Name: screening for functional non-coding genetic variants using electrophoretic mobility shift assay (emsa) and dna-affinity precipitation assay (dapa)
Uploaded: 2016-08-21T13:00:00
Description: Watch this Scientific Journal Video about Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA at JoVE.com

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.

1. Preparation of Solutions and Reagents
<ol>
	<li>Order custom DNA oligonucleotide probes for use in EMSA and DAPA.
	<ol>
		<li>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&#39; fluorophore. For DAPA oligos, add a 5&#39; biotin tag.</li>
		<li>Order both the sense strand and its reverse complement strand. Alternatively...

<ol>
	<li>Hindorff, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+etiologic+and+functional+implications+of+genome-wide+association+loci+for+human+diseases+and+traits.">Potential etiologic and functional implications of genome-wide association loci for human diseases and traits.</a> Proc Natl Acad Sci U S A. 106 (23), 9362-9367 (2009).</li><li>Maurano, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+localization+of+common+disease-associated+variation+in+regulatory+DNA.">Systematic localization of common disease-associated variation in regulatory DNA.</a> Science. 337 (6099), 1190-1195 (2012).</li><li>Ward, L. D., Kellis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+noncoding+genetic+variation+in+complex+traits+and+human+disease.">Interpreting noncoding genetic variation in complex traits and human disease.</a> Nat Biotechnol. 30 (11), 1095-1106 (2012).</li><li>Paul, D. S., Soranzo, N., Beck, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interpretation+of+non-coding+sequence+variation:+concepts+and+challenges.">Functional interpretation of non-coding sequence variation: concepts and challenges.</a> Bioessays. 36 (2), 191-199 (2014).</li><li>Zhang, F., Lupski, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-coding+genetic+variants+in+human+disease.">Non-coding genetic variants in human disease.</a> Hum Mol Genet. , (2015).</li><li>Lee, T. I., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+and+its+misregulation+in+disease.">Transcriptional regulation and its misregulation in disease.</a> Cell. 152 (6), 1237-1251 (2013).</li><li>Slatkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+disequilibrium--understanding+the+evolutionary+past+and+mapping+the+medical+future.">Linkage disequilibrium--understanding the evolutionary past and mapping the medical future.</a> Nat Rev Genet. 9 (6), 477-485 (2008).</li><li>Bush, W. S., Moore, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11:+Genome-wide+association+studies.">Chapter 11: Genome-wide association studies.</a> PLoS Comput Biol. 8 (12), e1002822 (2012).</li><li>1000 Genomes Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+map+of+genetic+variation+from+1,092+human+genomes.">An integrated map of genetic variation from 1,092 human genomes.</a> Nature. 491 (7422), 56-65 (2012).</li><li>Chang, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+PLINK:+rising+to+the+challenge+of+larger+and+richer+datasets.">Second-generation PLINK: rising to the challenge of larger and richer datasets.</a> Gigascience. 4, 7 (2015).</li><li>Purcell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLINK:+a+tool+set+for+whole-genome+association+and+population-based+linkage+analyses.">PLINK: a tool set for whole-genome association and population-based linkage analyses.</a> Am J Hum Genet. 81 (3), 559-575 (2007).</li><li>ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Crawford, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+DNase+hypersensitive+sites+using+massively+parallel+signature+sequencing+(MPSS).">Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS).</a> Genome Res. 16 (1), 123-131 (2006).</li><li>Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+native+chromatin+for+fast+and+sensitive+epigenomic+profiling+of+open+chromatin,+DNA-binding+proteins+and+nucleosome+position.">Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.</a> Nat Methods. 10 (12), 1213-1218 (2013).</li><li>Giresi, P. G., Kim, J., McDaniell, R. M., Iyer, V. R., Lieb, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAIRE+Formaldehyde-Assisted+Isolation+of+Regulatory+Elements)+isolates+active+regulatory+elements+from+human+chromatin.">FAIRE Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin.</a> Genome Res. 17 (6), 877-885 (2007).</li><li>Kent, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+genome+browser+at+UCSC.">The human genome browser at UCSC.</a> Genome Res. 12 (6), 996-1006 (2002).</li><li>Roadmap Epigenomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Martens, J. H., Stunnenberg, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLUEPRINT:+mapping+human+blood+cell+epigenomes.">BLUEPRINT: mapping human blood cell epigenomes.</a> Haematologica. 98 (10), 1487-1489 (2013).</li><li>Liu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cistrome:+an+integrative+platform+for+transcriptional+regulation+studies.">Cistrome: an integrative platform for transcriptional regulation studies.</a> Genome Biol. 12 (8), R83 (2011).</li><li>Griffon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+public+ChIP-seq+experiments+reveals+a+complex+multi-cell+regulatory+landscape.">Integrative analysis of public ChIP-seq experiments reveals a complex multi-cell regulatory landscape.</a> Nucleic Acids Res. 43 (4), e27 (2015).</li><li>Staudt, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lymphoid-specific+protein+binding+to+the+octamer+motif+of+immunoglobulin+genes.">A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes.</a> Nature. 323 (6089), 640-643 (1986).</li><li>Singh, H., Sen, R., Baltimore, D., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nuclear+factor+that+binds+to+a+conserved+sequence+motif+in+transcriptional+control+elements+of+immunoglobulin+genes.">A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes.</a> Nature. 319 (6049), 154-158 (1986).</li><li>Weirauch, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+and+inference+of+eukaryotic+transcription+factor+sequence+specificity.">Determination and inference of eukaryotic transcription factor sequence specificity.</a> Cell. 158 (6), 1431-1443 (2014).