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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(píng)價(jià)用RAP_PCR法從測(cè)序膠上得到深海細(xì)菌(Shewanella piezotolerance WP3)差異片段驗(yàn)證的最佳方法。方法：用RT_PCR，RNA點(diǎn)雜交，熒光實(shí)時(shí)定量PCR對(duì)不同鹽濃度的差異片段進(jìn)行對(duì)比實(shí)驗(yàn)。結(jié)果：3種方法均能檢測(cè)到片段的差異表達(dá)：RT_PCR靈敏性最強(qiáng)，RNA點(diǎn)雜交需要的RNA量最大，熒光實(shí)時(shí)定量PCR精確性最高。結(jié)論：在實(shí)驗(yàn)條件允許下，用熒光實(shí)時(shí)定量PCR對(duì)較少片段的驗(yàn)證較好。
【關(guān)鍵詞】 ShewanellapiezotoleranceWP3;差異顯示;RAP_PCR方法;差異片段，驗(yàn)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m.zxtf.net.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(zhǎng)溫度0～28℃，最適15℃，生長(zhǎng)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(zhǎng)的2個(gè)極限生長(zhǎng)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(diǎn)是得到的片段假陽(yáng)性比率較高。對(duì)于大規(guī)模差異片段的篩選，已有文獻(xiàn)報(bào)道的反向RNA點(diǎn)雜交方法[3]。但對(duì)于少量差異片段的篩選，用何種方法合適，目前少有報(bào)道。本研究用RAP_PCR法從測(cè)序膠上得到WP3的差異片段，旨在探討重新驗(yàn)證這些片段的最佳方法。
