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医学论文范文:不同方法鉴定差异显示技术RAP_PCR得到差异基因的比较
【摘要】 目的：评价用RAP_PCR法从测序胶上得到深海细菌(Shewanella piezotolerance WP3)差异片段验证的最佳方法。方法：用RT_PCR，RNA点杂交，荧光实时定量PCR对不同盐浓度的差异片段进行对比实验。结果：3种方法均能检测到片段的差异表达：RT_PCR灵敏性最强，RNA点杂交需要的RNA量最大，荧光实时定量PCR精确性最高。结论：在实验条件允许下，用荧光实时定量PCR对较少片段的验证较好。
【关键词】 ShewanellapiezotoleranceWP3;差异显示;RAP_PCR方法;差异片段，验证
Comparison of Three Methods for Confirmation of Differential Fragments from
RAP_PCRLI Sheng_kang1， 2，WANG Yu_qiao2
(1 Department of Biology，College of Science of Shantou University，Shantou 515063，China，2 Key Laboratory of Marine Biogenetic Resources，Third Institute of Oceanography State Oceanic Administration，Xiamen 361005，China)
[Abstract]Objective： To test the best method for confirming differential fragments from the sequencing gels. Methods： RNA samples of the cultures from the different salt concentrations were taken as a model. RT_PCR， RNA dot hybridization and real_time PCR were used for confirmation of differential fragments. Results： Three methods could produce good results. RT_PCR method had high sensibility，RNA dot hybridization needed huge quantitative RNA sample and the real_time PCR had high accuracy. Conclusion： Real_time PCR which can quantify the change folds can be a better choice医.学.全.在.线www.med126.com.
[Key Words]Shewanella piezotolerance WP3，differential display，RAP_PCR，differential fragment,confirmation
ShewanellapiezotoleranceWP3分离自西太平洋1914m的深海沉积物中，是一株革兰氏阴性、兼性厌氧的杆状深海细菌，耐冷、压，生长温度0～28℃，最适15℃，生长压力0～50MPa，最适20MPa，是研究极端微生物适应极端环境的理想材料[1]。作者选用WP3生长的2个极限生长盐浓度构建RNA池，以不同盐浓度的RNA，在WP3中建立差异显示RAP_PCR法[2]，但这种技术最突出的特点是得到的片段假阳性比率较高。对于大规模差异片段的筛选，已有文献报道的反向RNA点杂交方法[3]。但对于少量差异片段的筛选，用何种方法合适，目前少有报道。本研究用RAP_PCR法从测序胶上得到WP3的差异片段，旨在探讨重新验证这些片段的最佳方法。
