Hybridization buffers using low molecular weight dextran sulfate and methods for their use

Abstract

This invention demonstrates that lower molecular weight dextran sulfate is an effective volume exclusion agent for hybridization reactions. The hybridization buffers of this invention utilize smaller polymers of dextran sulfate, and thus are of a lower viscosity than conventional hybridization buffers that use higher molecular weight dextran sulfate polymers as volume exclusion agents. The lower viscosity of these low molecular weight dextran sulfate polymer buffers makes them useful for automated hybridization processes as in dispensing, automated flow, and mixing on slide.

1 - 8 . (cancelled).  
     
     
         9 . A method of automatically hybridizing a polynucleotide probe to a target, comprising the steps of 
 preparing a section of tissue or cells to be examined;    hybridizing the tissue section or cellular preparation with a polynucleotide probe composition in the presence of low molecular weight dextran sulfate wherein said probe composition contains at least one sequence complementary to a coding region of the target;    removing unhybridized probe from said tissue section or cellular preparation; and    detecting the hybridized probe-target combination.    
     
     
         10 . The method of  claim 9  wherein said polynucleotide probe composition is selected from the group consisting of DNA probes and RNA probes.  
     
     
         11 . The method of  claim 9  wherein said tissue section is a paraffin-embedded tissue section.  
     
     
         12 . The method of  claim 9  wherein said tissue section is a fresh-frozen tissue section.  
     
     
         13 . The method of  claim 9  wherein said polynucleotide probe composition is labeled with a detectable label.  
     
     
         14 . The method of  claim 9  wherein said label is selected from the group consisting essentially of fluorophores, haptens and chromogens.  
     
     
         15 . The method of  claim 9  wherein the step of preparing a section of tissue or cells to be examined comprises a liquid-based preparation step.  
     
     
         16 . The method of  claim 9  wherein the step of preparing a section of tissue or cells to be examined comprises contacting the target RNA or DNA with blocking DNA to suppress background cross-reactive signal.  
     
     
         17 . The method of  claim 9  wherein said hybridization, removal and detection steps are performed by an automated tissue staining instrument.  
     
     
         18 . The method of  claim 9  wherein said probe composition is arrayed on a solid substrate.