</li><li>Ward, L. D., Kellis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HaploReg:+a+resource+for+exploring+chromatin+states,+conservation,+and+regulatory+motif+alterations+within+sets+of+genetically+linked+variants.">HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants.</a> Nucleic Acids Res. 40 (Database issue), D930-D934 (2012).</li><li>Boyle, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annotation+of+functional+variation+in+personal+genomes+using+RegulomeDB.">Annotation of functional variation in personal genomes using RegulomeDB.</a> Genome Res. 22 (9), 1790-1797 (2012).</li><li>Hume, M. A., Barrera, L. A., Gisselbrecht, S. S., Bulyk, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniPROBE,+update+2015:+new+tools+and+content+for+the+online+database+of+protein-binding+microarray+data+on+protein-DNA+interactions.">UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein-DNA interactions.</a> Nucleic Acids Res. 43 (Database issue), D117-D122 (2015).</li><li>Mathelier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JASPAR+2014:+an+extensively+expanded+and+updated+open-access+database+of+transcription+factor+binding+profiles.">JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles.</a> Nucleic Acids Res. 42 (Database issue), 142-147 (2014).</li><li>Smith, M. F., Delbary-Gossart, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+Mobility+Shift+Assay+(EMSA).">Electrophoretic Mobility Shift Assay (EMSA).</a> Methods Mol Med. 50, 249-257 (2001).</li><li>Franza, B. R., Josephs, S. F., Gilman, M. Z., Ryan, W., Clarkson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cellular+proteins+recognizing+the+HIV+enhancer+using+a+microscale+DNA-affinity+precipitation+assay.">Characterization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity precipitation assay.</a> Nature. 330 (6146), 391-395 (1987).</li><li>. BCA Protein Assay Kit: User Guide Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf">https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf</a> (2014)</li><li>Wijeratne, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphopeptide+separation+using+radially+aligned+titania+nanotubes+on+titanium+wire.">Phosphopeptide separation using radially aligned titania nanotubes on titanium wire.</a> ACS Appl Mater Interfaces. 7 (21), 11155-11164 (2015).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fastest+Western+in+Town:+A+Contemporary+Twist+on+the+Classic+Western+Blot+Analysis.">The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis.</a> J. Vis. Exp. (84), (2014).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lupus+Risk+Variant+Increases+pSTAT1+Binding+and+Decreases+ETS1+Expression.">Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression.</a> Am J Hum Genet. 96 (5), 731-739 (2015).</li><li>Ramana, C. V., Chatterjee-Kishore, M., Nguyen, H., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+roles+of+Stat1+in+regulating+gene+expression.">Complex roles of Stat1 in regulating gene expression.</a> Oncogene. 19 (21), 2619-2627 (2000).</li><li>Fillebeen, C., Wilkinson, N., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+Mobility+Shift+Assay+(EMSA)+for+the+Study+of+RNA-Protein+Interactions:+The+IRE/IRP+Example.">Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example.</a> J. Vis. Exp. (94), e52230 (2014).</li><li>Heng, T. S., Painter, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+Genome+Project,+C.+The+Immunological+Genome+Project:+networks+of+gene+expression+in+immune+cells.">Immunological Genome Project, C. The Immunological Genome Project: networks of gene expression in immune cells.</a> Nat Immunol. 9 (10), 1091-1094 (2008).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioGPS:+an+extensible+and+customizable+portal+for+querying+and+organizing+gene+annotation+resources.">BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.</a> Genome Biol. 10 (11), R130 (2009).</li><li>Wu, C., Macleod, I., Su, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioGPS+and+MyGene.info:+organizing+online,+gene-centric+information.">BioGPS and MyGene.info: organizing online, gene-centric information.</a> Nucleic Acids Res. 41 (Database issue), D561-D565 (2013).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+features+and+chromatin+structure+around+the+genomic+regions+bound+by+119+human+transcription+factors.">Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.</a> Genome Res. 22 (9), 1798-1812 (2012).</li><li>Holden, N. S., Tacon, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+problems+of+the+electrophoretic+mobility+shift+assay.">Principles and problems of the electrophoretic mobility shift assay.</a> J Pharmacol Toxicol Methods. 63 (1), 7-14 (2011).</li><li>Xu, J., Liu, H., Park, J. S., Lan, Y., Jiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osr1+acts+downstream+of+and+interacts+synergistically+with+Six2+to+maintain+nephron+progenitor+cells+during+kidney+organogenesis.">Osr1 acts downstream of and interacts synergistically with Six2 to maintain nephron progenitor cells during kidney organogenesis.</a> Development. 141 (7), 1442-1452 (2014).</li><li>Yang, T. -. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genevar:+a+database+and+Java+application+for+the+analysis+and+visualization+of+SNP-gene+associations+in+eQTL+studies.">Genevar: a database and Java application for the analysis and visualization of SNP-gene associations in eQTL studies.</a> Bioinformatics. 26 (19), 2474-2476 (2010).</li><li>Fort, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+liver+enhancer+in+the+fibrinogen+gene+cluster.">A liver enhancer in the fibrinogen gene cluster.</a> Blood. 117 (1), 276-282 (2011).</li><li>Solberg, N., Krauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luciferase+assay+to+study+the+activity+of+a+cloned+promoter+DNA+fragment.">Luciferase assay to study the activity of a cloned promoter DNA fragment.</a> Methods Mol Biol. 977, 65-78 (2013).</li><li>Rahman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+repressor+element+in+the+5'-untranslated+region+of+human+Pax5+exon+1A.">A repressor element in the 5'-untranslated region of human Pax5 exon 1A.</a> Gene. 263 (1-2), 59-66 (2001).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Guided+Human+Genome+Engineering+via+Cas9.">RNA-Guided Human Genome Engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li></ol>

