Fig. 5: Development of a PAM-free platform.
a Schematic diagram of the PAM sequence introduced at different positions on the RPA forward primers. b Comparison between the expected sequence and the Sanger sequencing data of the amplification products of three targets. The green sequence represents the amplified product sequence after the PRA forward primer. The lower case red sequence represents the Sanger sequencing data, which does not match the expected sequence. The right side of the figure represents the distance between the amplification product after the PRA primer and PAM (TTTV), while ‘U’ represents the unmodified sequence. Y, yes; N, no. The sequences marked in yellow represent the corresponding PAM-containing RPA forward primer that we selected for sensitivity analysis in the subsequent analysis. All the RPA primer sequences are provided in Supplementary Data 5. c Analytical sensitivity of two-step reactions of the three targets using a PAM-containing RPA forward primer and a PAM-free target activator. The target substrate was tenfold serially diluted to concentrations ranging from 106 to 100 copies per reaction. Error bars represent mean ± SD, n = 3. ***P < 0.001, ****P < 0.0001; ns not significant.
