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Methylation-specific PCR


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#1 XCI

XCI

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Posted 19 August 2010 - 04:53 AM

Hi

I知 new in the field of methylation-specific PCR (MS-PCR).
I知 using MS-PCR to investigate methylation at imprinted loci.
I use EZ DNA methylation-Gold Kit (Zymo Research) to bisulfite convert my DNA (a 2.5 hour reaction as recommended by the company). I have obtained the primer sequences from another laboratory and they appear to work well in my hands as well. However I知 wondering how reproducible my MS-PCR results are.

I知 interested in calculating the unmethylated/methylated ratio of my DNA samples. How big differences in this ratio should I expect/tolerate if in run 2 identical PCR reactions with DNA from the same sample (and from the same bisulfate reaction)?

Some of my results are quite reproducible but sometimes I see differences in the ratio of 0.8-1. Is that way too much? And if so, what can be the explanation?

Thanks in advance

XCI

#2 methylnick

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Posted 20 August 2010 - 05:37 PM

I don't think it is reliable to calculate a methylation ratio using MSPCR as you suggest,

I would expect the variation between reactions (PCR) and the efficiency of the methyl specific and non-methyl specific are different and could mask the real changes or states you are trying to measure.

A direct sequencing approach or even pyrosequencing would be a better way to go because both alleles are measured in the same reaction

nick

All comments and communication are my own personal ones, and are not tied to any of my affiliations. 





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