| 王锋超,王军平,粟永萍,高京生,楼淑芬,刘晓宏,任泂,章波.增强型RACE技术克隆辐射诱导小鼠肠上皮新基因RS1[J].中华放射医学与防护杂志,2003,23(5):324-326 |
| 增强型RACE技术克隆辐射诱导小鼠肠上皮新基因RS1 |
| Cloning of radiation-induced new gene RS1 expressed in mouse intestinal epithelium by enhanced RACE |
| 投稿时间：2003-04-10 |
| DOI： |
| 中文关键词: 增强RACE 逆转录PCR 探针杂交 RS1基因 |
| 英文关键词:Enhanced RACE PCR RT-PCR Probe hybridization RS1 gene |
| 基金项目:国家自然科学基金资助项目(39970232,30230360) |
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| 全文下载次数: 1968 |
| 中文摘要: |
| 目的 在获得了辐射诱导小鼠肠上皮新基因RS1EST序列的基础上获得全长cDNA。方法 RT-PCR方法对RS1基因进行表达谱分析,找到表达丰度较高的组织提取总RNA作为模板,应用增强RACE技术即结合生物素探针富集靶cDNA的方法克隆RS1的cDNA末端。结果 克隆到RS1片段3′端约2kb的序列。该序列5′端包含已知的RS1片段,3′端具有明显的polyA加尾信号,有编码区的终止密码子。明确了RS1的正、负链及其蛋白编码方向,为RS15′端的克隆提供了必要信息。结论 结果与本实验的设计完全一致,说明这一方法对从表达丰度低,难以设计最佳基因特异性引物的EST序列克隆其对应的全长cDNA是可行的,同时为下一步研究RS1在小鼠肠上皮辐射应激反应中的作用和被辐射诱导表达的调控模式奠定了基础。 |
| 英文摘要: |
| Objective To obtain full-length cDNA of radiation-induced new gene RS1 expressed in mouse intestinal epithelium. Methods The tissue expression profile of RS1 was analyzed by semi-quantitative RT-PCR to find the target tissue which highly expresses RS1.The total RNA extracted from the corresponding tissue was taken as the template for reverse-transcription.Enhanced RACE PCR was used to clone the full-length cDNA of RS1,including enrichment of the target gene through biotin-labeled probe for magnetic bead purification and nested PCR. Results About a 2 kb long 3′end was successfully cloned and cloning of the 5′end proceeded well. Conclusion The result is consistent with our experiment design.The set of combined techniques has been identified with the cloning of full-length cDNA from EST sequence especially when the optimal gene-specific primers are not available or the expression level of target gene is low. |
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