methylation in CpG island - (Jun/08/2005 )

there is a question: were the whole CpG's C methylated when the CpG island has been methylated and the expression of the gene was interfered?

Is this your question: To silence a gene, do all CpG sites need to be methylated in the CpG island?

If that's your question, the answer is no.

[quote=pcrman,Jun 14 2005, 12:39 AM]
Is this your question: To silence a gene, do all CpG sites need to be methylated in the CpG island?

sorry for my poor english!
we use methylation specific PCR to detect the methylation status of a gene. and the primer of the reaction can only contain 1 or 2 CpG site, we actruely can not know about the methylation status of a gene or its CpG island. how can we predicate if a gene's CpG island was methylated with the result of MSP.

I would say, combine your MSP with a restriction digest. Use a diagnostic restriction enzyme to determine if other sites within your MSPCR amplicon are methylated.

If there were hpaII sites within your amplicon digest with TaqI as all HpaII sites containing a methylated CpG convert to a TaqI site, the presence of a digestion (two or more bands bepending on the number of sites) will tell you that the sites encompassing the restriction enzyme are indeed methylated.

The better option is to bisulfite PCR and sequence your island.

Nick

Ideally, before picking up a MSP primer, bisulfite genomic sequencing should be done to map the whole CpG island for methylation. From the detailed mapping data, pick up some CpG sites whose methylation status correlates well with gene transcription. Then design MSP primers on those sites for screening of large number of samples. It all depends on the purpose of your study. Now the literature is flooded with MSP data which are mostly derived from randomly picked primers.