Method and nucleic acids for the analysis of colon cell proliferative disorders

Abstract

The invention provides methods and nucleic acids for detecting, differentiating or distinguishing between colon cell proliferative disorders by analysis of one or more of the genes Versican, TPEF, H-Cadherin, Calcitonin, and EYA4. The invention further provides novel nucleic acid sequences useful for the cell proliferative disorder specific analysis of said genes as well as methods, assays and kits thereof.

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 6 to SEQ ID NO 25 and sequences complementary thereto.  
     
     
         2 . An oligomer, in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising at least one base sequence having a length of at least 9 nucleotides which is complementary to, or hybridises under moderately stringent or stringent conditions to a pretreated genomic DNA according to one of the SEQ ID NO 6 to SEQ ID NO 25 or 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 , characterised in that the cytosine of the CpG, or the thymine of the TpG or the adenosine of the CpA dinucleotide is located approximately in the middle third of the oligomer.  
     
     
         5 . A set of oligomers, comprising at least two oligomers according to any of  claims 2  to  4 .  
     
     
         6 . A set of at least two oligonucleotides as recited in  claims 2  to  5 , which can be used as primer oligonucleotides for the amplification of DNA sequences of one of SEQ ID NO 6 to SEQ ID NO 25 and sequences complementary thereto.  
     
     
         7 . A set of oligonucleotides as recited in  claims 2  to  5 , characterised in that at least one oligonucleotide is bound to a solid phase.  
     
     
         8 . Use of a set of oligomer probes comprising at least four of the oligomers according to any of claims  5  through  7  for detecting the cytosine methylation state and/or single nucleotide polymorphisms (SNPs) within one of the sequences according to SEQ ID NO 1 to SEQ ID NO 5, or sequences complementary thereto.  
     
     
         9 . A method for manufacturing an arrangement of different oligomers (array) fixed to a carrier material for analysing colon cell proliferative disorders associated with the methylation state of the CpG dinucleotides of one of SEQ ID NO 1 to SEQ ID NO 5, and sequences complementary thereto wherein at least one oligomer according to any of the claims  2  through  5  and  7  is coupled to a solid phase.  
     
     
         10 . An arrangement of different oligomers (array) obtainable according to  claim 7 .  
     
     
         11 . An array of different oligonucleotide- and/or PNA-oligomer sequences as recited in  claim 10 , characterised in that these are arranged on a solid phase in the form of a rectangular or hexagonal lattice.  
     
     
         12 . The array as recited in any of the claims  10  or  11 , characterised in that the solid phase surface is composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold.  
     
     
         13 . A method for detecting, differentiating or distinguishing between colon cell proliferative disorders associated with at least one gene and/or their regulatory regions from the group comprising Versican, TPEF, H-Cadherin, Calcitonin and EYA4 in a subject, said method comprising contacting a target nucleic acid in a biological sample obtained from said subject with at least one reagent or a series of reagents, wherein said reagent or series of reagents, distinguishes between methylated and non methylated CpG dinucleotides within the target nucleic acid.  
     
     
         14 . A method according to  claim 13 , comprising: 
 a) obtaining, from a subject, a biological sample having subject genomic DNA;    b) treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridisation properties;    c) contacting the treated genomic DNA, or the treated fragment thereof, with an amplification enzyme and at least two primers comprising, in each case a contiguous sequence at least 16 nucleotides in length that is complementary to, or hybridises under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO 6 to SEQ ID NO 25, or complements thereof, wherein the treated DNA or a fragment thereof is either amplified to produce one or more amplificates, or is not amplified; and    d) determining, based on the presence or absence of, or on a property of said amplificate, the methylation state of at least one CpG dinucleotide sequence of SEQ ID NO 1 to SEQ ID NO 5, or an average, or a value reflecting an average methylation state of a plurality of CpG dinucleotide sequences of SEQ ID NO 1 to SEQ ID NO 5, whereby at least one of detecting or distinguishing between colon cell proliferative disorders is, at least in part, enabled.    
     
