Primers for analyzing methylated sequences and methods of use thereof
Primers having abasic regions or mismatches for amplifying sequences suspected of having methylation. Primers having abasic regions or mismatches for amplifying sequences adjacent to suspected or known methylated sequences. Methods of using primers having abasic regions or mismatches for identification of methylated sequences or sequences adjacent to suspected or known methylation sequences.
1. A primer that it is able to specifically hybridize to either a methylated or an unmethylated CpG site of a template nucleic acid, wherein the primer comprises an abasic region that interacts with the methylated or the unmethylated CpG site of the template nucleic acid wherein the primer comprises the nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.
2. The primer according to claim 1 , wherein the abasic region spans at least one base of the template.
3. The primer according to claim 1 , wherein the abasic region is covalently linked to a guanine moiety.
4. The primer according to claim 1 , wherein the abasic region comprises a moiety selected from the group consisting of:
O-dimethoxytrityl-1′,2′-dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;
O-dimethoxytrityl-1′-methoxy-2′-dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;
[4-(4,4′-Dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite;
O-Dimethoxytrityl-1′-Deoxyribose-2′-O-Triisopropylsilyloxymethyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;
(4,4′-Dimethoxytrityloxy)-dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite);
O-Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;
O-Dimethoxytrityl-triethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; and
3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
5. The primer according to claim 1 , wherein the primer comprises at least one mismatched nucleotide that has similar annealing characteristics to both uracil and cytosine such that the primer hybridizes to either the methylated or the unmethylated CpG site of the template nucleic acid.
6. The primer according to claim 5 , wherein the primer comprises the nucleotide sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.
7. The primer according to claim 1 , wherein the primer further comprises an adaptor sequence.
8. The primer according to claim 7 , wherein the adaptor sequence comprises a homopolymer region.