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. Preparación de soluciones y reactivos <ol><li> Ordenar sondas de oligonucleótidos de ADN personalizados para su uso en la EMSA y la DAPA. <ol><li> Para reducir la proteína de unión no específica, diseñar oligos cortos (entre 35-45 pares de bases (pb) de longitud) 30, y colocar la variante de interés directamente en el centro, flanqueado por su secuencia genómica endógena 17 pb. Para oligos EMSA, añadir un &#39;fluoróforo 5. Para oligos DAPA, añadir una etiqueta 5 &#39;con biotina. </li><li> O...

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<html> A pesar de los avances en las tecnologías de secuenciación y genotipado han mejorado enormemente nuestra capacidad para identificar variantes genéticas asociadas con la enfermedad, nuestra capacidad para comprender los mecanismos funcionales afectadas por estas variantes se está quedando. Una fuente importante del problema es que muchas variantes asociados a la enfermedad se encuentran en n en la codificación de las regiones del genoma, lo que probablemente afectará más difícil de predecir mecanismos que c...

Secuenciación y genotipado basado en estudios, incluyendo estudios de genoma completo (GWAS Association), estudios candidato locus, y profundo de secuenciación de los estudios, han identificado muchas variantes genéticas que están estadísticamente asociados con una enfermedad, rasgo o fenotipo. Contrariamente a las predicciones iniciales, la mayoría de estas variantes (85-93%) se localizan en regiones no codificantes y no cambian la secuencia de aminoácidos de las proteínas de 1,2. Interpretación de la función de estas variantes no codificantes y la determinación de los mecanismos biológicos que los conectan a la enfermedad asociada, rasgo, o fenotipo ha demostrado ser un desafío 3-6. Hemos desarrollado una estrategia general para identificar los mecanismos moleculares que enlazan variantes a un fenotipo intermedio importante - la expresión génica. Esta tubería está específicamente diseñado para identificar la modulación de la unión por variantes genéticas TF. Esta estrategia combina los enfoques computacionales y técnicas de biología molecular destinadas a predecirefectos biológicos de las variantes candidatos in silico, y verificar estas predicciones empíricamente (Figura 1). <img alt="Figura 1" src="/files/ftp_upload/54093/54093fig1.jpg" /> Figura 1:.. Enfoque estratégico para el análisis de los pasos no codificantes de variantes genéticas que no están incluidos en el protocolo detallado asociado a este manuscrito están sombreados en gris <a href="https://www.jove.com/files/ftp_upload/54093/54093fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> En muchos casos, es importante comenzar mediante la ampliación de la lista de variantes para incluir a todos los de alta desequilibrio de unión (LD) con cada variante estadísticamente asociado. LD es una medida de asociación no aleatoria de los alelos en dos posiciones cromosómicas diferentes, que se puede medir por el r 2 Estadística 7. r 2 es una medida de la linKage desequilibrio entre dos variantes, con una unión perfecta r 2 = 1 denota entre dos variantes. Alelos en alto LD se encuentran a co-segregar en el cromosoma a través de poblaciones ancestrales. matrices de genotipos actuales no incluyen todas las variantes conocidas en el genoma humano. En su lugar, se aprovechan de la LD dentro del genoma humano e incluyen un subconjunto de las variantes conocidas que actúan como representantes de otras variantes dentro de una región particular del LD 8. Por lo tanto, una variante sin ninguna consecuencia biológica puede estar asociado con una enfermedad en particular, ya que es en LD con la variante de la causal variante con un efecto biológico significativo. Del procedimiento, se recomienda para convertir la última versión de los 1.000 genomas Proyecto 9 archivos variante llamada (VCF) en archivos binarios compatibles con PLINK 10,11, una herramienta de código abierto para el análisis de asociación de genoma completo. Posteriormente, todas las demás variantes genéticas con LD r 2&gt; 0,8 con cada entrada va genéticaRiant puede ser identificado como candidatos. Es importante la utilización de la población de referencia apropiada para este paso- por ejemplo, si una variante fue identificada en sujetos de ascendencia europea, los datos de los sujetos de ascendencia debe emplearse la misma para la expansión LD. expansión LD a menudo resulta en variantes de candidatos docenas, y es probable que sólo una pequeña fracción de estos contribuyen a mecanismo de la enfermedad. A menudo, no es factible para examinar