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编号:10924713
PolⅡ-ChIP-PE-MS方法的建立及其在SNP功能研究中的应用
http://www.100md.com 晏泽辉, 邓国宏, 王宇明
单核苷酸多态性; RNA多聚酶Ⅱ; 染色质免疫沉淀; 引物延伸; 质谱,晏泽辉,&
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     晏泽辉, 邓国宏, 王宇明, 第三军医大学西南医院全军感染病研究所 重庆市 400038

    晏泽辉, 男, 1978-12-16生, 湖北省罗田县人, 汉族, 2003年第三军医大学西南医院硕士生, 医师.

    国家自然科学基金资助, No.30470964

    通讯作者: 王宇明, 400038, 重庆市, 第三军医大学西南医院全军感染病研究所. wym417@mail.tmmu.com.cn

    电话: 023-68754141

    收稿日期: 2005-08-29 接受日期: 2005-09-06

    Construction of RNA polymerase II-Chromatin immunoprecipitation-primer extension-mass spectrum and its application in function research of single nucleotide polymorphism

    Ze-Hui Yan, Guo-Hong Deng, Yu-Ming Wang

    Ze-Hui Yan, Guo-Hong Deng, Yu-Ming Wang, Institute of Infectious Diseases of Chinese PLA, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China

    Supported by National Natural Science Foundation of China, No. 30470964

    Correspondence to: Yu-Ming Wang, Institute of Infectious Diseases of Chinese PLA, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China. wym417@mail.tmmu.com.cn

    Received: 2005-08-29 Accepted: 2005-09-06

    Abstract

    AIM: To construct a new method named PolII-ChIP-PE-MS (RNA polymerase II-Chromatin immunoprecipitation-primer extension-mass spectrum), and to validate the reliability of this method in the study of single nucleotide polymorphism (SNP) allele-specific quantification of RNA polymerase loading.

    METHODS: The C/T SNP of SNRPN at nt 1654312 (numbered according to NT_026446) was genotyped in a genomic DNA sample and a cDNA sample of HepG2 cell line by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). After the ChIP assay was performed for phosphorylated Pol II using antibodies highly specific for certain phosphorylated serine residues of the CTD, the extension of short primer was carried out and then the samples were analyzed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF).

    RESULTS: Comparison of genomic DNA and cDNA of HepG2 cells conformed that, in cell lines heterozygous with respect to this SNP, only one of the two alleles present in the genomic DNA produced an mRNA transcript. When chromatin from heterozygous cell lines was analyzed for the SNP at nt1654312 in SNRPN, the pro-immunoprecipitated starting material and the genomic DNA contained similar amounts of each allele, whereas the material (chromatin to which phosphorylat-ed Pol II was bound) contained predominantly one alle-le, corresponding to the single allele that produced an mRNA transcript.

    CONCLUSION: Pol II-ChIP-PE-MS is successfully co-nstructed and it is an applicable and reliable method to detect the SNP allele-specific quantification of RNA polymerase loading. It can be used in detecting the function of disease-related gene SNP.

    Key Words: Single nucleotide polymorphism; RNA polymerase II; Chromatin immunoprecipitation; Primer extension; Mass spectrum ......

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