Methods and nucleic acids for analyses of cellular proliferative disorders
The invention provides methods, nucleic acids and kits for detecting, or for detecting and distinguishing between or among liver cell proliferative disorders or for detecting, or for detecting and distinguishing between or among colorectal cell proliferative disorders. The invention discloses genomic sequences the methylation patterns of which have utility for the improved detection of and differentiation between said class of disorders, thereby enabling the improved diagnosis and treatment of patients.
1. A method for detecting CpG dinucleotide methylation in genomic DNA of the Septin 9 gene from colorectal cells, comprising:
a) extracting or otherwise isolating genomic DNA comprising colorectal cell genomic DNA comprising a Septin 9 gene from a biological sample obtained from a subject;
b) treating the genomic DNA of a), or a fragment thereof comprising the Septin 9 gene, 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 genomic DNA of b), or the treated fragment thereof, with an amplification enzyme and at least one oligonucleotide or peptide nucleic acid (PNA) oligomer comprising a contiguous sequence of at least 9 nucleotides that is identical to, complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NOs: 4 to 15, and the complements thereof, wherein the treated colorectal cell genomic DNA or the fragment thereof is amplified to produce at least one amplificate; and
d) detecting by determining a presence of, or a property of, the at least one amplificate, the methylation state or level of at least one CpG dinucleotide of the Septin 9 gene, or an average, or a value reflecting an average methylation state or level of a plurality of CpG dinucleotides of the Septin 9 gene.
2. The method of claim 1 , wherein the at least one oligonucleotide or peptide nucleic acid (PNA) oligomer of step c) is not identical or complementary to all or a portion of the sequences according to SEQ ID NOs: 1 to 3.
3. The method of claim 1 , wherein the biological sample obtained from the subject is selected from the group consisting of cell lines, histological slides, biopsies, paraffin-embedded tissue, body fluids, urine, blood plasma, blood serum, whole blood, cells isolated from the blood, and any combinations thereof.
4. The method of claim 1 , wherein treating the genomic DNA, or the fragment thereof in step b) comprises use of a reagent selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, and any combinations thereof.
5. The method of claim 1 , wherein the at least one oligonucleotide or peptide nucleic acid (PNA) oligomer of step c) is a methylation-unspecific primer.
6. The method of claim 1 , wherein determining of step d) comprises the use of at least one nucleic acid molecule or peptide nucleic acid molecule, in each case the at least one nucleic acid molecule or peptide nucleic acid molecule comprising a contiguous sequence of at least 9 nucleotides in length that is identical to, complementary to, or hybridizes under moderately stringent or stringent conditions to, a sequence selected from the group consisting of SEQ ID NOs: 4 to 15, and complements thereof, wherein the at least one nucleic acid molecule or peptide nucleic acid molecule suppresses amplification of the nucleic acid to which it is hybridized.
7. The method of claim 1 , wherein determining in step d) comprises hybridization of at least one nucleic acid molecule or peptide nucleic acid molecule, in each case the at least one nucleic acid molecule or peptide nucleic acid molecule comprising a contiguous sequence of at least 9 nucleotides in length that is identical to, complementary to, or hybridizes under moderately stringent or stringent conditions to, a sequence selected from the group consisting of SEQ ID NOs: 4 to 15, and complements thereof.
8. The method of claim 1 , wherein the at least one hybridizing nucleic acid molecule or peptide nucleic acid molecule is bound to a solid phase.
9. The method of claim 8 , further comprising extending the at least one hybridized nucleic acid molecule or peptide nucleic acid molecule by at least one nucleotide base.
10. The method of claim 1 , wherein determining in step d), comprises sequencing of the amplificate.
11. The method of claim 1 , wherein the oligonucleotide or peptide nucleic acid (PNA) oligomer of step c) is a methylation-specific primer.
12. The method of claim 1 , wherein the contacting of 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); and generation of an amplificate nucleic acid molecule carrying a detectable label.
13. The method of claim 1 , wherein the subject has colorectal cancer or a colorectal cell proliferative disorder and the method is carried out repeatedly.
14. The method of claim 1 , wherein the subject has colorectal cancer or a colorectal cell proliferative disorder.
15. The method of claim 14 , wherein the subject has a neoplastic colorectal cell proliferative disorder.