Method for methylation analysis
Aspects of the invention relate to composition and methods for the providing of DNA for methylation analysis that is in particular suitable to be applied in reference laboratories. Further 5 aspects of the invention relate to composition and methods for the highly specific and sensitive methylation analysis of the Septin 9 gene also in particular suitable to be applied in reference laboratories.
1. A method for methylation analysis, comprising
a) treating genomic DNA with one or more reagents to convert unmethylated cytosine bases to uracil sulfonate or to another base having a different binding behavior than cytosine, while methylated cytosine remains unchanged;
b) amplifying the treated DNA by means of
i) at least one oligonucleotide comprising or consisting essentially of a sequence as defined by SEQ ID NO: 5 or sequence variants derived from said sequence by 5′-terminal and/or 3′-terminal deletion of 1, 2, 3, 4 or 5 nucleotides; and
ii) at least one oligonucleotide comprising or consisting essentially of a sequence as defined by SEQ ID NO: 63 or sequence variants derived from said sequence by 5′-terminal and/or 3′-terminal deletion of 1, 2, 3, or nucleotides;
wherein said oligonucleotides are suitable for use as primers;
and optionally, a polymerase; and
c) deducing the presence or absence of methylation of the CpG dinucleotides amplified in step b) from the results of step b).
2. The method according to claim 1 , wherein the amplifying of the treated DNA additionally involves
iv) at least one oligonucleotide comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO: 3, 7, 9, 23-30, 49-57, 73-76, 91, and 97-101, wherein said one or more oligonucleotides are suitable for use as blockers; and
v) optionally, at least one oligonucleotide comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO: 4, 8, 31-42, 58-61, 83 and 84, wherein said one or more oligonucleotides are suitable for use as probes and/or at least one oligonucleotide combination comprising or consisting essentially of either the sequences of SEQ ID NO: 77 and 78, of SEQ ID NO: 79 and 80, of SEQ ID NO: 81 and 82, of SEQ ID NO: 102 and 103, or of SEQ ID NO: 104 and 105, wherein said one or more oligonucleotide combinations are suitable for use as probe combinations.
3. A method for detecting and/or classifying cellular proliferative disorders, comprising:
a) treating genomic DNA with one or more reagents to convert unmethylated cytosine bases to uracil sulfonate or to another base having a different binding behavior than cytosine, while methylated cytosine remains unchanged;
b) amplifying the treated DNA by means of
i) at least one oligonucleotide comprising or consisting essentially of a sequence as defined by SEQ ID NO: 5 or sequence variants derived from said sequence by 5′-terminal and/or 3′-terminal deletion of 1, 2, 3, 4 or 5 nucleotides; and
ii) at least one oligonucleotide comprising or consisting essentially of a sequence as defined by SEQ ID NO: 63 or sequence variants derived from said sequence by 5′-terminal and/or 3′-terminal deletion of 1, 2, 3, or 4 nucleotides;
wherein said oligonucleotides are suitable for use as primers;
and optionally, a polymerase; and
c) deducing the presence or absence of methylation of the CpG dinucleotides amplified in step b) from the result of step b), wherein at least one of detecting and classifying cellular proliferative disorders is, at least in part, afforded.
4. The method according to claim 3 , wherein the amplifying of the treated DNA additionally involves
iv) at least one oligonucleotide comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO: 3, 7, 9, 23-30, 49-57, 73-76, 91, and 97-101, wherein said one or more oligonucleotides are suitable for use as blockers; and
v) optionally, at least one oligonucleotide comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO: 4, 8, 31-42, 58-61, 83 and 84, wherein said one or more oligonucleotides are suitable for use as probes and/or at least one oligonucleotide combination comprising or consisting essentially of either the sequences of SEQ ID NO: 77 and 78, of SEQ ID NO: 79 and 80, of SEQ ID NO: 81 and 82, of SEQ ID NO: 102 and 103, or of SEQ ID NO: 104 and 105, wherein said one or more oligonucleotide combinations are suitable for use as probe combinations.
5. Kit, comprising
a) at least one oligonucleotide comprising or consisting essentially of a sequence as defined by SEQ ID NO: 5 or sequence variants derived from said sequence by 5′-terminal and/or 3′-terminal deletion of 1, 2, 3, 4 or 5 nucleotides; and
b) at least one oligonucleotide comprising or consisting essentially of a sequence as defined by of SEQ ID NO: 63 or sequence variants derived from said sequence by 5′-terminal and/or 3′-terminal deletion of 1, 2, 3, or 4 nucleotides;
and optionally, a polymerase.
6. The kit according to claim 5 further comprising
(d) at least one oligonucleotide comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO: 3, 7, 9, 23-30, 49-57, 73-76, 91, and 97-101;
(e) optionally, at least one oligonucleotide comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO: 4, 8, 31-42, 58-61, 83, and 84, and/or at least one oligonucleotide combination comprising or consisting essentially of either the sequences of SEQ ID NO: 77 and 78, of SEQ ID NO: 79 and 80, of SEQ ID NO: 81 and 82, of SEQ ID NO: 102 and 103, or of SEQ ID NO: 104 and 105.