, if desired, by adding another set of electrodes operated in the third dimension,
Additional separation capabilities can be added.

The state of the equipment and methodologies for performing one- and two-dimensional electrophoretic separations have been substantially improved. At least one commercially available device (CHEF gel device, Bi
The o-Rad Life Science Research Product Catalog, 1997, pp. 175-182) can perform two-dimensional electrophoretic separations with programmed automatic control of electric field orientation and pulse duration.

Processes and Means for Changing at least One Other Property of an Electrophoretic Medium Simultaneously with a Change in Electric Field Orientation of Electrophoresis The electrophoretic medium comprises one or more properties of the electrophoretic medium (eg, temperature , PH
(Or ionic strength) can be reversibly repeated between at least two different states determined by the user. In one situation, the ligand has a relatively large affinity for the analyte of interest. In other situations, the ligand has a relatively low binding affinity for the analyte. In a preferred embodiment, the non-analyte sample components have a low affinity for the ligand in both situations. In a more preferred embodiment of the invention, the change in media state and electric field orientation is simultaneously regulated, so that electrophoresis of non-analyte material occurs under all conditions and electric field orientations, but the electrophoresis of the analyte is It occurs only under limited combination of conditions and electric field orientation. Thus, the analyte and non-analyte molecules have different net mobilities for each cycle, and their separation in the media increases with each cycle.

Varying the medium temperature is one preferred means for adjusting the analyte-ligand binding affinity. Because the temperature can be changed with little or no manipulation of the electrophoretic medium, and a great deal of equipment is commercially available for temperature control. However, other media characteristics can also be used. Non-limiting listings of possible properties that are known to affect non-covalent chemical bonding include changes in medium pH, changes in ionic strength of the medium, and other chemical compositions of the medium. Change.

In one particularly preferred embodiment of the invention, the affinity ligand is a nucleic acid and the analyte is a nucleic acid sample having at least one region complementary to the nucleic acid of the affinity ligand. In this case, the binding between the analyte and the ligand is
It can be adjusted effectively by changing the temperature of the gel. For example,
If the temperature is higher than the Td of the ligand-analyte complex, the binding affinity will be lower.
Similarly, if the temperature is lower than Td, the binding affinity will be substantially greater.

Processes and means for circulating an electrophoretic medium between two temperatures are well known to those skilled in the art. For example, temperature controlled devices for performing vertical or horizontal electrophoresis are commercially available (Bio-Rad Life Science Research Product Catalog (1997), pp. 127-133; Pharmacia Biotech BioDirectory). (1997), 345, 309,
334). In some devices, temperature is controlled by circulating water (or an appropriate coolant) through the device. In such a device, temperature cycling may be achieved by switching the coolant supply between two regulated reservoirs set to the desired temperature. In some electrophoresis devices,
The medium is in thermal contact with a programmable thermocycler that relies on the Petier effect for heating and cooling. (The thermocycler is MJ
Research (Watertown MA)). For example, an electrophoresis unit with a heating / cooling device by the Peltier effect is available from Pharmacia (Pharmacia Biotech Bio Directory (1997), p. 334).

Another method of adjusting the analyte-ligand binding affinity is by changing the ionic strength of the electrophoresis buffer. The ionic strength of the buffer that promotes binding depends on the analyte and the type of affinity ligand. Generally, buffers with higher ionic strength promote binding. Where the analyte binds to the affinity ligand, a buffer having an ionic strength of about 100 mM to 1 M is preferred. Where the analyte does not bind to the affinity ligand, a buffer having an ionic strength of about 10 mM or less is preferred. Equilibrium dialysis or hybridization experiments can be used to reasonably predict the stability of a particular analyte / affinity ligand binding complex at a particular ionic strength.

Another way to adjust the analyte-ligand binding affinity is by changing the electrophoresis buffer to a denaturing buffer. The denaturing buffer contains a chemical agent that disrupts the binding of the analyte to the affinity ligand (hereinafter "denaturing agent"). For example, formamide or urea can be a component of a denaturing buffer. The amount of denaturing agent required depends on the type of target molecule, the type of affinity binding interaction, the electric field strength, the ionic strength, and the temperature of the electrophoresis. Generally, denaturing buffers can have a very wide concentration range of formamide or urea. Formamide can be used at concentrations up to 95% (vol / vol) and urea can be used at concentrations up to 8M. Equilibrium dialysis or hybridization experiments can also be used to reasonably predict the stability of an analyte / affinity ligand binding complex in a particular denaturing buffer.

