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Kamchatka crab duplex-specific nuclease-mediated transcriptome subtraction method for identifying long cDNAs of differentially expressed genes

文献类型： 外文期刊

作者： Peng, Ri-He 1 ; Xiong, Ai-Sheng 1 ; Xue, Yong 1 ; Li, Xian 1 ; Liu, Jin-ge 1 ; Cai, Bin 1 ; Yao, Quan-Hong 1 ;

作者机构： 1.Shanghai Acad Agr Sci, Agrobiotechnol Res Inst, Shanghai Key Lab Agr Genet & Breeding, Shanghai, Peoples R China

关键词： kamchatka crab duplex-specific nuclease;suppression subtractive hybridization;full-length cDNAs;complicate tissue or cell types;POLYMERASE-CHAIN-REACTION;NORMALIZATION;HYBRIDIZATION;SUPPRESSION;LIBRARIES;DISPLAY;DISCOVERY;CLONING;SAGE

期刊名称：ANALYTICAL BIOCHEMISTRY （ 影响因子：3.365； 五年影响因子：3.049 ）

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收录情况： SCI

摘要： The subtraction method is a quick and economical technique to scan differential gene expression. However, most subtraction methods are limited by the complexity and length of cDNA samples. To overcome this problem, we developed a novel method to identify the unique full-length cDNAs in two complicated tissue or cell types. This method, duplex-specific nuclease (DSN)-mediated transcriptome subtraction (DTS), is based on the normalization strategy of the crab duplex-specific nuclease and the subtraction method of suppression subtractive hybridization. DSN eliminates nearly all of the common sequences in the tester and driver cDNA samples after the first hybridization step, ensures accurate discrimination between the tester and the driver cDNA samples, and enriches the full-length differential cDNAs from the tester. Using the DTS method, we have successfully identified an 1812-bp additional GUS gene from the complicated Arabidopsis seedling cDNA library. We also employed DTS to detect the differences in mRNA expression of salt-treated Arabidopsis seedlings to illustrate further the efficiency of the subtraction method. (C) 2007 Elsevier Inc. All rights reserved.

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