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. 2014 May 29;7:9. doi: 10.1186/1756-8935-7-9

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Copyright © 2014 Gagne et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Schematic of Dlk1-Gtl2 imprinting cluster and regions analyzed. (A)Dlk1-Gtl2 imprinting cluster, including transcriptional start sites (arrows), transcription units (gray boxes) and differentially methylated regions (black boxes). (B, C) The 600 bp (B) and 220 bp (C) regions of the Dlk1-DMR analyzed by bisulfite mutagenesis and DNA sequencing in this study, corresponding to positions 109,459,577-109,460,173 and 109,459,680-109,459,900, NC_000078.6, respectively. The C/T polymorphism (*) between C57BL/6 J and Mus musculus castaneus is located at 109,459,746. (D) Sequence flanking the C/T polymorphism (red text) and schematic representing ligation of the hairpin linker to BglI-digested genomic DNA. The hairpin linker was designed to anneal to itself, forming a hairpin structure, and to the 3′ overhang generated following BglI digestion. Ligation of the hairpin linked to BglI-digested DNA results in the covalent attachment of the complimentary strands of DNA. Primers (block arrows) were designed to anneal to the bisulfite-mutagenized genomic DNA in order to amplify the region of interest.