Optionally, the analyte-ligand binding affinity can also be adjusted by changing the pH of the buffer. For example, nucleic acids hybridize at approximately neutral pH (eg, pH 6-8) with complementary nucleic acid affinity ligands. DNA affinity ligands hybridized to a DNA target can be destroyed at acidic pH (eg, below pH 5) or at basic pH (eg, above pH 11). Changing the pH of the electrophoresis buffer is a preferred method of breaking the binding of the protein analyte to the affinity ligand. Equilibrium dialysis experiments can be used to estimate the pH range for binding and dissociation of a particular protein to an analyte.

To obtain maximum separation efficiency, switching the media conditions must switch the analyte between fully bound and completely unbound. This complete distinction between bound and unbound analyte states is made using a single-stranded nucleic acid analyte-ligand system, as exemplified in a later section of this disclosure. Can be. However, such an absolute two-state behavior is not required for a satisfactory application of the present invention. Generally, it is sufficient that the mobility of the analyte be substantially changed by changes in media conditions. Here, “substantially” means that the change in mobility observed in the medium containing the ligand is similar for a similar medium containing no ligand or chemically similar to the original ligand. Mean greater than the change in mobility observed for media containing ligands that cannot form a specific binding complex with the analyte. Where such substantial changes in analyte mobility occur, even weak specific ligand / analyte interactions can be used successfully to practice the invention. Because any number of purification cycles can be repeated in an automated manner. In such cases, each cycle provides a small, but measurable, separation between analyte and non-analyte sample components, and the purification cycle can be repeated until the required level of separation is achieved. .

Applications of the Invention The present invention can be used to purify analytes and use them for subsequent characterization and other preparation purposes. Further, the present invention can be used to detect an analyte.

For preparative purposes, the present invention allows many repetitive cycles of affinity purification to be performed using a single, automated, programmable device using easily prepared and inexpensive affinity media. To show the power. Preferably, elution of the purified product is performed by Gombocz et al., US Pat. No. 5,217,591, Kra.
gt and Ballen can be performed electrophoretically using a modification of the method disclosed in US Pat. No. 3,989,612. The teachings of which are incorporated herein by reference in their entirety. Other elution methods are possible and are well known to those skilled in the art.

For detection purposes, the present invention shows its power because non-analyte sample components that can contribute to undesirable levels of background can be removed. The general format of such an assay involves the following steps: 1) preparation of a sample to be tested; 2) purification of a possible sample analyte by the method of the invention; and 3) analysis of the product of the purification process of step 2. Object assay. As a non-limiting example of a useful detection application using the present invention, this general method 3
Two variants are shown below. These examples are in no way intended to limit the scope of the invention. Other detection methods may be more useful for other types of analytes. These all relate to the detection of bacteria in blood samples.

A) Detection by Dye Binding In step 1, total RNA is prepared from a blood sample (Chomczyn)
Ski et al., Anal. Biochem. (1987), 162: 156; Cho
mczynski, Biotechniques (1993), 15: 532;
TRI Reagent BD, Sigma (1999), Catalog No. T3809, 1545
page). In step 2, the RNA is subjected to the method of the invention using a specific affinity medium to bind the bacterial ribosomal RNA. In step 3, the product of the purification process is a dye that fluoresces brightly when bound to nucleic acid (eg, Acridine Orange, SYBR Green II, TO-TO or YO-YO
) Is used to check for the presence of RNA. Another example of a suitable dye is Hau
Grand "Fluorescent Probes and Research Reagents Handbook, 6th Edition" (Mole
cultural Probes, Eugene, OR, (1996), 144-1-1.
56).

B) Detection by Co-Purification of Enzyme Label In step 1, total RNA is prepared from a blood sample, and bacterial ribosomal RNA analytes in the sample are alkalinized by means that are not destroyed during the purification process of step 2. Attached to an enzyme reporter molecule such as phosphatase, horseradish peroxidase or luciferase. Preferably, the binding of the RNA analyte to the enzyme reporter is mediated by a nucleic acid sandwich hybridization probe to which the enzyme is attached (Ranki and Soderlan).
d, U.S. Patent No. 4,486,539; Engelhardt and Ra
bbani, US Patent No. 5,288,609, the teachings of which are incorporated herein by reference in their entirety). In the assay of step 3, the enzyme reporter molecule is detected using a chromogenic, chemiluminescent or chemiluminescent substrate.

