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J Vis Exp. 2016 Aug 21;(114). doi: 10.3791/54093.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA).

Author information

1
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital.
2
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital; Medical Scientist Training Program, University of Cincinnati; Immunology Graduate Program, University of Cincinnati.
3
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital; Immunology Graduate Program, University of Cincinnati.
4
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital; Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital.
5
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital; Leah.Kottyan@cchmc.org.

Abstract

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

PMID:
27585267
PMCID:
PMC5091922
DOI:
10.3791/54093
[Indexed for MEDLINE]
Free PMC Article

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