菌落PCR差异筛选cDNA片段文库

Differential Screening cDNA Fragment Library by Colony PCR

目的探索菌落PCR差异筛选cDNA片段文库的可行性。方法在用抑制性PCR构建cDNA片段文库的同时,分别制备正、反向减法杂交探针并用碱性磷酸酶直接标记。随后用这些探针与菌落PCR的产物杂交,从而筛选出差异表达克隆,并再次用RT-PCR证实其差异表达。结果建立的菌落PCR反应体系经济实用,方法稳定,并成功地从500个克隆中筛选出差异表达克隆。结论菌落PCR可用于差异筛选构建于质粒载体的cDNA片段文库。

Objective To explore the possibility of differential screening cDNA fragment library by means of colony PCR. Methods Forward and backward cDNA probes were synthesized and directly labeled with alkaline phosphatase, while cDNA fragment library was constructed using suppression PCR. The probes were subsequently hybridized to the colony PCR products arrayed on a membrane. Differentially expressed colonies were screened and confirmed by RT-PCR. Results Colony PCR to screen differentially expressed clones was not only economical and convenient, but also safe and reliable. Several differentially expressed genes among 500 clones were found in our study. Conclusion Colony PCR can be used for differential screening cDNA fragment library constructed in plasmid vector.