Electrophoretic analysis of molecules using immobilized probes

This application claims priority to Provisional Application No. 60/046,108, filed May 16, 1997, the teachings of which are herein incorporated, in their entirety, by reference.

Nucleic acid base pairing is an extremely high affinity and specific interaction. For this reason, nucleic acid hybridization assays have been devised for a variety of diagnostic purposes.

Under laboratory conditions, hybridization assays can be extraordinarily sensitive, detecting femtogram amounts of a specific molecule. However, several technical limitations have prevented widespread use of hybridization analysis in commercial diagnostic techniques.

First, use of high activity hybridization probes requires stringent procedures for separating unhybridized for improperly hybridized) and hybridized probe. This separation can be facilitated by the use of solid phase hybridization formats, in which either the sample nucleic acid or the probe that is complementary to the desired target is immobilized on a solid support. In the latter strategy, the immobilized probe, hereafter referred to as the “capture” probe, is usually unlabeled, and the hybridization is detected by a second hybridization probe that binds the sample at a position separate from that recognized by the capture probe. Hybridized and unhybridized species can be separated by washing the support.

A second limitation of hybridization assays is that efficient hybridization of samples containing low concentrations of target nucleic acids frequently requires lengthy incubations (up to several hours) under carefully controlled conditions. Unfortunately, use of solid phase assays exacerbates this problem, since immobilized nucleic acids virtually always hybridize with slower kinetics than nonimmobilized ones.

For these reasons, a number of workers have sought methods to perform solid phase hybridizations with better kinetics and efficiency. Several groups have found that inclusion of high molecular weight polymers such as dextran sulfate or polyethylene glycol improves solid phase assay performance, albeit modestly. (Wieder and Wetmur, Biopolymers, 20:1537 (1981); Wetmur, Biopolymers, 14:2517 (1975); Yokota and Oishi, Proc. Natl. Acad Sci. USA, 87:6398 (1990)). Several groups have developed chromatographic solid phase hybridization methods that show improvements. In general, it has been found that flowing the solution phase nucleic acid strand over (or through) the solid support bearing the immobilized strand improves both kinetics and efficiency of hybridization. MacMahon and Gordon, U.S. Pat. No. 5,310,650, describes immobilized target molecules on nitrocellulose filters, with labeled probe flowing through the immobilized target regions by capillary action. In a similar experiment, Reinhartz et al., Gene, 136:221-226 (1993)) immobilized capture probes on paper filters and flowed labeled single-stranded PCR products through the capture probe region, again using capillary action. Others have demonstrated improved hybridization assays by passing samples through an HPLC column containing silica particles covalently modified with capture probes. (Tsurui et al., Gene, 88:233-239 (1990)).

However, despite these advances, there remains a need for a hybridization analysis method that is not only accurate, but fast, efficient and simple to use.

The present invention relates to the discovery that nucleic acids and nucleic acid analogs can be covalently attached (immobilized) to an electrophoretic medium and that electrophoresis can be used to separate, purify or analyze target molecules that specifically bind to (e.g., associate with), or are specifically bound by, the immobilized nucleic acids, or nucleic acid analogs. The immobilized nucleic acids, or nucleic acid analogs, are referred to herein as capture probes. These immobilized capture probes can be used to analyze a variety of molecules. One specific binding reaction encompassed by the present invention is hybridization. With hybridization, capture probes are typically nucleic acids comprising nucleotide sequences that are substantially complementary to the nucleotide sequences of the target nucleic acid so that specific hybridization results. Additionally, nucleic acid analogs such as peptide nucleic acids (PNA) can be covalently attached to the electrophoretic medium for use as capture probes. The capture probes, being immobilized within the medium used for electrophoretic separation, results in the target nucleic acid that specifically hybridizes with the capture probe also becoming immobilized in the matrix. As used herein, the term “matrix” refers to the immobilized polymeric components of the electrophoretic medium which provide the molecular sieving properties of the medium, and also provide the means for immobilization of the capture probes. Examples of suitable matrix materials include gel-forming polymers such as cross-linked polyacrylamide, agarose, and starch. Non-gel-forming polymers such as linear polyacrylamide, poly(N,N-dimethylacrylamide), poly(hydroxyethylcellulose), poly(ethyleneoxide) and poly(vinlyalcohol), as commonly used in capillary electrophoresis applications, can also serve as suitable matrices.

The present invention specifically relates to methods, and apparatus to carry out the methods of analysis described herein, in which electrophoresis is used to move solution phase target molecules into contact with a capture probe that is immobilized on a suitable electrophoresis matrix.