C) Detection by Simultaneous Purification of an Amplifiable Reporter Label In step 1, total RNA is prepared from a blood sample and the bacterial ribosomal RNA analyte in the sample is not destroyed during the purification process of step 2. By a substrate for Q-beta replicase, such as the MDV-1 substrate for Q-beta replicase (Lizardi et al., Bio / Technology 6:11).
97-1202, 1988). Preferably, binding of the RNA analyte to the enzyme reporter is mediated by a nucleic acid sandwich hybridization probe to which the amplifiable reporter is attached. By the assay of step 3, the amplifiable reporter molecule is detected using a suitable enzymatic amplification and detection process.

It is obvious to those skilled in the art that the above examples (a) to (c) are described in order of increasing sensitivity. The theoretical sensitivity of example (c) is limited only by the loss of analyte and / or amplifiable reporter during sample preparation and electrophoretic purification. This is because the Q-beta replicase system can detect one MDV-1 substrate in a sample.

The present invention is illustrated by the following examples. However, the following examples are not intended to be limiting in any way.

Example 1 Separation of Nucleic Acid Samples Examples of the invention used to separate a mixture of nucleic acids are shown in FIG. 1A and FIG. 1B. In this example, the ligand was prepared in US patent application Ser.
Nucleic acid GTA CC covalently immobilized in a standard polyacrylamide gel using the methods of 8 / 812,105 and 08 / 971,845.
A TAA CAG CAA GCC TCA (SEQ ID NO: 2). The teachings of which are incorporated herein by reference in their entirety.

A fluorescently labeled nucleic acid 5 that is non-complementary to the ligand (indicated by a square)
'-Fluorescein-ATT ACG TTG ATA TTG CTG AT
TA-3 ′ (SEQ ID NO: 3) was loaded in lane 1. 5'-Fluorescein-TGA GGC TTT, a fluorescently labeled nucleic acid complementary to a ligand
CTG TTA TGG TAC-3 '(SEQ ID NO: 1) was loaded in lane 2. This preparation contains trace amounts of chemical species (indicated by open circles). This species exhibits a slightly greater mobility than the major species (indicated by a closed circle) for unknown reasons. Lane 3 contained a mixture of the two fluorescein-disabled nucleic acids.

FIG. 1A and FIG. 1B correspond to step 1 in the first cycle.
And the gel after step 2 is completed. In step 1, the gel was maintained at 45 ° C. At this temperature, binding of the ligand to the complementary nucleic acid is prevented and 1
The electric field applied at 00V for 43 minutes had the polarity shown in Figure 1. In step 2, the gel was maintained at 25 ° C., a temperature that allowed binding of the ligand to complementary nucleic acids. The electric field applied at 100 V for 50 minutes had the opposite polarity to that used in step 1. At the end of step 1, the mobilities of the two fluorescently labeled nucleic acids were similar, as shown in Figure 1A. At the end of step 2, separation of the two nucleic acids was observed (FIGURE 1B).
). Figure 1C shows the same gel after a total of 3 cycles. Each cycle consisted of steps 1 and 2 (down / heat and up / cool) as described above for Figure 1A and Figure 1B. After the third cycle, the separation between complementary and non-complementary nucleic acids has increased. In particular, complementary nucleic acids migrated further down the gel, while non-complementary nucleic acids migrated in the opposite direction.

Preparation of RNA Analyzes Used in Examples 2 and 3 Three RNA analytes were used for the experiments shown in FIGURE 2 and FIGURE 3. All three are T7 polymerase (Boehringer
Enzymes from Mannheim, catalog # 881, 767; Promega
a) (catalog # P1420) and fluorinated ribonucleotide triphosphate (Boehringer Mann) as a label.
fluorescein-labeled nucleotides from Nheim, catalog # 1,68
5,619) produced by an in vitro transcription reaction. The three RNA molecules are: 1.16S Hha, 502 nucleotides long, non-specific sample component 2.16S Alu, 255 nucleotides long, non-specific sample component 3. E. coli RNase P RNA, 377 nucleotides long, specific RNA
The analyte was These RNA species were generated as detailed below.

The 16S Hha and 16S Alu plasmids pSCH038 were converted to American Type Culture.
Obtained from Collection (ATCC 87435). This plasmid is a PCRII plasmid vector (Invitrogen, Carlsba) containing the E. coli 16S ribosomal RNA (rRNA) sequence at positions 674 to 1411.
d, California, catalog number K2050-01). Perfect one
The 6S molecule is 1541 nucleotides in length. This vector is 5 'of the insert.
Flanked by a T7 RNA polymerase promoter site, so that in vitro transcription using such an enzyme results in RNA of the same sense strand as native 16S rRNA.