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...

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 molecular mechanisms that link variants to an important intermediate phenotype &#160;&#8211; gene expression. This pipeline is specifically designed to identify modulation of TF binding by genetic variants. This strategy combines computational approaches and molecular biology techniques aimed to predict biological effects of candidate variants in silico, and verify these predictions empirically (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/54093/54093fig1.jpg" /> 
Figure 1: Strategic approach for the analysis of non-coding genetic variants. Steps that are not included in the detailed protocol associated with this manuscript are shaded in grey. <a href="https://www.jove.com/files/ftp_upload/54093/54093fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In many cases, it is important to begin by expanding the list of variants to include all those in high linkage-disequilibrium (LD) with each statistically associated variant. LD is a measure of non-random association of alleles at two different chromosomal positions, which can be measured by the r2 statistic7. r2 is a measure of the linkage disequilibrium between two variants, with an r2 = 1 denoting perfect linkage between two variants. Alleles in high LD are found to co-segregate on the chromosome across ancestral populations. Current genotyping arrays do not include all known variants in the human genome. Instead, they exploit the LD within the human genome and include a subset of the known variants that act as proxies for other variants within a particular region of LD8. Thus, a variant without any biological consequence may be associated with a particular disease because it is in LD with the causal variant-the variant with a meaningful biological effect. Procedurally, it is recommended to convert the latest release of the 1,000 genomes project9 variant call files (vcf) into binary files compatible with PLINK10,11, an open-source tool for whole genome association analysis. Subsequently, all other genetic variants with LD r2 &#62;0.8 with each input genetic variant can be identified as candidates. It is important to use the appropriate reference population for this step- e.g., if a variant was identified in subjects of European ancestry, data from subjects of similar ancestry should be used for LD expansion.
LD expansion often results in dozens of candidate variants, and it is likely that only a small fraction of these contribute to disease mechanism. Often, it is infeasible to experimentally examine each of these variants individually. It is therefore useful to leverage the thousands of publically available functional genomic datasets as a filter to prioritize the variants. For example, the ENCODE consortium12 has performed thousands of ChIP-seq experiments describing the binding of TFs and co-factors, and histone marks in a wide range of contexts, along with chromatin accessibility data from technologies such as DNase-seq13, ATAC-seq14, and FAIRE-seq15. Databases and web servers such as the UCSC Genome Browser16, Roadmap Epigenomics17, Blueprint Epigenome18, Cistrome19, and ReMap20 provide free access to data produced by these and other experimental techniques across a wide range of cell types and conditions. When there are too many variants to examine experimentally, these data can be used to prioritize those located within likely regulatory regions in relevant cell and tissue types. Further, in cases where a variant is within a ChIP-seq peak for a specific protein, these data can provide potential leads as to the specific TF(s) or co-factors whose binding might be affecting.
Next, the resulting prioritized variants are screened experimentally to validate predicted genotype-dependent protein binding using EMSA21,22. EMSA measures the change in the migration of the oligo on a non-reducing TBE gel. Fluorescently labeled oligo is incubated with the nuclear lysate, and binding of nuclear factors will retard the movement of the oligo on the gel. In this manner, oligo that has bound more nuclear factors will present as a stronger fluorescent signal upon scanning. Notably, EMSA does not require predictions about the specific proteins whose binding will be affected.
