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Biology

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

Published: August 21st, 2016

DOI:

10.3791/54093

1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, 2Medical Scientist Training Program, University of Cincinnati, 3Immunology Graduate Program, University of Cincinnati, 4Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital
* These authors contributed equally

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....

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1. Preparation of Solutions and Reagents

  1. Order custom DNA oligonucleotide probes for use in EMSA and DAPA.
    1. To reduce non-specific protein binding, design short oligos (between 35-45 base pairs (bp) in length)30, and place the variant of interest directly in the center flanked by its 17 bp endogenous genomic sequence. For EMSA oligos, add a 5' fluorophore. For DAPA oligos, add a 5' biotin tag.
    2. Order both the sense strand and its reverse complement strand. Alternatively.......

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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.......

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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.......

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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.

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Name Company Catalog Number Comments
Custom DNA Oligonucleotides Integrated DNA Technologies http://www.idtdna.com/site/order/oligoentry
Potassium Chloride Fisher Scientific BP366-500 KCl, for CE buffer
HEPES (1M) Fisher Scientific 15630-080 For CE and NE buffer
EDTA (0.5M), pH 8.0 Life Technologies R1021 For CE, NE, and annealing buffer
Sodium Chloride Fisher Scientific BP358-1 NaCl, for NE buffer
Tris-HCl (1M), pH 8.0 Invitrogen BP1756-100 For annealing buffer
Phosphate Buffered Saline (1X) Fisher Scientific MT21040CM PBS, for cell wash
DL-Dithiothreitol solution (1M) Sigma 646563 Reducing agent
PMSF Thermo Scientific 36978 Protease Inhibitor
Phosphatase Inhibitor Cocktail  Thermo Scientific 78420 Prevents dephosphorylation of TFs
Nonidet P-40 Substitute IBI Scientific IB01140 NP-40, for nuclear extraction
BCA Protein Assay Kit Thermo Scientific 23225 For measuring protein concentration
Odyssey EMSA Buffer Kit Licor 829-07910 Contains all necessary EMSA buffers
TBE Gels, 6%, 12 Wells Invitrogen EC6265BOX For EMSA
TBE Buffer (10X) Thermo Scientific B52 For EMSA
FactorFinder Starting Kit Miltenyi Biotec 130-092-318 Contains all necessary DAPA buffers
Licor Odyssey CLx Licor Recommended scanner for DAPA/EMSA
Antibiotic-Antimycotic Gibco 15240-062 Contains 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Fungizone® Antimycotic
Fetal Bovine Serum Gibco 26140-079 FBS, for culture media
RPMI 1640 Medium Gibco 22400-071 Contains L-glutamine and 25mM HEPES

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