Two 5 mg plasmids were digested with the restriction enzymes HhaI or AluI.
The digested plasmid sample was used as a template for in vitro transcription using T7 polymerase and fluorescein labeled nucleotides. After synthesis,
The DNA template was removed by digestion with Dnase I and the transcript was
Unincorporated nucleotides were purified using a Pharmacia G25 spin column.

The first 68 nucleotides of both transcripts are derived from the cloning vector PCRII: there are 68 nucleotides between the T7 start site and the 16S insert. A
luI cleaves between nucleotide 860 and nucleotide 861 of the 16S rRNA sequence. Thus, the transcript (16S Alu) made from the template cut with AluI is 255 nucleotides in length.

HhaI cleaves the 16S sequence between nucleotides 1107 and 1108 of the 16S rRNA sequence. Thus, the length of the 16S Hha transcript is 502 nucleotides.

E. coli Rnase P RNA The naturally occurring E. coli Rnase P RNA is 377 nucleotides in length (Altman et al., IntRNA: Structure, Biosyn).
thesis, and Function (1995), pp. 67-78, So
ll and Raj Bhandary eds., American Society o
f Microbiology, Washington, D.C. C. ). A PCR fragment containing this sequence was generated from E. coli genomic DNA. The amplification primer 5 'of the gene contained the T7 RNA polymerase promoter. Thus, the resulting 396 bp DNA amplification product contained the T7 promoter 5 ′ immediately from the gene sequence of Rnase P RNA. This DNA is converted to T
7 polymerase was used as a template in an in vitro transcription reaction to generate fluorescently labeled Rnase P RNA. In vitro transcripts generated from this template were identical to the published sequence of E. coli Rnase P RNA.

Example 2. Isolation of Escherichia coli Rnase P RNA from 16S Hha RNA and 16S Alu (See Figures 2A, 2B, 2C and 2D) In this example, the ligands are derived from US patent application Ser. Nucleic acid 5'-CCA TCG GCG GTT TG covalently immobilized in a standard polyacrylamide gel using the methods of U.S. Pat.
C TCT CTG TTG-3 ′ (SEQ ID NO: 4). This ligand is
Complementary to the sequence contained within the E. coli Rnase P RNA. The ligand bound via its 5 'end and was present on the gel at a concentration of 10 mM (strand). The gel contained 5% polyacrylamide (29: 1, monomer: bisacrylamide), 45 mM tris borate (pH 8.3) and 2 mM EDTA (0
. 5 × TBE buffer). The gel is converted into a temperature controlled mini gel device (
Penguin Vertical Gel Box, Owl Scientific, Wobu
rn, MA). The temperature of the gel was controlled by water pumped from an external recirculation bath. For all electrophoresis steps, the applied electric field was 2
00 volts.

The left lane was loaded with fluorescently labeled E. coli Rnase P RNA. The right lane contains a total of three fluorescently labeled transcripts, Rnase P, 16
A mixture of S Hha and 16SAlu was loaded.

FIG. 2A shows that the fluorescent product after electrophoresis down the gel for 10 minutes at 54 ° C., the gel temperature at which the binding of the gel ligand to the specific analyte Rnase P RNA is broken, Indicates a pattern. The gel was prepared using Molecular Dynamics Fluorim without removing the glass plate.
Imaged directly using ager. The gel was put back into the gel box and equilibrated at 41 ° C., the gel temperature that allowed hybridization of the nucleic acid ligand to Rnase P RNA. Gel ligands are not complementary to 16S transcripts. At this temperature, the sample was electrophoresed upward for 20 minutes. The electric field was turned off and the gel temperature was changed to 59 ° C. Next, the sample was electrophoresed downward for 5 minutes, and a new gel image of the sample shown in FIG. 2B was taken. At this time, only the Rnase P RNA analyte remains in the gel. This indicates that the 16S transcript was efficiently removed during the upward electrophoresis step. The gel was returned to the apparatus and electrophoresed upward at 41 ° C. for 10 minutes and electrophoresed downward at 59 ° C. for 10 minutes. Another gel image shown in FIG. 2C was taken. Then, the gel was electrophoresed downward at 59 ° C. for 25 minutes to obtain F
The final image shown in FIG. 2D was obtained. The Rnase P RNA sample
As shown in FIG. 2C and FIG. 2D, the gel is sequentially moved downward in the downward direction. This indicates that the specific RNA analyte is being released during the downward electrophoresis step at elevated temperatures.

