Rapid one-step construction of hairpin RNA.
Hairpin RNA (hpRNA) is commonly used for gene-function exploration and gene engineering. In this study, a novel method was developed to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently based on Overlap Extension PCR (OE-PCR). This method, Mixed One-step OE-PCR (MOOE-PCR), can amplify two inverted repeats of DNA fragments and a spliceable intron in parallel, and then assemble them to generate ihpRNA constructs in the same tube without the purification of intermediate products. This method required a PCR process of 38-40 cycles and ordinary PCR reagents. A total of 10 ihpRNA constructs were amplified successfully using this method, with the stems ranging from 50bp to 484bp in length. Our results suggest that this novel method is a useful strategy for constructing ihpRNA.
Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, 571101 Hainan, China.
Pu
P
Yan
Wentao
W
Shen
Xinzheng
X
Gao
Jun
J
Duan
Peng
P
Zhou
D001483:Base Sequence; D007438:Introns; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry*; D034622:RNA Interference
D012313:RNA