MethyLight TaqMan® Assay Design
The MethyLight TaqMan® probe design involves the following two steps:
a) Predicting CpG islands
b) Designing MethyLight assays
a) Prediction of CpG islands: Beacon Designer™ predicts CpG islands in accordance with Gardiner-Garden and Frommer guidelines. Just load the sequence in Beacon Designer™ and the program will automatically predict these regions. You can also specify the CpG regions manually in a sequence. The CpG islands are displayed in green in the Sequence view tab and the regions that are rich in GC are displayed in red.
b) Designing MethyLight assays: Beacon Designer™ designs TaqMan® probes & methyl sensitive PCR primers. The MethyLight assays involves treatment of genomic DNA with sodium bisulphite followed by an alkaline treatment. Beacon Designer™ displays the corresponding bisulphite treated strand when the genomic sequence is provided. The program designs primers and TaqMan® probes for the specified CpG islands. You can also design suitable control primers and probes by designing them for unmethylated and untreated DNA sequences. These primers and TaqMan® probes are highly specific as they are designed by avoiding regions of significant cross homology identified by automatically interpreting BLAST search results. Template structures are also avoided during the design.