Yan W Y, Gao Z R, Wu G Z, Liu S X, Zheng Z X
Institute of Genetics, Fudan University.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 1990;8(4):249-52.
The template mRNA was extracted from Schistosoma japonicum. The first strand of cDNA was synthesized by AMV-reverse transcriptase. The second strand cDNA was first digested by RNase H to remove mRNA and was then synthesized by AMV-reverse transcriptase, T4-DNA polymerase. Sizing of cDNA was applied on a NACS column to remove small fragments of less than 1 kb. Homopolymeric tailing of vector (pUC18) was done with dGTP and DNA terminal transferase and tailing of the cDNA with dCTP was carried out under the same conditions. After annealing, the plasmids with cDNA were transformed into E. coli MC1061. The efficiency of cloning was about 10(4)/micrograms mRNA with 30% of the transformants having the inserts of cDNA (Figs. 1-2).
从日本血吸虫中提取模板mRNA。用禽成髓细胞瘤病毒逆转录酶(AMV-逆转录酶)合成cDNA第一链。第二链cDNA先用核糖核酸酶H消化以去除mRNA,然后用AMV-逆转录酶、T4-DNA聚合酶合成。将cDNA进行大小分级,应用于NACS柱以去除小于1 kb的小片段。用脱氧鸟苷三磷酸(dGTP)和DNA末端转移酶对载体(pUC18)进行同聚物加尾,并在相同条件下用脱氧胞苷三磷酸(dCTP)对cDNA进行加尾。退火后,将带有cDNA的质粒转化到大肠杆菌MC1061中。克隆效率约为每微克mRNA 10⁴个,30%的转化体含有cDNA插入片段(图1-2)。