title: Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA.
abstract: Across vertebrate genomes methylation of cytosine residues within the context of CpG dinucleotides is a pervasive epigenetic mark that can impact gene expression and has been implicated in various developmental and disease-associated processes. Several biochemical approaches exist to profile DNA methylation, but recently an alternative approach based on profiling non-methylated CpGs was developed. This technique, called CxxC affinity purification (CAP), uses a ZF-CxxC (CxxC) domain to specifically capture DNA containing clusters of non-methylated CpGs. Here we describe a new CAP approach, called biotinylated CAP (Bio-CAP), which eliminates the requirement for specialized equipment while dramatically improving and simplifying the CxxC-based DNA affinity purification. Importantly, this approach isolates non-methylated DNA in a manner that is directly proportional to the density of non-methylated CpGs, and discriminates non-methylated CpGs from both methylated and hydroxymethylated CpGs. Unlike conventional CAP, Bio-CAP can be applied to nanogram quantities of genomic DNA and in a magnetic format is amenable to efficient parallel processing of samples. Furthermore, Bio-CAP can be applied to genome-wide profiling of non-methylated DNA with relatively small amounts of input material. Therefore, Bio-CAP is a simple and streamlined approach for characterizing regions of the non-methylated DNA, whether at specific target regions or genome wide.
authors.affiliation: Department of Biochemistry, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford University, Oxford, UK.
authors.forename: Neil P
authors.initials: NP
authors.lastname: Blackledge
authors.forename: Hannah K
authors.initials: HK
authors.lastname: Long
authors.forename: Jin C
authors.initials: JC
authors.lastname: Zhou
authors.forename: Skirmantas
authors.initials: S
authors.lastname: Kriaucionis
authors.forename: Roger
authors.initials: R
authors.lastname: Patient
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Klose
mesh_terms: D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D018899:CpG Islands; D004247:DNA / Q000302:isolation & purification; D019175:DNA Methylation; D059014:High-Throughput Nucleotide Sequencing; D017422:Sequence Analysis, DNA; D016335:Zinc Fingers
chemical_list: D004247:DNA