从基因组DNA选择性捕获的5 - 羟甲基
Summary
描述的是一个两步骤的标记过程中使用的β-葡糖基转移酶（β-GT）来传输的叠氮化物5-HMC-葡萄糖，然后通过点击化学生物素链接器为容易和密度独立富集转移。这种高效的和具体的标记方法使富集5 – HMC具有极低的背景和高通量表观映射的通过新一代测序。
Abstract
5 – 甲基胞嘧啶（5-MC）构成约2-8％，总在人类基因组DNA胞嘧啶和影响广泛的生物学功能，包括基因表达，维持基因组的完整性，亲本印记，X染色体失活，调节发育，衰老和癌症。最近，氧化的5-MC，5 -羟甲基胞嘧啶（5-HMC），存在下，在哺乳动物细胞中被发现，特别是在胚胎干（ES）细胞和神经元细胞2-4。 TET家族铁（II）/α-酮戊二酸依赖性双加氧酶2，3 5-MC催化氧化所产生的5-HMC。 5-HMC提出参与胚胎干细胞（MES），正常的造血和恶性肿瘤，和受精卵开发2，5-10维护。为了更好地理解5-HMC的功能，可靠的和直接测序系统是必不可少的。传统的亚硫酸氢钠测序可以不区分5-HMC从5-MC 11 </sup>。为了解开生物5 – HMC，我们已经开发出一种高效，高选择性的化学标记方法和捕获5-HMC，利用噬菌体酶，葡萄糖部分，增加了一个5-HMC特别是12。
在这里，我们描述一个简单的两步骤程序选择性化学标记的5-HMC。在第一标记步骤中，基因组DNA中的5-HMC被标记为与6 – 叠氮-葡萄糖催化β-GT，葡糖基转移酶从T4噬菌体，在从一种方式，将6 – 叠氮-葡萄糖以5-HMC改性的辅助因子，：UDP-6-N3-Glc的（6-N3UDPG）。在第二步骤中，生物素化，二硫键的生物素连接器附着的叠氮基，通过点击化学。这两个步骤是非常具体的，高效的，从而完成标签，无论5-HMC丰富的基因组区域和极低的背景。生物素化的5 – HMC后，5-HMC-含DNA片段，然后选择性地捕获使用抗生蛋白链菌素珠粒的密度无关的方式。所得的5-HMC-富集的DNA片段可用于下游分析，包括下一代测序。
我们的选择性标记和捕获协议赋予高灵敏度，适用于任何变量/ 5-HMC不同丰度的基因组DNA的来源。虽然此协议的主要目的，是其下游的应用程序（ 即 ，下一代测序绘制出了在基因组的5-HMC分布），它是兼容的单分子实时SMRT（DNA）测序，这是能够提供单碱基分辨率的测序5 – HMC。
Protocol
1。基因组DNA碎片适合的全基因组测序平台使用所希望的大小范围内的超声处理的片段的基因组DNA。 （我们通常超声〜300个基点。）验证的粒度分布零散的基因组DNA，1％琼脂糖凝胶电泳（ 图1）。 2。 DNA的制备确定起始DNA金额5-HMC丰富的基因组DNA的基础上。由于5 – HMC水平的显着不同组织类型的不同，启动DNA金额的依赖样品…
Discussion
5 – 羟甲基胞嘧啶（5-HMC）是最近确定的表观遗传修饰在某些哺乳动物的细胞类型中的大量存在。这里介绍的方法是确定基因组中广泛分布的5-HMC。我们使用T4噬菌体的β-葡糖基转移酶，转移含有叠氮基团上的羟基基团的5-HMC一个精心设计的葡萄糖部分。叠氮基团，可以通过化学方法用生物素修饰，用于检测，亲和富集，和5-HMC-含在哺乳动物基因组的DNA片段测序。该协议具有优势超过5 HMC抗体为基础…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是支持的，部分由美国国立卫生研究院（GM071440 CH和NS051630/MH076090/MH078972的PJ）。
Materials
| Name | Company | Catalog # | Comment |
| Reagents | |||
| 5M Sodium chloride (NaCl) | Promega | V4221 | |
| 0.5M pH8.0 Ethylenediaminetetraacetic acid (EDTA) | Promega | V4231 | |
| 1M Trizma base (Tris) pH7.5 | Invitrogen | 15567-027) | |
| HEPES 1M, pH7.4 | Invitrogen | 15630 | |
| Magnesium chloride (MgCl2) 1M | Ambion | AM9530G | |
| Dimethyl sulfoxide (DMSO) | Sigma | D8418 | |
| Tween 20 | Fisher BioReagents | BP337-100 | |
| DBCO-S-S-PEG3-Biotin conjugate | Click Chemistry Tools | A112P3 | |
| 1,4-Dithiothreitol, ultrapure (DTT) Superpure | Invitrogen | 15508-013 | |
| QIAquick Nucleotide Removal Kit | Qiagen | 28304 | |
| Micro Bio-Spin 6 Column | Bio-Rad | 732-6222 | |
| Dynabeads MyOne | Invitrogen | 650-01 | |
| Streptavidin C1 | |||
| Qiagen MinElute PCR Purification Kit | Qiagen | 28004 | |
| UltraPure Agarose | Invitrogen | 16500500 | |
| UDP-6-N3-glucose | Active Motif | 55013 | |
| Enzyme | |||
| β-glucosyltransferase (β-GT) | New England Biolab | M0357 | |
| Equipment | |||
| Sonication device | Covaris | ||
| Desktop centrifuge | |||
| Water bath | Fisher Scientific | ||
| Gel running apparatus | Bio-Rad | ||
| NanoDrop1000 | Thermo Scientific | ||
| Labquake Tube Shaker | Barnstead | ||
| Labquake Tube Shaker | Thermolyne | ||
| Magnetic Separation Stand | Promega | Z5342 | |
| Qubit 2.0 Fluorometer | Invitrogen | ||
| Reagent setup 10 X β-GT Reaction Buffer (500 mM HEPES pH 7.9, 250 mM MgCl2) 2 X Binding and washing (B&W) buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween 20). |
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Tags
Selective Capture5-hydroxymethylcytosineGenomic DNA5-methylcytosineBiological FunctionsGene ExpressionGenome IntegrityParental ImprintingX-chromosome InactivationDevelopmentAgingCancerMammalian CellsEmbryonic Stem CellsNeuronal CellsTET Family Iron (II)/α-ketoglutarate-dependent DioxygenasesMES CellsNormal HematopoiesisMalignanciesZygote DevelopmentBisulfite SequencingLabeling And CaptureBacteriophage EnzymeGlucose MoietyGlucosyltransferase
Cite This Article
Li, Y., Song, C., He, C., Jin, P. Selective Capture of 5-hydroxymethylcytosine from Genomic DNA. J. Vis. Exp. (68), e4441, doi:10.3791/4441 (2012).