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《Academic Journal of Second Military Medical University》 2004-10
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Construction of full-length cDNA library of Candida albicans using SMART technology

XU Zheng 1, CAO Yong-Bing 1, GAO Ping-Hui 1, ZHANG Jun-Dong 1, CAO Ying-Ying 1,TANG Rong 2, YING Kang 2* , JIANG Yuan-Ying 1* (1.Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China; 2.Institute of United Gene Holding Ltd., Shanghai 200092)  
Objective:To rapidly construct a full-length cDNA library of Candida albicans with switching mechanism at 5′ end of RNA transcript (SMART) techniques, providing a base for the studying the novel multidrug resistance associated genes of Candida albicans. Methods: The total RNA was extracted from several Candida albicans (SC5314) samples of different growing phase and different drugs treatment by TRIzol reagent. The mRNA was subsequently separated with the Oligotex kit. The CDSⅢ/3′ PCR primer (containing SfiⅠB site) was used in the first-strand reaction and the SMART Ⅳ Oligonuleotide (containing SfiⅠA site) served as the short, extended template at the 5′ end of the mRNA. LD-PCR generated full-length double-strand cDNA, which was digested by SfiⅠ restriction enzyme and ligated to the SfiⅠ(AB)-digested pBSF vector. The ligated vector was then transformed by DH5α for the final construction of the cDNA library. The entire sequences of partial inserted cDNAs were obtained and the ratio of full-length cDNA was analysed. Results: The constructed Candida albicans cDNA library consisted of 1.1×10 6 independent clones with a recombination rate of 97.6%. Exogenous inserts of the recombinants was 0.75-3 kb in length, with most of them above 1.5 kb. Sequencing results and bioinformatics analysis of 60 clones indicated that the ratio of full-length cDNA was above 50%. Conclusion: A high-quality full-length cDNA library of Candida albicans has been constructed successfully, which is essential for screening and cloning of new multidrug resistance associated genes of Candida albicans.
【Fund】: 国家自然科学基金 ( 3 0 2 0 0 0 12 3 0 3 0 0 44 2 ) ;; 上海市基础研究重点项目 ( 0 3 JC14 0 0 6)
【CateGory Index】: R379.4
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