EDGAR STEVEN C.

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MedIP: Methylated DNA
immunoprecipitation
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 The Methylated DNA IP (MeDIP) is based on the affinity

purification of methylated DNA using an antibody directed
against 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-
hmC) in the case of hMeDIP.
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Sitios CpG
C: Citosina p:fosfato G:Guanina
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How it works

Methyl DNA IP is performed as follows: Genomic DNA from cultured cells

or tissues is prepared, sheared, and then denatured.

Then, immunoselection and immunoprecipitation can take place using the

antibody directed against 5 methylcytosine.

After isolation and purification is performed, the IP’d methylated DNA is

ready for any subsequent analysis as qPCR, amplification, hybridization on
microarrays or next generation sequencing.
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Bioruptor ultrasonicators
with adaptive cavitation
technology for highest
shearing performance
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For years, classical tumor-suppressor genes for which genetic

lesions had been described, such as VHL, p73, or p16 (INK4a),
constituted the only source of candidate genes used for
performing analysis on the methylation.

Vol. 44 ı No. 1 ı 2008 www.biotechniques.com ı BioTechniques ı 39

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A monoclonal antibody raised against 5-methylcytidine (5mC)

is used to purify methylated DNA

Sonication conditions must be optimized to yield fragments

ranging in size between 300 and 600 bp

 denatured at 95°C  DNA fragments

Use of protein G coupled to standard or magnetic beads as well as

multiple washes following incubation with the anti-5mC antibody
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For instance, colon carcinoma and prostate cancer cells have recently been analyzed using
MeDIP combined with a microarray containing over 13,000 promoters
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(126 genes) are enriched when immunoprecipitating this

colon cancer cell line with anti-5mC.

And subtle methylation changes do not seem to

be a Good target for such studies.

DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted

allows the unambiguous identification of CpG islands
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 Otro método que se ha utilizado recientemente, específicamente

en el estudio de la metilacion genómica de tejidos cancerosos,
es el análisis de la recuperación de islas CpG metiladas
(MIRA) [Raunch et al. 2006]. El MIRA se basa en la purificacion de ADN
metilado con columas de proteínas de unión a zonas CpG

 En el diseño experimental, se debe considerar que los métodos

MedIP y MIRA sulen tener poca sensibilidad en regiones
genómicas con baja densidad relativa de sitios CpG