     
         15 . A method according to  claim 13  comprising the following steps of 
 a) obtaining, from a subject, a biological sample having subject genomic DNA;    b) treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties;    c) amplifying one or more fragments of the treated DNA such that only or preferentially DNA originating from colon or colon cell proliferative disorder cells are amplified;    d) detecting the amplificates or characteristics thereof and thereby deducing on the presence or absence of a colon cell proliferative disorder.    
     
     
         16 . A method according to  claims 13  to  15  wherein said colon cell proliferative disorders are taken from the group comprising adenocarcinomas, polyps, squamous cell cancers, carcinoid tumours, sarcomas and lymphomas.  
     
     
         17 . The method of  claims 14  to  16 , wherein in step a) the biological sample obtained from the subject is selected form the group consisting of histological slides, biopsies, paraffin-embedded tissue, bodily fluid, stool, blood, serum, plasma, urine, sputum and combinations thereof.  
     
     
         18 . The method of one of  claims 14  to  17 , wherein step b) treating the genomic DNA, or the fragment thereof, comprises use of a bisulfite solution.  
     
     
         19 . The method of one of  claims 14  to  16 , wherein treating in b) is subsequent to embedding the DNA in agarose.  
     
     
         20 . The method of one of  claims 14  to  19 , wherein step b) treating the genomic DNA, comprises treating in the presence of at least one of a DNA denaturing agent or a radical scavenger.  
     
     
         21 . The method of one of  claims 14  to  20 , wherein contacting or amplifying in step c) comprises use of at least one method selected from the group consisting of: use of a heat-resistant DNA polymerase as the amplification enzyme; use of a polymerase lacking 5′-3′ exonuclease activity; use of a polymerase chain reaction (PCR); generation of a amplificate nucleic acid molecule carrying a detectable labels; and combinations thereof.  
     
     
         22 . The method of  claim 21 , wherein the detectable amplificate label is selected from the label group consisting of: fluorescent labels; radionuclides or radiolabels; amplificate mass labels detectable in a mass spectrometer; detachable amplificate fragment mass labels detectable in a mass spectrometer; amplificate, and detachable amplificate fragment mass labels having a single-positive or single-negative net charge detectable in a mass spectrometer; and combinations thereof.  
     
     
         23 . The method of  claim 22 , comprising in step d) use of mass spectrometry for detecting the amplificate, or detachable amplificate fragment mass labels.  
     
     
         24 . The method according to one of  claims 14  to  23  wherein in step c) of the method 2 or more different fragments are amplified.  
     
     
         25 . The method according to one of  claims 14  to  24  wherein one or more of said primers comprise sequences taken from the group according to SEQ ID NO 34 to SEQ ID NO 49, 96, 97, 101, 102, 106 and 107.  
     
     
         26 . The method according to one of  claims 14  to  24  wherein one or more of said primers comprise one or more CpG, TpG or CpA dinucleotides.  
     
     
         27 . The method of  claim 26  wherein said primers comprise between 2 to 5 CpG, TpG or CpA dinucleotides.  
     
     
         28 . The method according to one of claims  26  or  27  wherein said one or more CpG, TpG or CpA dinucleotides are located within the 3′ half of the primer.  
     
     
         29 . The method according to one of  claims 26  to  28  wherein said primers comprise one or more bases which hybridise to positions that were converted in the treatment of step b) in  claim 14 , or of step c) in  claim 15 .  
     
     
         30 . The method of  claim 29  wherein at least one of said bases are located within the 3′ half of the primer.  
     
     
         31 . The method according to one of  claims 14  to  30  wherein said amplificates obtained in step d) comprise at least one 20 base pair sequence that comprises 3 or more CpG, TpG or CpA dinucleotides.  
     
     
         32 . The method according to one of  claims 14  to  31 , further comprising in step c) the use of at least one nucleic acid molecule or peptide nucleic acid molecule at least 18 base pairs in length comprising one or more CpG, TpG or CpA dinucleotides and wherein the sequence of said molecule is complementary or identical to a sequence selected from the group consisting of SEQ ID NO 6 to SEQ ID NO 25, and complements thereof, and wherein said nucleic acid molecule or peptide nucleic acid molecule suppresses amplification of the nucleic acid to which it is hybridised.  
     
