Identification of differentially methylated and mutated nucleic acids

Abstract

The present invention provides Methyl- (or Mutant-) Differential Display (MDD) methods and nucleic acid probes for detecting mutations and the methylation patterns of nucleic acids. Regions of the genome are differentially methylated or mutated in different cell types, including cancerous cell types. The TSP50 gene is thereby identified and found to be differentially expressed in breast cancer cells. The present invention provides methods and compositions for identifying aberrant expression of the TSP50 gene and of tsp50 protein. Antibodies of the present invention can be used for diagnosis and treatment of diseases characterized by aberrant tsp50 expression.

1 - 49 . (canceled)  
     
     
         50 . A method for detecting whether a genomic DNA is hypomethylated or hypermethylated in the region which comprises TSP50 that is present in a test sample of cells which comprises: 
 a) cleaving said genomic DNA isolated from said test sample of cells with a restriction enzyme to generate a cleaved test-cell DNA;    b) hybridizing a probe to said cleaved test-cell DNA to form a hybridization complex, wherein said probe comprises a nucleic acid which hybridizes to said region which comprises TSP50; and    c) determining the size of the hybridization complex;    wherein said restriction enzyme cleaves a nonmethylated DNA but does not cleave a methylated DNA.    
     
     
         51 . The method of  claim 50 , wherein said test sample comprises breast tissue or ovarian tissue, or testicular tissue or a body fluid.  
     
     
         52 . The method of  claim 50 , wherein said test sample comprises cancer cells.  
     
     
         53 . The method of  claim 50 , wherein said region of genomic DNA which comprises TSP50 is within 10 kb of transcribed TSP50 nucleotides.  
     
     
         54 . The method of  claim 50 , wherein said region of genomic DNA which comprises TSP50 is within 2 kb of transcribed TSP50 nucleotides.  
     
     
         55 . The method of  claim 50 , wherein said restriction enzyme is Msp I.  
     
     
         56 . The method of  claim 50 , which further comprises: 
 a) cleaving genomic DNA isolated from a control sample of cells with said restriction enzyme to generate a cleaved control-cell DNA,    b) hybridizing said probe to said cleaved control-cell DNA to form a control-hybridization complex; and    c) determining the size of the control-hybridization complex.    
     
     
         57 - 59 . (canceled)  
     
     
         60 . A method of identifying human TSP50 mRNA in a test sample, which comprises: 
 a) contacting test sample RNA with a nucleic acid probe comprising a nucleotide sequence which is complementary to a portion of a human TSP50 mRNA; and    b) determining whether said probe hybridizes to said TSP50 mRNA.    
     
     
         61 . The method of  claim 60 , wherein said test sample comprises breast tissue or ovarian tissue or testicular tissue or a body fluid.  
     
     
         62 . The method of  claim 60 , wherein said test sample comprises cancer cells.  
     
     
         63 . The method of  claim 60 , which further comprises identifying TSP50 mRNA in a control sample, said control sample comprising a mammalian tissue sample in which TSP50 is not expressed.  
     
     
         64 - 66 . (canceled)  
     
     
         67 . A method of identifying TSP50 mRNA in a test sample, said method comprising the steps of making a cDNA to said TSP50 mRNA and identifying said cDNA.  
     
     
         68 . The method of  claim 67 , wherein said test sample comprises breast tissue or ovarian tissue or testicular tissue or a body fluid.  
     
     
         69 . The method of  claim 67 , wherein said test sample comprises cancer cells.  
     
     
         70 . The method of  claim 67 , which further comprises identifying TSP50 mRNA in a control sample, said control sample comprising a tissue sample in which TSP50 is not expressed.  
     
     
         71 . The method of  claim 67 , wherein said cDNA is made by reverse transcription.  
     
     
         72 . (canceled)  
     
     
         73 . The method of  claim 67 , wherein said cDNA is made by polymerase chain reaction.  
     
     
         74 . The method of  claim 73 , wherein said polymerase chain reaction employs a first primer comprising SEQ ID NO:12 and a second primer comprising SEQ ID NO: 11.  
     
     
         75 . (canceled)  
     
     
         76 . A method for identifying testicular tissue in a test sample which comprises detecting whether TSP50 mRNA is present in said test sample.  
     
     
         77 . (canceled)