Paul R. Rohde
P.Rohde at mailbox.uq.edu.au
Mon Oct 13 08:04:01 EST 1997
"Dr. Duncan Clark" <duncan at genesys.demon.co.uk> writes: > In article <6154gl$hgb$1 at nargun.cc.uq.edu.au>, Paul Rohde
> <P.Rohde at mailbox.uq.edu.au> writes
> >Sorry for my ignorant responce, and I don't know if it is applicable
> >to you, but there is a chemical method that transforms the methylated
> >base from one type of base to another.
> >So you PCR half your sample and sequence it.
> >On the other sample, you treat it, PCR and sequence, then look
> >for the base change differences (transformed bases).
> Good idea, the only snag is that all PCR products are always
> unmethylated unless you give the reaction methylated nucleotides. You
> therefore automatically lose whatever methylation you started from.
I don't want to be in a position where I have to defend something I said
about a subject area that isn't mine. I just heard it at a seminar
a fair while ago. But still, you missed the point!!
Yes, its true that the PCR product is unmethylated, but I didn't say to
measure the methylation on the PCR product!! Read what I said again.
Thankfully, Aimee answered me -- I wasn't to help her, but the little she
fed back to me will help me now to re-explain to you. Again:
A) You have you DNA sample that is methylated somehow.
B) Take half of this sample and treat it by bisulphate modifications.
This transforms any methylated C to T.
Ci) PCR this modified sample for sequencing
Cii) On the other half of the DNA sample that hasn't been treated
by bisulphate modifications, just PCR this for sequencing.
D) Compare the two sequences. The "untreated" sequence will be the
true DNA sequence. The "treated" sequence could show some differences
ie some T now exist that have replaced a C compared to the "untreated"
sequence. These Ts show the Cs that were methylated. Hey Presto!
Duncan, you must of read my posting way too late in the night. ;-)
If any body knows more about specifically methylation detection in
A/T rich genomes (I'm not sure what this implies really) please post
something because Aimee sounded pretty desperate!
> The problem with being on the cutting edge is that you occasionally get
> sliced from time to time....
> Duncan Clark
> DNAmp Ltd.
> TEl/FAX 01252376288
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