     
         33 . The method according to  claim 32  wherein the sequence of said nucleic acid(s) or peptide nucleic acid(s) is selected from the group consisting SEQ ID NO 85 to SEQ ID NO 87, 98, 103 and 108 and sequences complementary thereto.  
     
     
         34 . The method of  claim 32 , wherein amplification of DNA that was unmethylated prior to treatment of step b) is suppressed.  
     
     
         35 . The method of one of  claims 32  to  34 , wherein said nucleic acid molecule or peptide nucleic acid molecule is in each case modified at the 5′-end thereof to preclude degradation by an enzyme having a 5′-3′ exonuclease activity.  
     
     
         36 . The method of one of  claims 32  to  35 , wherein said nucleic acid molecule or peptide nucleic acid molecule in each case lack a 3′ hydroxyl group.  
     
     
         37 . The method of one of  claims 32  to  36 , wherein the amplification enzyme is a polymerase lacking 5′-3′ exonuclease activity.  
     
     
         38 . The method of one of  claims 32  to  37 , wherein the binding site of the oligonucleotide or PNA oligomer is identical to, or overlaps with that of the primer and thereby hinders hybridisation of the primer to its binding site.  
     
     
         39 . The method of one of  claims 32  to  38 , wherein the binding sites of at least two of the oligonucleotides or PNA oligomers are identical to, or overlap with those of at least two of the primers, and thereby hinder hybridisation of the primers to their binding site.  
     
     
         40 . The method of  claim 39 , wherein hybridisation of at least one of the oligonucleotides or peptide nucleic acid oligomers hinders hybridisation of a forward primer, and the hybridisation of at least one of the oligonucleotides or peptide nucleic acid oligomers hinders the hybridisation of a reverse primer that binds to the elongation product of said forward primer.  
     
     
         41 . The method of one of  claims 32  to  38 , wherein said oligonucleotide or peptide nucleic acid oligomer hybridises between the binding sites of the forward and reverse primers.  
     
     
         42 . The method of one of claims  14  or  15 , wherein determining in step d), comprises hybridisation of at least one nucleic acid molecule or peptide nucleic acid molecule in each case comprising a contiguous sequence at least 9 nucleotides in length comprising one or more CpG, TpG or CpA dinucleotides and wherein the sequence of said molecule that is complementary or identical to a sequence selected from the group consisting of SEQ ID NO 6 to SEQ ID NO 25.  
     
     
         43 . The method of  claim 42 , wherein at least one such hybridising nucleic acid molecule or peptide nucleic acid molecule is bound to a solid phase.  
     
     
         44 . The method of  claim 43 , wherein a plurality of such hybridising nucleic acid molecules or peptide nucleic acid molecules are bound to a solid phase in the form of a nucleic acid or peptide nucleic acid array selected from the array group consisting of linear, hexagonal, rectangular, and combinations thereof.  
     
     
         45 . The method of one of claims  14  or  15 , wherein determining in step d), comprises sequencing of the amplificate.  
     
     
         46 . The method of one of claims  14  or  15 , wherein determining in step d), comprises: hybridising at least one nucleic acid molecule comprising a contiguous sequence at least 9 nucleotides in length that is complementary to, or hybridises under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO 6 to SEQ ID NO 25, and complements thereof; and extending at least one such hybridised nucleic acid molecule by at least one nucleotide base.  
     
     
         47 . The method according to  claim 42  wherein the sequence of said nucleic acid(s) or peptide nucleic acid(s) is selected from the group consisting SEQ ID NO 50 to SEQ ID NO 84, SEQ ID NO 88 to SEQ ID NO 90, 99, 100, 104, 105, 109, 110 and sequences complementary thereto.  
     
     
         48 . The method according to  claim 42  wherein said oligonucleotides or PNA oligomers are fluorescently labelled, and wherein detection thereof is by either an increase or a decrease in fluorescence or fluorescence polarisation.  
     
