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

Miller, Daniel E.; Patel, Zubin; Lu, Xiaoming; Lynch, Arthur T.; Weirauch, Matthew T.; Kottyan, Leah

doi:10.3791/54093-v

Cited by 2 publications

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“…First, we employed pulldown and silver staining in SNPs that had exhibited allele-specific differential binding by EMSA, excluding rs2814955 because of its low luciferase activity, again with 31bp oligonucleotides for each allele but now using nuclear extract from Daudi cells [26]. In two biological repeats, four high-probability functional variants again showed differential binding (red circles) ( Supplementary Figure 6 ).…”

Section: Resultsmentioning

confidence: 99%

“…Pulldown assay and Mass Spectrometry. Pulldown assay is modified from a published protocol for DNA Affinity Purification Assay (DAPA) [26]. Similar to SNP-seq, 500 ng of different alleles (31 bp, biotinylated) from a SNPs were attached to 25 μl streptavidin-Dynabeads (Invitrogen) and then incubated with 100 μg of Daudi nuclear extract for 1 h with or without non-biotinylated competitor (the same alleles).…”

Section: Snp-seq Oligos and Primersmentioning

confidence: 99%

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High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus

Wang,

Kim,

Martínez-Bonet

et al. 2023

Preprint

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Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKϵ. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a new regulatory pathway involving rs2297550, Ikaros, and IKKϵ implicated by human genetics in risk for SLE.

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Section: Resultsmentioning

confidence: 99%

Section: Snp-seq Oligos and Primersmentioning

confidence: 99%

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus

Wang,

Kim,

Martínez-Bonet

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

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Genetics of eosinophilic esophagitis

Kottyan

Rothenberg

2017

Mucosal Immunology

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Eosinophilic esophagitis (EoE) is a chronic, allergic disease associated with marked mucosal eosinophil accumulation. EoE disease risk is multifactorial and includes environmental and genetic factors. This review will focus on the contribution of genetic variation to EoE risk, as well as the experimental tools and statistical methodology used to identify EoE risk loci. Specific disease-risk loci that are shared between EoE and other allergic diseases (TSLP, LRRC32) or unique to EoE (CAPN14), as well as Mendellian Disorders associated with EoE, will be reviewed in the context of the insight that they provide into the molecular pathoetiology of EoE. We will also discuss the clinical opportunities that genetic analyses provide in the form of decision support tools, molecular diagnostics, and novel therapeutic approaches.

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

Cited by 2 publications

References 0 publications

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus

Genetics of eosinophilic esophagitis

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