Methylated CpG island amplification (MCA)
View Patent ↗The present invention provides a method for identifying a methylated CpG containing nucleic acid by contacting a nucleic acid with a methylation sensitive restriction endonuclease that cleaves unmethylated PcG sites and contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, which cleaves both methylated and unmethylated CpG sites. The method also includes amplification of the CpG-containing nucleic acid using CpG-specific oligonucleotide primers. A method is also provided for detecting an age associated disorder by identification of a methylated CpG containing nucleic acid. A method is further provided for evaluating the responses of a cell to an agent. A kit is useful for detection of a CpG containing nucleic acid is also provided. Nucleic acid sequences encoding novel methylated clones.
1. A kit comprising a carrier means containing one or more containers comprising a membrane, wherein the membrane has a nucleic acid sequence, and the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 (MINT1, MINT2, MINT4, MINT6, MINT5, MINT 9, MINT10, MINT14, MINT15, MINT17, MINT19, MINT20, MINT22, MINT23, MINT24, MINT25, MINT27, MINT30, MINT31, MINT32, and MINT33) and is immobilized on the membrane.
2. An isolated nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 (MINT1, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINT15, MINT17, MINT19, MINT20, MINT22, MINT23, MINT24, MINT25, MINT27, MINT30, MINT31, MINT32, and MINT33).
3. The nucleic acid of claim 2 , wherein the nucleic acid is methylated.
4. The nucleic acid of claim 3 , wherein said nucleic acid is unmethylated.
5. The nucleic acid of claim 2 , wherein the nucleic acid is operatively linked to an expression control sequence.
6. The nucleic acid of claim 5 , wherein the expression control sequence is a promoter.
7. The nucleic acid of claim 6 , wherein the promoter is tissue specific.
8. An expression vector containing the nucleic acid of claim 2 .
9. The vector of claim 8 , wherein the vector is a plasmid.
10. The vector of claim 8 , wherein the vector is a viral vector.
11. The vector of claim 10 , wherein the viral vector is a retroviral vector.
12. A host cell containing the vector of claim 8 .
13. The host cell of claim 12 , wherein the cell is an eukaryotic cell.
14. The host cell of claim 12 , wherein the cell is a prokaryotic cell.
15. An isolated nucleic acid sequence comprising at least one methylated CpG dinucleotide, wherein the isolated nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-15 and 17-33.