摘要
目的:建立RNA多聚酶Ⅱ-染色质免疫沉淀-引物延伸-飞行时间质谱技术(PolⅡ-ChIP-PE-MS),并利用该方法检测SNP等位特异磷酸化PolⅡ结合量,为活体状态下研究疾病关联基因SNP的功能研究提供一种高通量的研究手段.方法:采用PCR-RFLP方法对HepG2细胞印迹基因SNRPN(仅父源等位基因表达)基因组DNA和cDNA进行基因分型,利用PolⅡCTD末端Ser5特异性抗体进行染色质免疫沉淀,针对SNP位点两侧序列设计PCR引物和延伸引物,以外侧引物进行PCR扩增,采用延伸引物进行VLET引物延伸,产物纯化后经MALDI-TOF质谱鉴定.结果:对SNRPN杂合子细胞的SNRPNC1654312TSNP进行PolⅡ-ChIP-PE-MS分析发现,基因组DNA样本和染色质免疫沉淀前样本在C、T等位点都包含相同PolⅡ结合量,但免疫沉淀后标本(被磷酸化RNA多聚酶Ⅱ结合的染色质)就仅在T等位点有结合,与产生mRNA转录的等位点相对应.结论:成功建立起PolⅡ-ChIP-PE-MS方法,利用该方法检测SNP等位特异磷酸化PolⅡ结合量是可靠的,可以利用PolⅡ-ChIP-PE-MS策略鉴定疾病关联基因SNP的功能.
AIM： To construct a new method named PolⅡ-ChlP-PE- MS （RNA polymerase Ⅱ-Chromatin immunoprecipitationprimer 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-offlight （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 phosphorylated Pol Ⅱ was bound） contained predominantly one allele, corresponding to the single allele that produced an mRNA transcript. CONCLUSION： Pol Ⅱ-ChIP-PE-MS is successfully constructed 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.
出处
《世界华人消化杂志》
CAS
北大核心
2005年第20期2431-2436,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助
No.30470964~~
