摘要
目的建立Has-miR-17编码基因启动子区的PCR扩增及鉴定体系。方法提取人源细胞系基因组DNA为实验样本,以Has-miR-17编码基因上游启动子区序列为目标序列,行序列特征分析并设计特异PCR扩增及测序引物,利用添加DMSO和使用5’加尾引物的体系优化策略建立Has-miR-17基因启动子区PCR扩增及鉴定体系并行性能评估。同时另以10例人源细胞系为样本初步验证该PCR及鉴定体系的应用效果。结果经电泳鉴定与测序分析确认在联合使用5'加尾PCR引物和5%DMSO的条件下Has-miR-17基因启动子区PCR扩增及鉴定体系已成功建立,其检测下限达10 ng/μl且特异度好、重复性佳。利用该体系成功实现对10例人源细胞株Has-miR-17基因启动子区的PCR扩增及鉴定。结论已成功建立Has-miR-17编码基因启动子区的PCR扩增及鉴定体系。
Objective To establish the PCR amplification and sequencing system of Hsa-miR-17 gene promoter region. Methods To establish the PCR amplification and sequencing system of Hsa-miR-17 gene promoter region and evaluate its performance by analyzing the sequence characteristics, designing PCR and sequencing primers, extracting sample genome DNA from different human cell lines and using optimization strategy of adding DMSO to the final concentration of 5% and making use of 5＇ tailed PCR primers;Then the established system was further verified in 10 other cell line samples. Results The PCR amplification and sequencing system of hsa-miR-17 gene promoter region, which had good repeatability, good specificity and its detection limit was 10 ng/μl, was established successfully under the condition of using 5＇ tailed PCR primers and 5% DMSO and that was confirmed by electrophoresis analysis and Sanger sequencing. And fragment of Has-miR-17 gene promoter region in 10 human cell lines were successfully achieved and verified by this system. Conclusion The PCR system of Hsa miR-17 gene promoter region was established successfully.
出处
《现代检验医学杂志》
CAS
2017年第5期4-7,12,共5页
Journal of Modern Laboratory Medicine
基金
广州市卫计委医药卫生科技项目(No.20161A011086)
