Published: August 21st, 2016
We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.
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.
Sequencing and genotyping based studies, including Genome-Wide Association Studies (GWAS), candidate locus studies, and deep-sequencing studies, have identified many genetic variants that are statistically associated with a disease, trait, or phenotype. Contrary to early predictions, most of these variants (85-93%) are located in non-coding regions and do not change the amino acid sequence of proteins1,2. Interpreting the function of these non-coding variants and determining the biological mechanisms connecting them to the associated disease, trait, or phenotype has proven challenging3-6. We have developed a general strategy to identify the molec....
1. Preparation of Solutions and Reagents
In this section, representative results of what to expect are provided when performing an EMSA or DAPA, and the variability with regards to the quality of lysate is characterized. For example, it has been suggested that freezing and thawing protein samples multiple times may result in denaturation. In order to explore the reproducibility of EMSA analysis in the context of these "freeze-thaw" cycles, two 35 bp oligos differing at one genetic variant were incubated with a single bat.......
Although advances in sequencing and genotyping technologies have greatly enhanced our capacity to identify genetic variants associated with disease, our ability to understand the functional mechanisms impacted by these variants is lagging. A major source of the problem is that many disease-associated variants are located in n on-coding regions of the genome, which likely affect harder-to-predict mechanisms controlling gene expression. Here, we present a protocol based on the EMSA and DAPA techniques, valuable molecular t.......
We thank Erin Zoller, Jessica Bene, and Lindsey Hays for input and direction in protocol development. MTW was supported in part by NIH R21 HG008186 and a Trustee Award grant from the Cincinnati Children's Hospital Research Foundation. ZHP was supported in part by T32 GM063483-13.....
|Custom DNA Oligonucleotides
|Integrated DNA Technologies
|KCl, for CE buffer
|For CE and NE buffer
|EDTA (0.5M), pH 8.0
|For CE, NE, and annealing buffer
|NaCl, for NE buffer
|Tris-HCl (1M), pH 8.0
|For annealing buffer
|Phosphate Buffered Saline (1X)
|PBS, for cell wash
|DL-Dithiothreitol solution (1M)
|Phosphatase Inhibitor Cocktail
|Prevents dephosphorylation of TFs
|Nonidet P-40 Substitute
|NP-40, for nuclear extraction
|BCA Protein Assay Kit
|For measuring protein concentration
|Odyssey EMSA Buffer Kit
|Contains all necessary EMSA buffers
|TBE Gels, 6%, 12 Wells
|TBE Buffer (10X)
|FactorFinder Starting Kit
|Contains all necessary DAPA buffers
|Licor Odyssey CLx
|Recommended scanner for DAPA/EMSA
|Contains 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Fungizone® Antimycotic
|Fetal Bovine Serum
|FBS, for culture media
|RPMI 1640 Medium
|Contains L-glutamine and 25mM HEPES
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