Methods and nucleic acids for analyses of colorectal cell proliferative disorders

A method for determining CpG methylation status of NGFR in colon cell genomic DNA in a human subject comprising the steps of:

• a) obtaining from the human subject a biological sample comprising colon cell genomic DNA, the biological sample selected from the group consisting of blood plasma, blood serum, whole blood, stool, colonic effluent, colon tissue, and colon cells;
• b) treating the colon cell genomic DNA, or a fragment thereof, with one or more reagents to convert cytosine bases that are unmethylated in the 5′-position thereof to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties;
• c) contacting the treated colon cell genomic DNA of b), or the treated fragment thereof, within NGFR gene sequence (SEQ ID NO:
• 7) with an amplification enzyme and at least two primers that are complementary to, or hybridize under moderately stringent or stringent conditions to, a sequence selected from the group consisting of SEQ ID NOs:
• 316, 317, 432, and 433, and complements thereof, wherein the treated colon cell genomic DNA or the fragment thereof is either amplified to produce at least one amplificate, or is not amplified;
• and
• d) determining the methylation state of at least one CpG dinucleotide of a sequence selected within NGFR gene sequence SEQ ID NO:
• 7, based on
• a presence or absence of the at least one amplificate or on a property of the at least one amplificate, or
• an average methylation state or a value reflecting an average methylation state of a plurality of CpG dinucleotides within the NGFR gene sequence according to SEQ ID NO:
• 7.

A method for determining CpG methylation status of NGFR in colon cell genomic DNA in a human subject comprising:

• a) obtaining a biological sample from the human subject, the biological sample comprising colon cell genomic DNA, the biological sample selected from the group consisting of blood plasma, blood serum, whole blood, stool, colonic effluent, colon tissue, and colon cells;
• b) extracting, or otherwise isolating the colon cell genomic DNA from the biological sample;
• c) contacting the colon cell genomic DNA, or a fragment thereof comprising at least 16 contiguous nucleotides of SEQ ID NO:
• 7, with one or more methylation-sensitive restriction enzymes, wherein the colon cell genomic DNA is either cleaved thereby to produce cleavage fragments, or not cleaved thereby;
• and
• d) determining the CpG methylation status of SEQ ID NO:
• 7 based on
• the presence or absence of at least one of the cleavage fragments produced in step c), or
• an average methylation status or a value reflecting an average methylation status of a plurality of CpG dinucleotides of target CpG dinucleotide sequences within SEQ ID NO:
• 7.

A kit comprising:

• a methylation-sensitive restriction enzyme;
• and
• at least one nucleic acid molecule or peptide nucleic acid molecule, comprising a contiguous sequence of at least 16 nucleotides that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO:
• 7, and complements thereof.

A kit comprising:

• a bisulfite reagent;
• and
• at least one nucleic acid molecule or peptide nucleic acid molecule, comprising a contiguous sequence of at least 16 nucleotides that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NOS:
• 316, 317, 432, and 433, and complements thereof.