After every cycle of DNA replication, several modifications occur in the DNA. DNA methylation is one such post-synthesis modification. DNA methylation has been proven by research to be manifested in a number of biological processes such as regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in cancerous cells. It also acts as a protection mechanism adopted by the pathogen DNA (mainly bacterial against the endonuclease activity that destroys any foreign DNA.
In DNA, methylation usually occurs in the CpG islands, a CG rich region, upstream of the promoter region. The letter "p" here signifies that the C and G are connected by a phosphodiester bond. In humans, DNA methylation is carried out by a group of enzymes called DNA methyltransferases. These enzymes not only determine the DNA methylation patterns during the early development, but are also responsible for copying these patterns to the strands generated from DNA replication.
MethyLight assay, a TaqMan® probes based methylation profiling technique, can be effectively used for DNA methylation profiling, measure DNA methylation or detect methylated alleles in presence of the unmethylated ones. Due to the specificity of TaqMan® based assays, it is much more accurate than the end point detection assay such as methyl specific PCR (MSP).
The DNA sample is first treated with sodium bisulphite which converts the unmethylated cytosine bases to uracil. The methylated cytosines on the other hand, remain unaffected. Methyl Specific primers are designed to anneal with bisulphite converted DNA. This sample is amplified by PCR replacing the uracil with thymine. A TaqMan® probe is then used to detect the methylated strand.
a) Prediction of CpG islands in the DNA sequence.
b) Design methyl specific primers and TaqMan® probes.
c) Design suitable control primers and TaqMan® probes for corresponding unmethylated and untreated sequences.
d) Analyze pre-designed/published primers and/or TaqMan® probes and design of compatible oligos.
e) Primers and TaqMan® probes are designed by avoiding regions of homology identified by automatically interpreting BLAST search results.
Campbell, M.K., (1995) Biochemistry. Saunders College: Philadelphia, pgs. 615-16, 181.
Maclean, N., S.P. Gregory, and R.A. Flavell (1993) Eukaryotic Genes. Butterworth and Co., London, pgs. 53-67.
Xu, DG., HZ He, GG Zhang, B. Ganswendt, and H. Peter, (1993) DNA methylation of monohalogenated methanes of F344 rats, J. Tongi Med. Univ.,13(2)
Freiefelder, D., (1987). Molecular Biology. Jones and Bartlett: Boston, pgs 550-559.
Beacon Designer™ automates the design of real time primers and probes. It is used by molecular biologists worldwide to design successful real time PCR assays. It saves the time and the money involved in failed experiments. Beacon Designer™ is a flexible solution to your real time primer and probe design needs and pays for itself many times over.
You can BLAST search sequences and search for template structures from within the program. The results of both these searches are used while designing primers and probes. The regions that exhibit significant cross homologies and template structures are avoided automatically during the design.