人胃癌组织和对应正常胃粘膜cDNA文库的构建

Establishment of cDNA Libraries from Human Gastric Cancer Tissue and Its Normal Mucosal Counterpart

作者从一例人高转移型原发性胃癌组织及其对应的正常胃粘膜中分别提取总RNA和分离多聚(A)^+RNA,进而合成cDNA。cDNA第一条链合成效率前者为58.8%,后者为29.8%;第二条链合成效率前者为98.3%,后者为93.3%。cDNA分子大小在0.5～8Kb之间。cDNA接上含有NotI切点和EcoRI粘性末端的接头,重组到经EcoRI酶解5端脱磷酸化的pT7T318U质粒上,转化大肠杆菌NM522,构建了二个cDNA文库,文库的容量分别为1.2×10~5与1.0×10~5重组子,重组率均为80%,克隆效率分别为2.4×10~6克隆/lμg cDNA与2.0×10~6克降/lμg cDNA,插入片段的大小在0.5Kb～4Kb之间。

Total cellular RNAs were extracted from a case of hurrah primary gastric cancer tissue and its surrounding normal mucosa with guanidinium isothiocyanate/CsCl ultracentrifugation technique. Then, poly(A)^+ RNAs were recovered through a 2-cycle oligo(dT)cellulose column. The undegraded poly(A)^+ RNAs were used to synthesize the cDNA The synthesis efficiency of the first-strand cDNAs from tumor poly (A)^+ RNA and normal poly (A)^+ RNA was 58.8% and 29.8%, and that of the second-strand cDNA was 98.3% and 93.3% respectively. The cDNA molecules ranged in size from 0.5 kb to 8 kb. EcoRI-ended cDNAs were ligated to the EcoRI-digested anddephosphorylatedplasmid pT7T318U, then transforrned the competent cells of EcoRI NM^522. Results showed: (1) 1.2×10~5 and 1.0×10~5 recombinant clones were present in the 2 libraries respectively and their efliciency was 2.4×10~6 and 2.0×10~6 clones/μg cDNA respectively. (2) The recombinant rote of both were approximately 80%. (3) The length of the inserted foreign DNA fragments in the recombinants varied from 0.5 kb to 4.0 kb.