The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3'end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.
Keywords: Loop-mediated isothermal amplification; RNA measurement; RNase H-dependent PCR; Rolling circle amplification; Splint R ligase.
©2021 Hasegawa et al.