methylation - primier design (Nov/13/2007 )

I read about methylation detection kit vi Na bisulfate. the think is I don't understand how you can detect unmethylated site using
Primers to amplify either the methylated strand or unmethylated strand.

How the primer will recognize the methylated strand or unmethylated strand.\
how it works?


let ' say I have this sequence:

ACCCGch3CpGTAGTch3CpGGCpGAAGch3CpGGACpGTT

after Na bisulfate:

AUUUGch3CpGTAGTch3CpGGUpGAAGch3CpGGAUpGTT


then how the pcr step work to indentified the methylation

thanks

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and if it us unmethylated the sequence would be

AUUUGUGTAGTUGGUGAAUGGAUGTT

so then you make one primer to the methylated and one primer to the unmethylated sequence which are different...

Not sure how to handle multiple CpGs with this method, what we do is design primers in regions with no CpGs then use sequencing to determine if it was methylated or not (C=methylated U/T=unmethylated)

hope this helps you.

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well my understanding is that unmethylated sequence should be

after Na bisulfate:

AUUUGch3CpGTAGTch3CpGGUpGAAGch3CpGGAUpGTT
meaning:
AUUUGCGTAGTCGGUGAAGCGGAUGTT and not the one you suggest right?

AUUUGUGTAGTUGGUGAAGUGGAUGTT

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ok but even if I use different set of primer how do I know the C methylated

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hi there,

you pick a primer that does not bind to any methyl CpG's because you don't know if they are methylated or not.

you perform the PCR and then sequence the amplicon, so all unmethylated C's become T after sequencing and methylated C's remain as C's in the sequence chromatogram.

Nick

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