     
         49 . The method according to  claim 42  wherein the hybridisation of the oligonucleotides or PNA oligomers is detectable by fluorescence resonance energy transfer, and wherein the detection is by either an increase or a decrease in fluorescence.  
     
     
         50 . The method of one of claims  14  or  15 , wherein the background DNA concentration is at between 100 to 1000 fold excess of the concentration of the DNA to be investigated.  
     
     
         51 . A method according to  claim 13 , comprising: 
 a) obtaining, from a subject, a biological sample having subject genomic DNA;    b) extracting the genomic DNA;    c) contacting the genomic DNA, or a fragment thereof, comprising SEQ ID NO 1 to SEQ ID NO 5 or a sequence that hybridises under stringent conditions to SEQ ID NO 1 to SEQ ID NO 5, with one or more methylation-sensitive restriction enzymes, wherein the genomic DNA is either digested thereby to produce digestion fragments, or is not digested thereby; and    d) determining, based on a presence or absence of, or on property of at least one such fragment, the methylation state of at least one CpG dinucleotide sequence of SEQ ID NO 1 to SEQ ID NO 5, or an average, or a value reflecting an average methylation state of a plurality of CpG dinucleotide sequences of SEQ ID NO 1 to SEQ ID NO 5, whereby at least one of detecting the prostate cell proliferative disorder, or distinguishing between a transitional and a peripheral zone of origin of the prostate cell proliferative disorder is, at least in part, afforded.    
     
     
         52 . The method of  claim 51 , further comprising, prior to determining in step d), amplifying of the digested or undigested genomic DNA.  
     
     
         53 . The method of  claim 52 , wherein amplifying comprises use of at least one method selected from the group consisting of: use of a heat resistant DNA polymerase as an amplification enzyme; generation of a amplificate nucleic acid carrying a detectable label; and combinations thereof.  
     
     
         54 . The method of  claim 53 , wherein the detectable amplificate label is selected from the label group consisting of: fluorescent labels; radionuclides or radiolabels; amplificate mass labels detectable in a mass spectrometer; detachable amplificate fragment mass labels detectable in a mass spectrometer; amplificate, and detachable amplificate fragment mass labels having a single-positive or single-negative net charge detectable in a mass spectrometer; and combinations thereof.  
     
     
         55 . The method of  claim 54 , comprising use of mass spectrometry for detecting amplificate, or detachable amplificate fragment mass labels.  
     
     
         56 . The method of  claim 55 , wherein the mass spectrometry is selected from the group consisting of matrix assisted laser desorption/ionisation mass spectrometry (MALDI), electron spray mass spectrometry (ESI), and combinations thereof.  
     
     
         57 . The method of  claim 51 , wherein the biological sample obtained from the subject is selected from the group consisting of histological slides, biopsies, paraffin-embedded tissue, bodily fluid, stool, blood, serum, plasma, urine, sputum and combinations thereof.  
     
     
         58 . A kit useful for detecting, differentiating or distinguishing between colon cell proliferative disorders, comprising: 
 i) a bisulfite reagent; and    ii) 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 hybridises under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 25, and complements thereof.    
     
     
         59 . The kit of  claim 58 , 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.  
     
     
         60 . The use of a nucleic acid according to  claim 1 , of an oligonucleotide or PNA-oligomer according to one of the claims  2  through  7 , of a kit according to claims  58  or  59 , of an array according to one of the claims  11  through  12 , of a set of oligonucleotides according to one of claims  5  through  7 , or a method according to  claims 13  to  57 , for the classification, differentiation and/or diagnosis of colon cell proliferative disorders or the predisposition to colon cell proliferative disorders.  
     
     
         61 . The use of a nucleic acid according to  claim 1 , of an oligonucleotide or PNA-oligomer according to one of the claims  2  through  7 , of a kit according to claims  58  or  59 , of an array according to one of the claims  11  through  12 , of a set of oligonucleotides according to one of claims  5  through  7 , or a method according to  claims 13  to  57 , for the therapy of colon cell proliferative disorders.