Once variants are identified that are located within predicted regulatory regions and are capable of differentially binding nuclear factors, computational methods are employed to predict the specific TF(s) whose binding they might affect. We prefer to use CIS-BP23,24, RegulomeDB25, UniProbe26, and JASPAR27. Once candidate TFs are identified, these predictions can be specifically tested using antibodies against these TFs (EMSA-supershifts and DAPA-Westerns). An EMSA-supershift involves the addition of a TF-specific antibody to the nuclear lysate and oligo. A positive result in an EMSA-supershift is represented as a further shift in the EMSA band, or a loss of the band (reviewed in reference28). In the complementary DAPA, a 5&#39;-biotinylated oligo duplex containing the variant and the 20 base-pair flanking nucleotides are incubated with nuclear lysate from relevant cell type(s) to capture any nuclear factors specifically binding the oligos. The oligo duplex-nuclear factor complex is immobilized by streptavidin microbeads in a magnetic column. The bound nuclear factors are collected directly through elution29,48. Binding predictions can then be assessed by a Western blot using antibodies specific for the protein. In cases where there are no obvious predictions, or too many predictions, the elutions from variant pull-downs of the DAPA experiments can be sent to a proteomics core to identify candidate TFs using mass-spectrometry, which can subsequently be validated using these previously described methods.
In the remainder of the article, the detailed protocol for EMSA and DAPA analysis of genetic variants is provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Custom DNA Oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>http://www.idtdna.com/site/order/oligoentry</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>KCl, for CE buffer</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Fisher Scientific</td>
			<td>15630-080</td>
			<td>For CE and NE buffer</td>
		</tr>
		<tr>
			<td>EDTA (0.5M), pH 8.0</td>
			<td>Life Technologies</td>
			<td>R1021</td>
			<td>For CE, NE, and annealing buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td>NaCl, for NE buffer</td>
		</tr>
		<tr>
			<td>Tris-HCl (1M), pH 8.0</td>
			<td>Invitrogen</td>
			<td>BP1756-100</td>
			<td>For annealing buffer</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>Fisher Scientific</td>
			<td>MT21040CM</td>
			<td>PBS, for cell wash</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol solution (1M)</td>
			<td>Sigma</td>
			<td>646563</td>
			<td>Reducing agent</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td>Protease Inhibitor</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail&#160;</td>
			<td>Thermo Scientific</td>
			<td>78420</td>
			<td>Prevents dephosphorylation of TFs</td>
		</tr>
		<tr>
			<td>Nonidet P-40 Substitute</td>
			<td>IBI Scientific</td>
			<td>IB01140</td>
			<td>NP-40, for nuclear extraction</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>For measuring protein concentration</td>
		</tr>
		<tr>
			<td>Odyssey EMSA Buffer Kit</td>
			<td>Licor</td>
			<td>829-07910</td>
			<td>Contains all necessary EMSA buffers</td>
		</tr>
		<tr>
			<td>TBE Gels, 6%, 12 Wells</td>
			<td>Invitrogen</td>
			<td>EC6265BOX</td>
			<td>For EMSA</td>
		</tr>
		<tr>
			<td>TBE Buffer (10X)</td>
			<td>Thermo Scientific</td>
			<td>B52</td>
			<td>For EMSA</td>
		</tr>
		<tr>
			<td>FactorFinder Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-318</td>
			<td>Contains all necessary DAPA buffers</td>
		</tr>
		<tr>
			<td>Licor Odyssey CLx</td>
			<td>Licor</td>
			<td></td>
			<td>Recommended scanner for DAPA/EMSA</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>Contains 10,000 units/mL of penicillin, 10,000 &#181;g/mL of streptomycin, and 25 &#181;g/mL of Fungizone&#174; Antimycotic</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>FBS, for culture media</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>22400-071</td>
			<td>Contains L-glutamine and 25mM HEPES</td>
		</tr>
	</tbody>
</table>

