采用简单高效的cDNA合成技术制备Wistar大鼠脑cDNA基因文库,以纯化的poly( A)+-RNA为模板,含Not I切点的oligo-(dT)15为引物,在反转录酶的作用下,合成第一股单链cDNA;用E.coli RNase H除去模板RNA,并以E.coli DNA聚合酶I,E.coli DNA连接酶和T4 DNA聚合酶催化合成cDNA第二条链,即成为双链cDNA;此双股cDNA除0.5μg用于插入pSPORT I载体,转入E.coli DH5a,建成cDNA文库外,其余保存在-20℃,以此cDNA为模板,应用PCR方法,先后克隆了谷氨酸脱羧酶(GAD,1800bp)、神经元特异性烯醇化酶(NSE,1340bp),甲状腺激素受体(T3-receptor,1230bp)、胆囊收缩素(CCK,345bp)的全编码基因.
A cDNA library of Wistar rat brain was constructed with a highly effcient and simplified method. The template Poly (A)+-RNA was extracted from Wistar rat brain. The first strand of cDNA was synthesized by SuperScript RNase H- reverse transcriptase with the oligo (dT)15 primer which contains Not I site. After the poly (A)+ -RNA was removed with E. coli RNase H, the second strand was synthesized by means of E. coli DNA polymerase I, E. coli DNA ligase and T4 DNA polymerase.Then Sal I adapter was added and the cDNA digested with Not I .Half of the cDNA was inserted into plasmid pSPORT I and transformed E .coli DH 5a. The other half was stored at -20℃. It was shown that the cDNA length ranged from 100 to 10 000 bp. Using this cDNA library as a template, four genes have been amplified successfully. They are glutamic acid decarboxylase (GAD, 1800 bp),neuron-specific enolase (NSE, 1340 bp),T3 receptor(1230 bp) and cholecystokinin(CCK, 345 bp).The method for storage of the cDNA library which can be kept for a longer period was also developed.
宋学文,赵崇,周仁,郭善一,张镜宇.大鼠脑cDNA文库的构建[J].生物化学与生物物理进展,1996,23(2):169-172
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