experimentalmente cada una de estas variantes de forma individual. Es por lo tanto útil para aprovechar los miles de conjuntos de datos de genómica funcional públicamente disponibles como un filtro para priorizar las variantes. Por ejemplo, el consorcio ENCODE 12 ha realizado miles de experimentos chip-ss que describen la unión de TFS y co-factores, y las marcas de las histonas en una amplia gama de contextos, junto con los datos de la cromatina accesibilidad de las tecnologías tales como DNasa-ss 13, ATAC -seq 14 y ss Faire-15. DatabASES y servidores web, tales como la UCSC Genome Browser 16, Hoja de Ruta Epigenómica 17, 18 Epigenoma Blueprint, Cistrome 19 y 20 de RECONFIGURACIÓN proporcionan acceso libre a los datos producidos por estas y otras técnicas experimentales a través de una amplia gama de tipos y condiciones de las celdas. Cuando hay demasiadas variantes para examinar experimentalmente, estos datos se pueden utilizar para dar prioridad a los situados dentro de las regiones reguladoras probables en los tipos de células y tejidos pertinentes. Además, en los casos en que es una variante dentro de un pico de chip-ss para una proteína específica, estos datos pueden proporcionar pistas potenciales en cuanto a la TF (s) específico o co-factores cuya unión podría estar afectando. A continuación, las variantes resultantes prioridad son examinados experimentalmente para validar la unión usando EMSA 21,22 proteína genotipo dependiente predicho. EMSA mide el cambio en la migración de la oligo en un gel de TBE no reductor. oligo marcado con fluorescencia se incuba con ellisado nuclear, y la unión de factores nucleares retardará el movimiento del oligo en el gel. De esta manera, oligo que se ha unido factores más nucleares presentará como una señal fluorescente más fuerte sobre la exploración. En particular, la EMSA no requiere predicciones acerca de las proteínas específicas cuya unión se verán afectados. Una vez que se identifican las variantes que se encuentran dentro de las regiones reguladoras predichos y son capaces de factores nucleares diferencialmente de unión, se emplean métodos computacionales para predecir el TF (s) específico cuya unión podrían afectar. Nosotros preferimos utilizar CIS-BP 23,24, RegulomeDB 25, Uniprobe 26 y 27 JASPAR. Una vez que se identifican candidato TFS, estas predicciones pueden ser probados específicamente el uso de anticuerpos contra estos TFS (EMSA-supershifts y DAPA-Oeste). Un EMSA-supershift implica la adición de un anticuerpo específico-TF al lisado nuclear y oligo. Un resultado positivo en un EMSA-supershift es represented como un cambio adicional en la banda de EMSA, o una pérdida de la banda (revisado en referencia 28). En la DAPA complementaria, un dúplex de oligo 5 &#39;biotinilado que contiene la variante y la 20 pares de bases que flanquean nucleótidos se incuban con lisado nuclear de tipo de célula relevante (s) para capturar cualquier factores nucleares se unen específicamente a los oligos. El complejo del factor nuclear-duplex oligo es inmovilizado por estreptavidina microperlas en una columna magnética. Los factores nucleares unidas se recogieron directamente a través de la elución 29,48. predicciones de unión pueden ser evaluados por una transferencia de Western usando anticuerpos específicos para la proteína. En los casos en que no hay predicciones obvias, o demasiadas predicciones, las eluciones de variantes desplegables de los experimentos DAPA se pueden enviar a un núcleo de la proteómica para identificar TFS candidatos utilizando la espectrometría de masa, que posteriormente se pueden validar el uso de estos descritos anteriormente métodos. En el resto de la article, se proporciona el protocolo detallado para el análisis EMSA y DAPA de variantes genéticas.

<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.

En esta sección, los resultados representativos de lo que cabe esperar se proporcionan al realizar una EMSA o DAPA, y la variabilidad en cuanto a se caracteriza la calidad de lisado. Por ejemplo, se ha sugerido que la congelación y descongelación muestras de proteínas varias veces puede dar lugar a la desnaturalización. Con el fin de explorar la reproducibilidad del análisis EMSA en el contexto de estos ciclos de &quot;congelación-descongelación&quot;, dos 35 oligos pb que difier...

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.

La detección de funcional no codificante-variantes genéticas Uso de movilidad electroforética Shift ensayo (EMSA) y el ADN afinidad Precipitación Ensayo (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.

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