Electrophoretic analysis of molecules using immobilized probes

Abstract

Methods of detecting target molecules using electrophoresis and media containing immobilized capture are described.

1 . A method of detecting one, or more, target molecules in a test sample, wherein the target molecule has a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the target molecule can form binding complexes with capture probes, comprising the steps of:
 a) immobilizing one or more capture probes within at least one region of a medium suitable for electrophoresis of target molecules;   b) introducing the test sample into the electrophoresis medium;   c) subjecting the electrophoresis medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes; and   d) detecting the presence of target molecules or target molecule/capture probe complexes immobilized in the medium.   
   
   
       2 . (canceled) 
   
   
       3 . The method of  claim 1  wherein in step d), the rate of target molecule migration, or a property which is related to the rate of target molecule migration, within the electrophoretic medium is measured, wherein the rate of target molecule migration is indicative of at least one chemical or structural property of the target molecule. 
   
   
       4 . The method of  claim 1  wherein the capture probe is selected from the group consisting of: nucleic acids, modified nucleic acids and nucleic acid analogs. 
   
   
       5 . The method of  claim 1  wherein the electrophoretic medium is a solution containing at least one type of polymer. 
   
   
       6 . The method of  claim 5  wherein the polymers are selected from the group consisting of: polyacrylamide, poly (N,N-dimethylacrylamide), poly(hydroxyethylcellulose), poly(ethyleneoxide) and poly(vinylalcohol). 
   
   
       7 . The method of  claim 1  wherein the medium is a gel formed from at least one type of polymer. 
   
   
       8 . The method of  claim 7  wherein the electrophoretic medium is formed using at least one class of polymers selected from the group consisting of: polyacrylamide, agarose and starch. 
   
   
       9 . (canceled) 
   
   
       10 . A method for purifying or concentrating target molecules from a complex test sample, comprising:
 a) immobilizing one or more capture probes within a medium suitable for electrophoresis;   b) introducing the test sample into the electrophoresis medium;   c) subjecting the electrophoresis medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes and for non-specific components of the sample to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium;   d) treating the electrophoresis medium to release at least one of the following:
 i) the capture probe, 
 ii) the target molecule, or 
 iii) the target/capture probe binding complex; 
   e) eluting the target molecules or target/capture probe complexes released in step d) from the medium,   whereby the released and eluted target molecules are substantially pure.   
   
   
       11 . The method of  claim 10  wherein the releasing treatment in step d) is accomplished by raising the temperature of the medium to a temperature sufficient to denature the target/capture probe complexes. 
   
   
       12 . The method of  claim 10  wherein the releasing treatment of step d) is accomplished by chemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and in step e) the released target molecule/capture probe complex is eluted from the medium. 
   
   
       13 . The method of  claim 10  wherein the releasing treatment of step d) is accomplished by photochemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and in step e) the released target molecule/capture probe complex is eluted from the medium. 
   
   
       14 . The method of  claim 10  wherein the releasing treatment of step d) is accomplished by increasing the electrophoretic field strength to a level sufficient to disrupt target/capture probe complexes. 
   
   
       15 . The method of  claim 1 , wherein the capture probes are immobilized in a discrete region or regions of the electrophoretic medium. 
   
   
       16 . The method of  claim 1 , wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 
   
   
       17 . A method of detecting one or more target molecules in a test sample, comprising the steps of:
 a) copolymerizing one or more nucleic acid aptamer capture probes with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing at least one immobilized nucleic acid aptamer capture probe covalently attached to the electrophoretic medium;   b) introducing a test sample into the electrophoretic medium;   c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe; and   d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample.   
   
   
       18 . The method of  claim 17 , wherein the capture probes are immobilized in a discrete region or regions of the electrophoretic medium. 
   
   
       19 . The method of  claim 17 , wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules.