人胎儿骨骼和关节RACE cDNA文库的构建

目的 建立人胎儿骨骼和关节快速扩增cDNA末端（rapid amplification of cDNA ends，RACE cDNA）文库，为分离骨骼和关节发育相关基因奠定基础。方法 采用改进的早硫氰酸胍－酚－氯仿－异戊醇一步法提取骨骼和关节总RNA，用TaKaRa公司生产的cDNA合成试剂盒合成平末端的双链cDNA,然后与衔接子连接，再用位于双链cDNA末端的通用引物扩增全部cDNA。结果 建立了从骨骼和关节构建RACE cDNA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 cDNA文库。结论 所构建的利用少量总RNA构建RACE cDNA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。

The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint

Objective To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone and joint specific development related genes.Methods Total RNA of bone and joint were extracted with the modified single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. The double stranded end blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers.Results A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed.Conclusion The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue specific genes.