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.
Yan
Pu
P
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.
Shen
Wentao
W
Gao
Xinzheng
X
Duan
Jun
J
Zhou
Peng
P
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