人胎儿骨骼和关节RACE cDNA文库的构建

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目的 建立人胎儿骨骼和关节快速扩增 c DNA末端 (rapid amplification of c DNA ends,RACE c DNA)文库 ,为分离骨骼和关节发育相关基因奠定基础。方法 采用改进的异硫氰酸胍 -酚 -氯仿 -异戊醇一步法提取骨骼和关节总 RNA,用 Ta Ka Ra公司生产的 c DNA合成试剂盒合成平末端的双链 c DNA,然后与衔接子连接。再用位于双链 c DNA末端的通用引物扩增全部 c DNA。结果 建立了从骨骼和关节构建 RACE c DNA文库的方法 ,并用该方法成功地构建了人胎儿骨骼和关节 RACE c DNA文库。结论 所构建的的利用少量总 RNA构建 RACE c DNA文库方法切实可行 ,所构建的文库适用于用 RACE方法从中分离骨骼和关节发育相关基因 OBJECTIVE: To establish a rapid amplification of c DNA ends cDNA library of human fetal bones and joints and lay a foundation for the isolation of genes related to bone and joint development. METHODS: Bone and joint total RNA was extracted by one-step method using guanidinium isothiocyanate-phenol-chloroform-isopentyl alcohol. The blunt-ended double-stranded cDNA was synthesized by c-DNA synthesis kit from Ta Ka Ra Company. Sub-connection. The entire c DNA was amplified using universal primers located at the ends of double-stranded c DNA. Results The method of constructing RACE cDNA library from bones and joints was established and the human fetal bone and joint RACE cDNA library was successfully constructed. Conclusion The constructed method of constructing RACE cDNA library with a small amount of total RNA is feasible and the constructed library is suitable for the isolation of bone and joint development related genes by RACE
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