Construction of siRNA/miRNA expression vectors based on a one-step PCR process.
BACKGROUND
RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems.


RESULTS
Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate.


CONCLUSIONS
This novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries.
Xu
Jun
J
Institute of Molecular Biology, Biology Faculty of Hubei University, Wuhan, Hubei Province, PR China. xujun.allen@gmail.com
Zeng
Jie Qiong
JQ
Wan
Gang
G
Hu
Gui Bin
GB
Yan
Hong
H
Ma
Li Xin
LX
D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection / Q000379:methods
D017931:DNA Primers; D035683:MicroRNAs; D034741:RNA, Small Interfering