Enhancer scanning to locate regulatory regions in genomic loci

Melissa Buckley, Anxhela Gjyshi, Gustavo Mendoza-Fandiño, Rebekah Baskin, Renato S. Carvalho, Marcelo A. Carvalho, Nicholas T. Woods, Alvaro N.A. Monteiro

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

This protocol provides a rapid, streamlined and scalable strategy to systematically scan genomic regions for the presence of transcriptional regulatory regions that are active in a specific cell type. It creates genomic tiles spanning a region of interest that are subsequently cloned by recombination into a luciferase reporter vector containing the simian virus 40 promoter. Tiling clones are transfected into specific cell types to test for the presence of transcriptional regulatory regions. The protocol includes testing of different single-nucleotide polymorphism (SNP) alleles to determine their effect on regulatory activity. This procedure provides a systematic framework for identifying candidate functional SNPs within a locus during functional analysis of genome-wide association studies. This protocol adapts and combines previous well-established molecular biology methods to provide a streamlined strategy, based on automated primer design and recombinational cloning, allowing one to rapidly go from a genomic locus to a set of candidate functional SNPs in 8 weeks.

Original languageEnglish (US)
Pages (from-to)46-60
Number of pages15
JournalNature protocols
Volume11
Issue number1
DOIs
StatePublished - Jan 3 2016

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Buckley, M., Gjyshi, A., Mendoza-Fandiño, G., Baskin, R., Carvalho, R. S., Carvalho, M. A., Woods, N. T., & Monteiro, A. N. A. (2016). Enhancer scanning to locate regulatory regions in genomic loci. Nature protocols, 11(1), 46-60. https://doi.org/10.1038/nprot.2015.136