The methods of the present invention are applicable to analysis of any chemical entity that can be electrophoresed (e.g., a charged molecule that has detectable mobility when placed in an electrophoretic field) and that binds to, or is bound by, nucleic acids. Such entities include, for example, DNA or RNA samples, nucleic acid binding proteins, and aptamer binding partners (aptamers are nucleic acids that are selected to bind to specific binding partners such as peptides, proteins, drugs, polysaccharides and small organic molecules, e.g., theophylline and caffeine; Jenison, et al., Science, 263:1425-1429 (1994)). For example, methods described herein can be used for analysis and purification of target nucleic acids using immobilized capture probes, where specific binding involves base pairing interactions between sample nucleic acids and the capture probe, as in nucleic acid hybridization. The methods described herein are also useful for purification of sequence-specific nucleic acid binding proteins, since synthetic nucleic acids of defined sequence can be immobilized in matrices commonly used for protein electrophoresis.

The test sample can be from any source and can contain any molecule that can form a binding complex with a capture probe. Specifically encompassed by the present invention are samples from biological sources containing cells, obtained using known techniques, from body tissue (e.g., skin, hair, internal organs), or body fluids (e.g., 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.

The test sample is treated in such a manner, known to those of skill in the art, so as to render the target molecules contained in the test sample available for binding. For example, if the target molecule is a nucleic acid present in a cell, a cell lysate is prepared, and a crude cell lysate (e.g., containing the target nucleic acid as well as other cellular components such as proteins and lipids) can be analyzed. Alternatively, the target nucleic acids can be isolated (rendering the target nucleic acids substantially free from other cellular components) prior to analysis. Isolation can be accomplished using known laboratory techniques. The target nucleic acid can also be amplified (e.g., by polymerase chain reaction or ligase chain reaction techniques) prior to analysis.

The test sample is then introduced into a suitable electrophoretic medium. The capture probes are immobilized within the electrophoresis matrix by direct attachment to the medium, or by attachment to particles that are suspended and trapped within the matrix. In either case, the capture probes are immobilized, that is, they do not migrate under the influence of the applied electric field.

The test sample containing the target molecule can be detectably labeled before, during, or after the electrophoresis step. Detecting the presence of target molecule/capture probe complexes immobilized in the matrix is indicative of the presence of the target molecule that specifically binds to, or is bound by, the capture probe. Once the test sample is introduced into the electrophoretic medium it is subjected to an electrical field resulting in the electrophoretic migration of the test sample through the matrix, under conditions and time sufficient for the target molecule, of the test sample, if present, to bind to one, or more, capture probes, resulting in target molecule/capture probe complexes immobilized in the matrix. Typical voltage gradients used in nucleic acid electrophoresis procedures range from approximately 1 V/cm to 100 V/cm. Other field strengths may be useful for certain highly specialized applications.

The target immobilization may be transient or stable for a substantial time period, depending on the strength and lifetime of the target/capture probe binding complex. In one embodiment of the present invention, the target molecule transiently binds to, or associates with, one or more capture probes immobilized in the matrix. In this embodiment the target molecule may bind and be released multiple times during migration through one more regions of the electrophoretic matrix containing immobilized capture probes. The electrophoretically induced migration is thereby hindered and delayed, such that the migration rate is slower than if no such binding occurred, and the time to migrate through the subject region and the matrix is increased. The rate of migration of the target molecule within the electrophoretic matrix is measured, and can be compared with migration rate of the target molecule in a reference (e.g. control) experiment. As used herein, the control experiment is an equivalent experiment with substantially similar, or equivalent, materials and conditions, except that no capture probes are immobilized in the matrix. Alternatively a control experiment can be a similar experiment corrected for differences from exact equivalence of the test experiment, or can be a sufficiently similar experiment that no such corrections are necessary to obtain analytical results.

If, in a particular experiment, the migration rate of a molecule is found to be slower than the migration rate of the molecule in a reference experiment, this delayed migration indicates that the molecule is the target molecule that associates with one or more capture probes contained in the matrix. Furthermore, the degree of reduction, or decrease, in the target molecule migration rate is indicative of the following: the affinity of binding of the target molecule with the one or more capture probes; the concentration of capture probes immobilized within the matrix; and the extent of the capture probe region or regions traversed in migration through the matrix.

Alternatively, the migration rate of the target molecule may be compared with the migration rate of a control molecule with known migration rate under the experimental conditions. The relative migration times of the target molecule migrating through a matrix with one or more immobilized capture probes associating with the target molecule, and the migration rate of the control molecule under substantially similar, or equivalent, conditions but with no such association, are empirically determined or calculated from their molecular properties.

If, under experimental conditions the similar, or relative migration rates of a putative target (e.g., test) molecule and the control molecule are substantially equivalent (e.g., in the correct or expected relationship) this indicates that the test molecule is actually the target molecule of interest and that the matrix contains one or more immobilized capture probes that associate with the target molecule. Alternatively, the same indication can be obtained from the relative migration distances achieved in the matrix containing the subject capture probes, for the target molecule and the test molecule, after similar migration times under substantially similar, or experimental conditions.