title: Enhancer scanning to locate regulatory regions in genomic loci.
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.
authors.affiliation: Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.|University of South Florida Cancer Biology PhD Program, Tampa, Florida, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Buckley
authors.affiliation: Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.|University of South Florida Cancer Biology PhD Program, Tampa, Florida, USA.
authors.forename: Anxhela
authors.initials: A
authors.lastname: Gjyshi
authors.affiliation: Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Mendoza-Fandiño
authors.affiliation: Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.
authors.forename: Rebekah
authors.initials: R
authors.lastname: Baskin
authors.affiliation: Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.|Department of Pharmacology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Renato S
authors.initials: RS
authors.lastname: Carvalho
authors.affiliation: Instituto Nacional de Câncer, Divisão de Farmacologia, Rio de Janeiro, Brazil.|Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Carvalho
authors.affiliation: Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.|Department of Oncologic Science, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Woods
authors.affiliation: Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.|Department of Oncologic Science, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA.
authors.forename: Alvaro N A
authors.identifier: http://orcid.org/0000-0002-8448-4801
authors.initials: AN
authors.lastname: Monteiro
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D056426:Genetic Loci / Q000235:genetics*; D023281:Genomics / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000235:genetics