汪海林团队提出基因水平碱基分析新方法（附原文）

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2 l  \0 R0 H7 |% R/ j# K日前，中科院生态环境中心汪海林研究组在5-羟甲基胞嘧啶（5hmC）分析方面取得重要进展，提出了对该物质进行分析的新方法。相关成果近日发表于《核酸研究》（Nucleic Acids Research）。
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3 v' i. q" V+ x; `3 ^据了解，5hmC是科学家发现的第六种碱基，已在多种哺乳动物组织和细胞中检出。另外，多种肿瘤中5hmC水平偏低，使其有可能成为肿瘤早期诊断的生物标志物。目前，基因水平5hmC的分析依赖于限制性内切酶方法，而相关方法受到酶切位点限制，并且难以区分多5hmC位点的序列。9 b: ^0 x# i5 b) h6 U! U& f

! C3 K, n) O: @5 r1 B8 z汪海林研究组提出利用硼酸与可葡萄糖基化的5hmC结合，发展新的基因水平5hmC分析方法。首先，采用T4噬菌体β-葡萄糖转移酶，在葡萄糖供体底物尿核苷二磷酸葡萄糖存在下，将葡萄糖基团转移至5hmC的羟基位置，从而生成葡萄糖基化5-羟甲基胞嘧啶。在此基础上，利用定量PCR分析。最终，研究人员发现硼酸可与葡萄糖基化5-羟甲基胞嘧啶中的邻二醇发生的共价作用，有效抑制聚合酶扩增反应的特点，可特异性地识别特定基因/片段中的5hmC。
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: q$ ^3 t9 D" w7 x  h业内专家表示，该方法不受酶切位点限制，可适用于各种细胞、各种序列中5hmC的分析鉴定，具有广阔的应用前景。; K. o2 J; s9 f4 [+ I- s% Y! T

" j+ ?( U" _# h  @+ i英文摘要
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  _" V9 t' c+ R$ v0 MNucl. Acids Res  doi：10.1093/nar/gku216
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* f& w0 o5 y# \: |3 @Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine
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' {# O& ~+ E2 K" ^Chao Zhao1， Hailin Wang1，*， Bailin Zhao1， Cuiping Li1， Ruichuan Yin1， Maoyong Song1， Baodong Liu1， Zhen Liu2 and Guibin Jiang17 _3 f! S' t0 G! r1 G1 ]
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The gene- or fragment-specific detection of newly recognized deoxyribonucleic acid (DNA) base 5-hydroxymethylcytosine (5hmC) will provide insights into its critical functions in development and diseases， and is also important for screening 5hmC-rich genes as an indicator of epigenetic states， pathogenic processes and pharmacological responses. Current analytical technologies for gene-specific detection of 5hmC are heavily dependent on glucosylated 5hmC-resistant restriction endonuclease cleavage. Here， we find that boronic acid (BA) can inhibit the amplification activity of Taq DNA polymerase for replicating glucosylated 5hmC bases in template DNA by interacting with their glucose moiety. On the basis of this finding， we propose for the first time a BA-mediated polymerase chain reaction (PCR) assay for rapid and sensitive detection of gene- or fragment-specific 5hmC without restriction-assay-like sequence limitations. To optimize the BA-mediated PCR assay， we further tested BA derivatives and show that one BA derivative， 2-(2′-chlorobenzyloxy) phenylboronic acid， displays the highest inhibitory efficiency. Using the optimized assay， we demonstrate the enrichment of 5hmC in an intron region of Pax5 gene (a member of the paired box family of transcription factors) in mouse embryonic stem cells. Our work potentially opens a new way for the screening and identification of 5hmC-rich genes and for high throughput analysis of 5hmC in mammalian cells.
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7 W+ I/ c, M8 Y2楼原文 感谢zb_ming 提供

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