screening for functional non-coding genetic variants using electrophoretic mobility shift assay (emsa) and dna-affinity precipitation assay (dapa)

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.

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 &#34;freeze-thaw&#34; cycles, two 35 bp oligos differing at one genetic variant were incubated with a single bat...

Watch this Scientific Journal Video about Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA at JoVE.com

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

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.

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

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...

The overall goal of this experimental strategy is to investigate the functionality of disease associated non-coding variants. This method can help answer key questions in genomics, such as how genetic variants that don't actually change amino acid sequence, still contribute to disease phenotype. The main advantage of this technique is that is provides a streamlined process for assessing genetic variance for function activity and gives insight into regulatory proteins and pathways effected.This technique can benefit the investigation of any disease with a candidate causal variant. Because the procedure for analyzing these variants is universal regardless of the phenotype being studied. Though this method can provide insight into human disease, it can also be applied to mutations in any organism.Nuclear lysates will be prepared from B-lymphoblastoid cells in this experiment, however this procedure can be applied to most cell lines. After counting cells using a hemocytometer, spin down the desired number of cells for lysing. Aspirate the media, add 10 milliliters of ice-cold PBS, and spin down the cells again.Wash the cells a second time with PBS, and remove the PBS after the spin. Resuspend the cell palate in ice-cold PBS using one milliliter of PBS per 10 to the seventh cells. Aliquot one milliliter of the cell suspension to 1.5 milliliter microcentrifuge tubes, so that each tube contains 10 to the seventh cells in PBS.Centrifuge for two minutes and aspirate off the PBS. Add to a working stock of CE buffer, one millimolar dithiothreitol, or DTT, 1X phosphatase inhibitor, and 0.5 millimolar Phenylmethanesulfonyl fluoride, or PMSF. Resuspend each cell palette with 400 microliters of this buffer, and incubate on ice for 15 minutes.Add 25 microliters of 10%Nonidet P-40 to each microcentrifuge tube, and mix by pipetting. Centrifuge at four degrees Celsius and maximum speed for three minutes. Decant and discard the supernatant.Next, add to a working stock of NE-buffer, one millimolar DTT, 1X phosphoaase inhibitor, and one millimolar PMSF. Resuspend each cell palate with 30 microliters of this buffer, and mix by vortexing. Incubate at 4 degrees Celsius, in a tube rotator, for 10 minutes.Centrifuge for two minutes. Collect the clear supernatant, which is the nuclear lysate. Aliquot the nuclear lysate before storing at minus 80 degrees Celsius to avoid multiple freeze thaw cycles that may degrade the protein.Begin this procedure by preparing a 50 nanomolar working stock of the oligonucleotide, as described in the protocol text. Place the oligos in a heat block at 90 degrees Celsius for five minutes. After five minutes, turn off the heat block and allow the oligos to slowly cool down to room temperature for at least one hour prior to use.Next prepare the electrophoretic mobility shift assay or EMSA gel. Remove the slide from a pre-cast, six percent tris-borate-EDTA, or TBE gel, and rinse under deionized water several times to remove any buffer from the wells. Assemble the gel electrophoresis apparatus, and check for leaks by filling the inner chamber with 0.5X TBE buffer.If no buffer leaks into the outer chamber, fill the outer chamber, roughly, two thirds of the way. Pre-run the gel at 100 volts for 60 minutes. When the pre-run is complete, flush each well with 200 microliters of 0.5X TBE buffer.Prepare a binding buffer master mix. In a microcentrifuge tube, mix these reagents that are common to all reactions. 