方法文章

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

DOI：

10.3791/54093

⸱

2016年8月21日

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

* These authors contributed equally

本文内容

摘要

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我们提出了一个战略计划和方案，用于识别影响转录因子 （TF） DNA 结合的非编码遗传变异。为基因型依赖性 TF DNA 结合的电泳迁移率变化测定 （EMSA） 和 DNA 亲和沉淀测定 （DAPA） 分析提供了详细的实验方案。

摘要

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基于群体和家系的遗传研究通常会导致鉴定出与临床疾病或表型在统计学上相关的遗传变异。对于许多疾病和性状，大多数变异是非编码的，因此可能通过影响微妙的、相对难以预测的控制基因表达的机制来发挥作用。在这里，我们描述了一种一般的战略方法，用于确定非编码变体的优先级，并筛选它们的功能。这种方法涉及使用功能基因组数据库进行计算优先级排序，然后对转录因子 （TFs） 与风险和非风险等位基因的差异结合进行实验分析。对于遗传变异的电泳迁移率变化测定 （EMSA） 和 DNA 亲和沉淀测定 （DAPA） 分析，使用合成 DNA 寡核苷酸 （oligo） 来鉴定疾病或表型相关细胞的核裂解物中的因子。对于 EMSA，通过在三硼酸盐-EDTA （TBE） 聚丙烯酰胺凝胶上进行非变性电泳分析具有或不具有结合核因子 （通常是 TFs） 的寡核苷酸。对于 DAPA，寡核苷酸与磁柱结合，特异性结合 DNA 序列的核因子通过质谱法或还原十二烷基硫酸钠聚丙烯酰胺凝胶电泳 （SDS-PAGE） 洗脱和分析，然后进行蛋白质印迹分析。这种通用方法可广泛用于研究与任何疾病、性状或表型相关的非编码遗传变异的功能。

引言

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Sequencing and genotyping based studies, including Genome-Wide Association Studies (GWAS), candidate locus studies, and deep-sequencing studies, have identified many genetic variants that are statistically associated with a disease, trait, or phenotype. Contrary to early predictions, most of these variants (85-93%) are located in non-coding regions and do not change the amino acid sequence of proteins1,2. Interpreting the function of these non-coding variants and determining the biological mechanisms connecting them to the associated disease, trait, or phenotype has proven challenging3-6. We have developed a general strategy to identify the molec....

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方案

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1. Preparation of Solutions and Reagents

  1. Order custom DNA oligonucleotide probes for use in EMSA and DAPA.
    1. To reduce non-specific protein binding, design short oligos (between 35-45 base pairs (bp) in length)30, and place the variant of interest directly in the center flanked by its 17 bp endogenous genomic sequence. For EMSA oligos, add a 5' fluorophore. For DAPA oligos, add a 5' biotin tag.
    2. Order both the sense strand and its reverse complement strand. Alternatively, order duplex (pre-annealed) oligos. When naming the oligos, base the nomenclature on an established reference genome.
      Note: "Risk" a....

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结果

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In this section, representative results of what to expect are provided when performing an EMSA or DAPA, and the variability with regards to the quality of lysate is characterized. For example, it has been suggested that freezing and thawing protein samples multiple times may result in denaturation. In order to explore the reproducibility of EMSA analysis in the context of these "freeze-thaw" cycles, two 35 bp oligos differing at one genetic variant were incubated with a single batch of nu.......

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讨论

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Although advances in sequencing and genotyping technologies have greatly enhanced our capacity to identify genetic variants associated with disease, our ability to understand the functional mechanisms impacted by these variants is lagging. A major source of the problem is that many disease-associated variants are located in n on-coding regions of the genome, which likely affect harder-to-predict mechanisms controlling gene expression. Here, we present a protocol based on the EMSA and DAPA techniques, valuable molecular t.......

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披露

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The authors have nothing to disclose.

致谢

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我们感谢 Erin Zoller、Jessica Bene 和 Lindsey Hays 在协议开发中提供的意见和指导。MTW 部分得到了 NIH R21 HG008186和辛辛那提儿童医院研究基金会的受托人奖资助。ZHP 部分由 T32 GM063483-13 提供支持。

....

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材料

本文使用的材料清单
姓名公司目录编号评论
定制 DNA 寡核苷酸集成 DNA 技术http://www.idtdna.com/site/order/oligoentry
钾Fisher ScientificBP366-500KCl，用于 CE 缓冲液
HEPES （1 M）FisherScientific15630-080用于 CE 和 NE 缓冲液
EDTA （0.5M），pH 值 8.0Life TechnologiesR1021用于 CE、NE 和退火缓冲液
氯化钠Fisher ScientificBP358-1NaCl，用于 NE 缓冲液
Tris-HCl （1M），pH 8.0InvitrogenBP1756-100用于退火缓冲液
磷酸盐缓冲盐水 （1x）Fisher ScientificMT21040CMPBS，用于细胞洗涤
DL-二硫苏糖醇溶液 （1 M）Sigma646563还原剂
蛋白酶抑制剂CocktailThermo Scientific87786防止 TFs
磷酸酶抑制剂混合物的分解代谢 Thermo Scientific78420防止 TF 去磷酸化
Nonidet P-40 替代IBI ScientificIB01140NP-40，用于细胞核提取
BCA 蛋白检测试剂盒Thermo Scientific23225用于测量蛋白浓度
Odyssey EMSA 缓冲液试剂盒Licor829-07910包含所有必要的 EMSA 缓冲液
TBE 凝胶，6%，12 孔用于 EMSAInvitrogenEC6265BOX
（10x）EMSAThermo ScientificB52
Miltenyi Biotec130-092-318包含所有必要的 DAPA 缓冲液
Licor Odyssey CLxLicor推荐用于 DAPA/EMSA
抗生素-抗真菌剂Gibco的扫描仪 15240-062含有 10,000 单位/ml 的青霉素，10,000 µg/ml 链霉素和 25 µ克/毫升 Fungizone®抗真菌性
胎牛血清Gibco26140-079FBS，用于培养基
RPMI 1640 培养基Gibco22400-071含有 L-谷氨酰胺和 25 mM HEPES
氯化TBE 缓冲液的 用于 FactorFinder 起始套件的

参考文献

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  1. Hindorff, L. A., et al. Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A. 106 (23), 9362-9367 (2009).
  2. Maurano, M. T., et al.

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标签

EMSA DAPA Western

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