Optimized method for methylated DNA immuno-precipitation
2015 (English)In: MethodsX, ISSN 1258-780X, E-ISSN 2215-0161, Vol. 2, e432-e439 p., eArticle in journal (Refereed) Published
Methylated DNA immunoprecipitation (MeDIP) is one of the most widely used methods to evaluate DNA methylation on a whole genome scale, and involves the capture of the methylated fraction of the DNA by an antibody specific to methyl-cytosine. MeDIP was initially coupled with microarray hybridization to detect local DNA methylation enrichments along the genome. More recently, MeDIP has been coupled with next generation sequencing, which highlights its current and future applicability. In previous studies in which MeDIP was applied, the protocol took around 3 days to be performed. Given the importance of MeDIP for studies involving DNA methylation, it was important to optimize the method in order to deliver faster turnouts. The present article describes optimization steps of the MeDIP method. The length of the procedure was reduced in half without compromising the quality of the results. This was achieved by:
• Reduction of the number of washes in different stages of the protocol, after a careful evaluation of the number of indispensable washes.
• Reduction of reaction times for detaching methylated DNA fragments from the complex agarose beads:antibody.
• Modification of the methods to purify methylated DNA, which incorporates new devices and procedures, and eliminates a lengthy phenol and chloroform:isoamyl alcohol extraction.
Place, publisher, year, edition, pages
Elsevier, 2015. Vol. 2, e432-e439 p., e
Other Basic Medicine Biological Sciences
IdentifiersURN: urn:nbn:se:liu:diva-123297DOI: 10.1016/j.mex.2015.10.006OAI: oai:DiVA.org:liu-123297DiVA: diva2:881219