Computer-assisted methods and apparatus for identification and characterization of biomolecules in a biological sample

Abstract

The present invention provides computer-assisted methods and apparatus for identifying, selecting and characterizing biomolecules in a biological sample. A two-dimensional array is generated by separating biomolecules present in a complex mixture. A computer-readable profile is constructed representing the identity and relative abundance of a plurality of biomolecules detected by imaging the two dimensional array. Computer-mediated comparison of profiles from multiple samples permits automated identification of subsets of biomolecules that satisfy pre-ordained criteria. Identified biomolecules can be automatically isolated from the two dimensional array by a robotic device in accordance with computer-generated instructions. A supported gel suitable for electrophoresis is provided that is bonded to a solid support such that the gel has two-dimensional spatial stability and the solid support is substantially non-interfering with respect to detection of a label, such as a fluorescent label, associated with one or more biomolecules in the gel.

We claim:  
     
         1 . A computer-assisted method for selecting and directing the isolation of one or more biomolecules present in a two-dimensional array, comprising: 
 imaging said two-dimensional array or a replica thereof to generate a computer-readable output comprising, for each of a plurality of biomolecules detected in said two-dimensional array, a pair of x,y coordinates and a signal value;    processing said output in at least one computer to select one or more of said detected biomolecules in accordance with previously ordained or operator-specified criteria; and    generating machine-readable instructions that direct a robotic device to isolate at least one of said selected biomolecules from said two-dimensional array.    
     
     
         2 . A supported polyacrylamide gel, wherein the gel is suitable for SDS-PAGE and is bonded to a rigid, generally planar solid support.  
     
     
         3 . The gel according to  claim 2 , wherein the gel is covalently bonded to the solid support.  
     
     
         4 . The gel according to  claim 3 , wherein the gel comprises sodium dodecyl sulfate.  
     
     
         5 . The gel according to  claim 3 , wherein the gel and the support are bonded via a bifunctional linker.  
     
     
         6 . The gel according to  claim 3 , wherein the solid support is glass.  
     
     
         7 . The gel according to  claim 3 , wherein the gel contains a plurality of separated proteins.  
     
     
         8 . The gel according to  claim 7 , wherein the proteins have been separated by isoelectric focusing followed by SDS-PAGE.  
     
     
         9 . The gel according to  claim 7 , wherein the proteins are held in a stable array.  
     
     
         10 . The gel according to  claim 9 , from which one or more portions containing one or more proteins have been removed without affecting spatial integrity of the remainder of the array.  
     
     
         11 . The gel according to  claim 3 , from which one or more portions have been removed without affecting spatial stability of the gel.  
     
     
         12 . The gel according to  claim 3 , wherein the solid support provides one or more reference points detectable by an imaging device.  
     
     
         13 . The gel according to  claim 3 , wherein the solid support provides a bar-code.  
     
     
         14 . A method of separating a plurality of proteins wherein said proteins are in a liquid sample, said method comprising steps of: 
 a) applying said liquid sample onto a surface of a gel medium, wherein said gel medium comprises a pH gradient and is attached to a backing material;    b) applying a voltage across said gel medium to effect a separation of said proteins;    c) subjecting said gel medium to conditions wherein the volume of said liquid sample is substantially decreased through imbibition of said liquid sample into said gel medium or through loss of water in said liquid sample to an insulating gaseous or a liquid environment of said gel, such that said proteins are substantially incorporated into said gel.    
     
     
         15 . A method of excising a plurality of small regions of an electrophoresis gel comprising resolved proteins, said method comprising steps of: 
 a) staining said gel;    b) scanning said gel to yield a digitized image;    c) inputting said image into an image processor for identifying a plurality of protein-containing regions in said gel;    d) excising said protein-containing regions from said gel by means of a computer-controlled movable punch device on the basis of coordinates supplied by said image processor, wherein said punch device comprises a tube-like cutter and a central piston within said cutter, said cutter and said piston being moved coaxially by a computer-controlled means to cut, retrieve and expel a plug of said gel; and    e) depositing said protein-containing regions in a plurality of vessels.    
     
     
         16 . An integrated system for two-dimensional electrophoresis, said system comprising: 
 a) means for preparing an isoelectric focusing gel;    b) means for loading said isoelectric focusing gel with a plurality of protein molecules;    c) means for applying a first voltage across said isoelectric focusing gel;    d) means for preparing a slab electrophoresis gel;    e) means for loading said isoelectric focusing gel onto said slab electrophoresis gel;    f) means for applying a second voltage across said slab electrophoresis gel; and    g) means for staining said slab electrophoresis gel.    
     
     
         17 . The integrated system according to  claim 16 , wherein said system is programmable.  
     
     
         18 . The integrated system according to  claim 17 , wherein said system can operate on a repeated basis.  
     
     
         19 . The integrated system according to  claim 17 , wherein said system comprises means for scanning said gel.  
     
     
         20 . The integrated system according to  claim 19 , wherein said system comprises computer software means for extracting estimates of protein abundance and position from an image generated by said means for scanning said gel.  
     
     
         21 . The integrated system according to  claim 19 , wherein said system comprises computer software means for inserting said estimates of protein abundance and position into a database.