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醫(yī)學(xué)論文范文:不同方法鑒定差異顯示技術(shù)RAP_PCR得到差異基因的比較
【摘要】 目的：評價用RAP_PCR法從測序膠上得到深海細(xì)菌(Shewanella piezotolerance WP3)差異片段驗證的最佳方法。方法：用RT_PCR，RNA點雜交，熒光實時定量PCR對不同鹽濃度的差異片段進(jìn)行對比實驗。結(jié)果：3種方法均能檢測到片段的差異表達(dá)：RT_PCR靈敏性最強(qiáng)，RNA點雜交需要的RNA量最大，熒光實時定量PCR精確性最高。結(jié)論：在實驗條件允許下，用熒光實時定量PCR對較少片段的驗證較好。
【關(guān)鍵詞】 ShewanellapiezotoleranceWP3;差異顯示;RAP_PCR方法;差異片段，驗證
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醫(yī).學(xué).全.在.線www.gydjdsj.org.cn.
[Key Words]Shewanella piezotolerance WP3，differential display，RAP_PCR，differential fragment,confirmation
ShewanellapiezotoleranceWP3分離自西太平洋1914m的深海沉積物中，是一株革蘭氏陰性、兼性厭氧的桿狀深海細(xì)菌，耐冷、壓，生長溫度0～28℃，最適15℃，生長壓力0～50MPa，最適20MPa，是研究極端微生物適應(yīng)極端環(huán)境的理想材料[1]。作者選用WP3生長的2個極限生長鹽濃度構(gòu)建RNA池，以不同鹽濃度的RNA，在WP3中建立差異顯示RAP_PCR法[2]，但這種技術(shù)最突出的特點是得到的片段假陽性比率較高。對于大規(guī)模差異片段的篩選，已有文獻(xiàn)報道的反向RNA點雜交方法[3]。但對于少量差異片段的篩選，用何種方法合適，目前少有報道。本研究用RAP_PCR法從測序膠上得到WP3的差異片段，旨在探討重新驗證這些片段的最佳方法。
