Method and Nucleic Acids for the Analysis of Colorectal Cell Proliferative Disorders

Abstract

The invention provides methods and nucleic acids for detecting, differentiating or distinguishing between colon cell proliferative disorders as well as therapy thereof by analysis of the gene EYA4 and its promoter and regulatory sequences. The invention further provides novel nucleic acid sequences useful for the cell proliferative disorder specific analysis of said gene as well as methods, assays and kits thereof.

What is claimed is: 
     
         1 . A nucleic acid comprising a sequence at least 18 bases in length of a segment of the chemically pretreated genomic DNA according to one of the sequences taken from the group comprising SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto. 
     
     
         2 . An oligomer, in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising in each case at least one base sequence having a length of at least 9 nucleotides which is complementary to, or hybridizes under moderately stringent or stringent conditions to a pretreated genomic DNA according to one of the SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto. 
     
     
         3 . The oligomer as recited in  claim 2 , wherein the base sequence includes at least one CpG, TpG or CpA dinucleotide. 
     
     
         4 . The oligomer as recited in  claim 3 , characterized in that the cytosine of the CpG dinucleotide is located approximately in the middle third of the oligomer. 
     
     
         5 . The oligomer as recited in  claim 2 , characterized in that it comprises a label. 
     
     
         6 . A set of oligomers, comprising at least two oligomers according to  claim 2 . 
     
     
         7 . A set of at least two oligonucleotides as recited in  claim 2 , which can be used as primer oligonucleotides for the amplification of DNA sequences of one of SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto. 
     
     
         8 . A set of oligonucleotides as recited in  claim 2 , characterized in that at least one oligonucleotide is bound to a solid phase. 
     
     
         9 . An arrangement of at least ten different oligomers according to  claim 2  (array). 
     
     
         10 . An array of different oligonucleotide- and/or PNA-oligomer sequences as recited in  claim 9 , characterized in that these are arranged on a plane solid phase in the form of a rectangular or hexagonal lattice. 
     
     
         11 . The array as recited in  claim 10 , characterized in that the solid phase surface is composed of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold. 
     
     
         12 . A composition of matter comprising the following:
 a nucleic acid comprising a sequence at least 18 bases in length of a segment of the chemically pretreated genomic DNA according to one of the sequences taken from the group comprising SEQ ID NO: 1 to SEQ ID NO: 5 and sequences complementary thereto, and   a buffer comprising at least one of the following substances: 1 to 5 mM Magnesium Chloride, 100-500 μM dNTP, 0.5-5 units of taq polymerase, an oligomer, in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising in each case at least one base sequence having a length of at least 9 nucleotides which is complementary to, or hybridizes under moderately stringent or stringent conditions to a pretreated genomic DNA according to one of the SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto.   
     
     
         13 . A kit useful for detecting, differentiating or distinguishing between colon cell proliferative disorders, comprising:
 a) a bisulfite reagent; and   b) at least one nucleic acid molecule or peptide nucleic acid molecule comprising, in each case a contiguous sequence at least 9 nucleotides in length that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NOS: 1 to 5, and complements thereof.   
     
     
         14 . The kit of  claim 13 , further comprising standard reagents for performing a methylation assay selected from the group consisting of MS-SNuPE, MSP, MethylLight™, HeavyMethyl™, COBRA, nucleic acid sequencing, and combinations thereof.