10X binding buffer, DTT polysorbate, Poly(dI-dC)and salmon sperm DNA.To set up the EMSA reactions, add nuclease-free water to each microcentrifuge tube, such that the final volume, following addition of all reagents, will be 20 microliters. Add the appropriate amount of master mix to each microcentrifuge tube. Add eight micrograms of nuclear lysate to the appropriate microcentrifuge tubes.Include tubes containing the oligo, without nuclear extract, as negative controls. Add 50 femtomoles of oligo to the appropriate microcentrifuge tubes, and flick to mix. Briefly spin the contents to the bottom of the tube.Incubate all tubes, for 20 minutes, at room temperature. Next, add two microliters of 10X orange loading dye to each microcentrifuge tube. Pipette up and down to mix.Load the samples into the pre-run, six percent, TBE gel by pipetting up and down to mix, and then expelling each sample into a separate well. Run the gel at 80 volts for approximately 60 to 75 minutes, until the orange dye has migrated two thirds to three quarters of the way down the gel. When the run is complete, use a gel knife to pry the plastic cassette open.Remove the gel from the cassette and place it in a container with 0.5 percent TBE buffer to keep it from drying out. Place the gel on the surface of an infrared and chemiluminescence imaging system. Eliminate any bubbles or contaminants that will disrupt the image.Scan the gel using the scanning system software as described in the protocol text. To begin this procedure, prepare a five micromolar oligo working stock as described in the protocol text. Place the oligos in a heat block at 95 degrees Celsius for five minutes.After five minutes, turn off the heat block and allow the oligos to slowly cool down to room temperature prior to use. Warm the following buffers to room temperature:binding buffer, low stringency wash buffer, high stringency wash buffer, and elution buffer. Prepare the binding mixtures for each variant.Mix one volume of nuclear lysate with two volumes of binding buffer. Add 1X phosphatase inhibitor, 0.5 millimolar PMSF, and 1X binding enhancer. Add 10 microliters of five micromolar biotinylated capture DNA to each binding mixture.Mix by flicking the tube several times. Incubate the binding mixtures for 20 minutes at room temperature. Next, add 100 microliters of streptavidin MicroBeads to each binding mixture.Incubate for 10 minutes at room temperature. For each oligo probe being tested, place a binding column in the magnetic separator. Make sure the columns are labelled with the variant oligo that was used in the binding mixture.Place a microcentrifuge tube directly under each binding column, and apply 100 microliters of binding buffer to rinse the column. Pipette the contents of each binding mixture into separate columns. Allow the liquid to flow completely through the column into the microcentrifuge tube.Apply 100 microliters of low stringency wash buffer to the column and wait until the column reservoir is empty. Apply 100 microliters of low stringency wash buffer to the column again. Apply 100 microliters of high stringency wash buffer to the column, and wait until the column reservoir is empty.Apply 100 microliters of high stringency wash buffer to the column again. Add 30 microliters of native elution buffer to the column and let stand for five minutes. Finally add an additional 50 microliters of native elution buffer to elute the bound transcription factors.In order to explore the reproducibility of EMSA results, and the consequences of freeze thaw cycles, oligos containing the reference, or non-reference allele of a genetic variant were used to probe the same preparation of B-lymphocyte nuclear extract after multiple cycles of freezing and thawing. The blue arrow indicates the free probe. Binding of the transcription factors to the oligo is seen as a band in the top half of the gel, indicated by the red arrow.These results indicate that freezing and thawing this particular nuclear extract up to five times has, seemingly, no effect on the integrity of the proteins. It is also important to optimize the signal to noise ratio for the EMSA. Various concentrations of oligos were used to probe a single preparation of nuclear lysate.An increase in the intensity of the band, up to 100 femtomoles of oligo was observed. Representative results from a study of a lupus associated risk allele are shown here. The transcription factor, STAT 1, was first identified by DAPA followed by proteomic analysis.A DAPA western blot then confirmed the identity of STAT 1, and the higher binding of the phosphorylated form of STAT 1 to the lupus risk oligo. Following this procedure, other techniques, such as mass spectrometry and western blot can be performed. This will help us to identify the regulatory protein showing allele dependent binding.Reporter assays like luciferase can also be used to investigate changes in gene expression as a function of allele.

Research

JoVE Journal

Biology

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...