Example 3. Isolation of Escherichia coli Rnase P RNA from 16S Hha RNA, 16S Alu RNA, and unlabeled total RNA from E. coli (Figu
(See re3A and FIGURE3B) Gels containing the ligand were prepared as in Example 2. Lane 1 contained E. coli Rnase P RNA; Lane 2 contained 16S Alu transcript; Lane 3 contained 16S Hha and 16S Alu; Lane 4 contained 16S Hha and Rnase P RNA; Lane 5 contained 6.3 mg of unlabeled total RNA from E. coli; lane 6 contained Rnase PR.
It contained 6.3 mg of unlabeled total RNA from NA, 16S Hha, 16S Alu, and E. coli. The gel was run with the specific analyte, Rnase PR.
50 ° C., the temperature at which hybridization of the ligand to NA is disrupted
And the sample was electrophoresed downward for 10 minutes. The gel image after Step 1 is shown in Figure 3A. The gel was equilibrated at 41 ° C. and electrophoresed upward for 40 minutes. At this temperature, Rnase P RNA can hybridize to the gel ligand. The gel image after step 2 is shown in FIG.
As shown in e3B, the 16S Hha and 16S Alu transcripts are completely removed from the gel. Rnase PR in gel
The specific retention of NA analyte was not affected by excess heterologous unlabeled RNA in lane 6. This further indicates that analyte capture is very specific.

Equivalents While the invention has been particularly shown and described with reference to preferred embodiments thereof, without departing from the spirit and scope of the invention as defined by the appended claims.
It will be understood by those skilled in the art that various modifications in form and detail may be made in the present invention. Such skilled artisan will be able to ascertain or ascertain using no more than routine experimentation, many equivalents, particularly to the particular embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1A, FIG. 1B and FIG. 1C are 5′-fluorescein-TG in electrophoretic media with covalently linked complementary nucleic acid strands.
A GGC TTT CTG TTA TGG TAC-3 '(SEQ ID NO: 1)
Shows the separation of non-complementary from non-complementary fluorescently labeled nucleic acids.

FIG. 2 FIG. 2A, FIG. 2B, FIG. 2C and FIG.
2D shows 16S Hha RNA and 16S Al in an electrophoretic medium having a nucleic acid sequence complementary to the sequence in covalently linked E. coli Rnase P RNA.
Figure 5 shows the separation of E. coli Rnase P RNA from uRNA.

FIG. 3A. FIG. 3A and FIG. 3B are used in electrophoretic media with nucleic acid sequences complementary to sequences in E. coli Rnase P RNA covalently bound.
2 shows the separation of E. coli Rnase P RNA from S Hha RNA, 16S Alu RNA, and unlabeled total RNA from E. coli.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] May 23, 2000 (2000.5.23)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

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[Claims]

30. The apparatus according to claim 29, wherein the direction of the electric field can be changed in one dimension.

[Procedure amendment]

[Submission date] July 3, 2001 (2001.7.3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

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[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Muir, Andrew, Earl. United States Massachusetts 02025 Cohasset, Jerusalem Road 481 (72) Inventor Balls, Tee. , Christian United States Massachusetts 02454 Waltham, Judith Lane Number Six 5

Claims (44)

