Fargion Silvia, Valenti Luca, Dongiovanni Paola, Fracanzani Anna Ludovica
Department of Internal Medicine, Universitá delgi Studi, Milan, Italy.
Methods Mol Med. 2004;98:47-58. doi: 10.1385/1-59259-771-8:047.
Polymorphisms in the promoter of the tumor necrosis factor alpha (TNFalpha) gene have been reported to affect the transcription rate and the release of this cytokine. Among the best studied, the -238 and -308 polymorphisms have been associated with an increased transcriptional activity and TNFalpha release, whereas the -863 polymorphism have been associated with a reduced transcriptional activity. A growing body of evidence indicates that these polymorphisms may affect susceptibility to different diseases. Here we describe a method to detect the -238, -308, and -863 TNFalpha polymorphisms by the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Briefly, DNA is amplified by PCR using mutagenic primers containing a single base-pair mismatch adjacent to the polymorphic site to introduce a restriction site into the wild-type nucleotide sequences after amplification. The PCR products are then digested with specific restriction enzymes and analyzed by agarose gel electrophoresis.
据报道，肿瘤坏死因子α（TNFα）基因启动子中的多态性会影响该细胞因子的转录速率和释放。在研究最多的多态性中，-238和-308多态性与转录活性增加和TNFα释放有关，而-863多态性与转录活性降低有关。越来越多的证据表明，这些多态性可能影响对不同疾病的易感性。在此，我们描述了一种通过聚合酶链反应（PCR）和限制性片段长度多态性（RFLP）分析来检测-238、-308和-863 TNFα多态性的方法。简而言之，使用含有与多态性位点相邻的单个碱基对错配的诱变引物通过PCR扩增DNA，以便在扩增后将限制性位点引入野生型核苷酸序列中。然后用特定的限制性酶消化PCR产物，并通过琼脂糖凝胶电泳进行分析。
