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
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	<li>Order custom DNA oligonucleotide probes for use in EMSA and DAPA.
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		<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...

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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...

Секвенирования и генотипирования на основе исследований, включая геномные исследования ассоциации (GWAS), кандидат исследований локуса, и глубокие секвенирования исследования, выявили множество генетических вариантов, которые статистически связаны с заболеванием, признаком, или фенотипа. Вопреки ранних предсказаний, большинство из этих вариантов (85-93%) расположены в некодирующих областей и не изменяют аминокислотную последовательность белков 1,2. Интерпретируя функции этих некодирующих вариантов и определения биологических механизмов , связывающих их с сопутствующей болезни, признак или фенотип оказался непростым 3-6. Мы разработали общую стратегию для выявления молекулярных механизмов, связывающих варианты к важному промежуточного фенотипа - экспрессии генов. Этот трубопровод разработан специально для идентификации модуляции TF связывания генетических вариантов. Эта стратегия сочетает в себе вычислительные подходы и методы молекулярной биологии, направленных предсказатьбиологические эффекты вариантов кандидатов в силикомарганца, и проверить эти предсказания эмпирически (рисунок 1). <img alt="Рисунок 1" src="/files/ftp_upload/54093/54093fig1.jpg" /> Рисунок 1:.. Стратегический подход для анализа некодирующих генетические варианты шагов, которые не включены в подробный протокол , связанный с этой рукописи заштрихованы серым цветом <a href="https://www.jove.com/files/ftp_upload/54093/54093fig1large.jpg" target="_blank">Пожалуйста</a> , <a href="https://www.jove.com/files/ftp_upload/54093/54093fig1large.jpg" target="_blank">нажмите здесь</a> , <a href="https://www.jove.com/files/ftp_upload/54093/54093fig1large.jpg" target="_blank">чтобы посмотреть увеличенную версию этой фигуры.</a> Во многих случаях, важно, чтобы начать путем расширения списка вариантов, чтобы включить все те, в высокой рычажной-неравновесия (LD) с каждым статистически связанным вариантом. ЛД является мерой неслучайной ассоциации аллелей двух различных хромосомных положениях, которые могут быть измерены с помощью R 2 статистики 7. R 2 представляет собой меру линКейдж неравновесие между двумя вариантами, с R 2 = 1 , обозначающее совершенной связи между двумя вариантами. Аллели в высокой LD оказываются совместно разделять на хромосоме через родовые популяции. Современные генотипирования массивы не включают в себя все известные варианты в геноме человека. Вместо этого они используют ЛД внутри генома человека и включают в себя подмножество известных вариантов , которые действуют в качестве прокси для других вариантов в пределах определенной области LD 8. Таким образом, вариант без какого-либо биологического последствие может быть связан с конкретным заболеванием, потому что это в LD с причинным вариантом: вариант с значимого биологического эффекта. С процедурной точки зрения рекомендуется преобразовать последний выпуск 1000 геномов проекта 9 файлов вариант вызова (VCF) в двоичные файлы , совместимые с Plink 10,11, инструмент с открытым исходным кодом для анализа всего генома ассоциации. Впоследствии все другие генетические варианты с LD г 2&gt; 0,8 с каждым входом генетической ваRiant могут быть идентифицированы в качестве кандидатов. Важно использовать соответствующий ссылочный населения для этого , например , Step - , если вариант был выявлен у пациентов европейского происхождения, данные от субъектов аналогичного происхождения должны быть использованы для расширения LD. Расширение LD часто приводит десятки вариантов кандидатов, и вполне вероятно, что лишь небольшая часть из них внести свой вклад в механизм заболевания. Часто, это неосуществимо экспериментально исследовать каждый из этих вариантов по отдельности. Поэтому полезно использовать тысячи доступных функциональных публично геномных наборов данных в качестве фильтра для определения приоритетности вариантов. Например, КОДИРОВАНИЯ консорциум 12 выполнил тысячи чиповых-сл экспериментов , описывающих связывание ТФ и сопутствующих факторов и гистонов марок в широком диапазоне контекстов, наряду с данными хроматина доступности от технологий , таких как ДНКазы сл 13, ATAC -seq 14 и FAIRE-15 сл. Databтузы и веб - серверы , такие как браузера УСК генома 16, Дорожная карта Epigenomics 17 Blueprint эпигеном 18, Cistrome 19 и ReMap 20 обеспечивают свободный доступ к данным , полученных с помощью этих и других экспериментальных методов в широком диапазоне типов и условий клеток. Когда есть слишком много вариантов, чтобы проверить экспериментально, эти данные могут быть использованы для приоритеты тех, которые находятся в пределах возможных регуляторных областей в соответствующих тканей и клеток типов. Кроме того, в тех случаях, когда вариант в микросхеме-сл пика для специфического белка, эти данные могут обеспечить потенциальных клиентов, как к специфическому TF (ами) или кофакторов, связывание может затрагивающий. Далее, полученные по приоритетам варианты экспериментально скринингу для проверки связывания с использованием EMSA 21,22 предсказанную генотип-зависимого белка. EMSA измеряет изменение миграции олиго на невосстановительной геле КЭ. Флуоресцентно меченных олиго инкубируют сядерный лизат, и связывание ядерных факторов замедлит движение олиго на геле. Таким образом, олиго-, который связан больше ядерных факторов представит в качестве более сильного флуоресцентного сигнала при сканировании. Следует отметить, что EMSA не требует предсказания о специфических белков, связывание будут затронуты. После того, как варианты идентифицируются, которые расположены в пределах прогнозируемых регуляторных областей и способны дифференцированно связывания ядерных факторов, вычислительные методы используются для прогнозирования конкретный TF (ы), связывание которого они могут повлиять. Мы предпочитаем использовать CIS-BP 23,24, RegulomeDB 25, UNIProbe 26 и 27 Джаспер. После того, как кандидат ТФ определены, эти прогнозы могут быть специально протестированы с использованием антител против этих ТФ (EMSA-supershifts и Dapa-вестерны). EMSA-supershift включает добавление антитела к TF специфичные к ядерной лизата и олиго. Положительный результат в EMSA-supershift является магнезииesented как дальнейший сдвиг в полосе EMSA или потери полосы (обзор в ссылке 28). В дополнительном Dapa, 5&#39;-биотинилированные олиго дуплекс, содержащий вариант и 20 пар оснований нуклеотидов фланговый инкубируют с ядерными лизата из соответствующего типа (ов) клеток, чтобы захватить любые ядерные факторы, специфически связывающих олигонуклеотидов. Фактор-комплекс олиго дуплексной ядерная скованной стрептавидином микрогранулы в магнитном колонке. Связанные ядерные факторы собираются непосредственно через элюирования 29,48. Связующие предсказания затем могут быть оценены с помощью Вестерн-блоттинга с использованием антител, специфичных к белку. В тех случаях, когда нет никаких очевидных предсказаний, или слишком много прогнозов, то элюции из вариантов отжимания экспериментов Dapa могут быть отправлены в активную зону протеомики для выявления кандидатов ТФ с использованием масс-спектрометрии, которые впоследствии могут быть проверены с помощью этих ранее описанных методы. В оставшейся части статьи обе, подробный протокол для EMSA и Dapa анализа генетических вариантов предусмотрена.

<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.

Скрининг для функционального Некодирующих генетических вариантов Использование электрофоретической подвижности Сдвиг анализа (EMSA) и ДНК-сродства осадков Анализ (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

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

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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

Электрофоретической подвижности сдвига анализа (EMSA) по изучению РНК-белковых взаимодействий: ИРЭ / IRP примера

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