[Claims] 1. a) contacting an electrophoretic medium having an immobilized affinity ligand with a test sample under conditions that the affinity ligand selectively binds to the analyte, referred to as initial conditions; b) in the test sample. Applying an electric field that is oriented in an initial direction for a time sufficient to cause the transfer of non-analyte material in the electrophoretic medium; and c) changing the direction of the electric field while simultaneously changing at least one property of the electrophoretic medium. Wherein said altering reduces the binding affinity of the affinity ligand for the analyte sufficiently to allow the analyte to move in the electric field, thereby separating the analyte from other components in the test sample; A method of separating an analyte from other components in a test sample. 2. The method according to claim 1, further comprising a step of repeating the step c) one or more times after repeating the step of returning to the initial condition and the step b). 3. The method of claim 2, wherein the direction of the electric field can be changed in one dimension. 4. The method according to claim 2, wherein the direction of the electric field can be changed in two dimensions. 5. The method according to claim 2, wherein the direction of the electric field can be changed in three dimensions. 6. The method of claim 1, wherein the property of the electrophoretic medium that is altered is temperature. 7. The method of claim 1, wherein the characteristic of the electrophoretic medium that is altered is a chemical composition.
The described method. 8. The method according to claim 7, wherein the chemical composition of the electrophoresis medium is changed by replacing the electrophoresis buffer. 9. The method of claim 7, wherein the change in chemical composition results in a change in pH of the electrophoretic medium. 10. The method of claim 7, wherein the chemical composition is changed by changing the concentration of at least one denaturing agent. 11. The method according to claim 10, wherein the denaturing agent is formamide. 12. The method according to claim 10, wherein the denaturing agent is urea. 13. The method of claim 7, wherein the change in chemical composition results in a change in ionic strength of the electrophoretic medium. 14. The affinity ligand may be a peptide, amino acid, nucleoside, nucleotide, nucleic acid, small organic molecule, lipid, hormone, drug, enzyme substrate,
Enzyme inhibitors, enzymes, coenzymes, inorganic molecules, chelating agents, polymer complexes, polysaccharides,
Or the method of claim 1, wherein the method is selected from the group consisting of monosaccharides. 15. The method according to claim 1, wherein the affinity ligand is a single-stranded nucleic acid sequence.
The described method. 16. The method of claim 15, wherein the analyte is a nucleic acid and a portion of the nucleic acid analyte is complementary to a portion of the nucleic acid affinity ligand. 17. The method of claim 16, wherein the initial conditions are high stringency hybridization conditions. 18. The method of claim 16, wherein the initial conditions are moderate stringency hybridization conditions. 19. The method according to claim 1, wherein the affinity ligand is a double-stranded nucleic acid sequence.
The described method. 20. The method according to claim 1, wherein the affinity ligand is an aptamer. 21. The method according to claim 1, wherein the affinity ligand is a protein. 22. The method according to claim 21, wherein the affinity ligand is an antibody. 23. The method according to claim 1, wherein the affinity ligand is a carbohydrate. 24. The method of claim 1, wherein the analyte is bound to a fluorescent dye prior to contacting the analyte with the electrophoretic medium. 25. The method of claim 1, wherein the analyte is bound to an enzyme reporter molecule prior to contacting the analyte with the electrophoretic medium. 26. The method according to claim 19, wherein the enzyme reporter molecule is alkaline phosphatase. 27. The method of claim 1, wherein the analyte is bound to an amplifiable reporter molecule prior to contacting the analyte with the electrophoretic medium. 28. The method according to claim 27, wherein the amplifiable reporter molecule is a substrate for Q-beta replicase. 29) a) an electrophoretic medium having immobilized affinity ligands; b) a power supply capable of generating an electric field sufficient to cause the migration of components in the test sample; c) connected to a power supply. At least one electrode system and an electrophoretic medium for directing an electric field gradient, wherein the direction of the electric field gradient of each electric system is reversible; d) a first buffer reservoir in contact with the electrophoretic medium; e) an electrophoretic medium. An apparatus for performing the method of claim 1, comprising: a second buffer reservoir in contact with the electrophoretic medium; and f) means for changing at least one property of the electrophoretic medium. 30. The apparatus according to claim 29, wherein the direction of the electric field can be changed in one dimension. 31. The apparatus according to claim 29, wherein the direction of the electric field can be changed in two dimensions. 32. The apparatus according to claim 29, wherein the direction of the electric field can be changed in three dimensions. 33. The apparatus of claim 29, wherein the property of the electrophoretic medium that is altered is a chemical composition. 34. The apparatus according to claim 33, wherein the chemical composition of the electrophoresis medium is changed by replacing the electrophoresis buffer. 35. The apparatus of claim 33, wherein the change in chemical composition results in a change in pH of the electrophoretic medium. 36. The apparatus according to claim 33, wherein the chemical composition is changed by changing the concentration of at least one denaturing agent. 37. The device according to claim 36, wherein the denaturing agent is formamide. 38. The device according to claim 36, wherein the denaturing agent is urea. 39. The apparatus of claim 33, wherein the change in chemical composition results in a change in ionic strength of the electrophoretic medium. 40. The apparatus of claim 29, wherein the property of the electrophoretic medium that is altered is temperature. 41. The apparatus according to claim 40, wherein the temperature is changed by circulating liquid from at least one temperature-regulated reservoir. 42. The apparatus according to claim 40, wherein the temperature is changed by a heating / cooling device based on the Peltier effect. 43. The apparatus of claim 40, wherein changes in the direction and temperature of the electric field gradient are controlled in an automated and programmable manner. 44. The apparatus of claim 40, wherein the temperature is changed by increasing a voltage generated by the power supply.