Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners

Abstract

Asymmetric PCR is one of the most utilized strategies in ssDNA generation towards DNA aptamer generation due to its low cost, robustness and the low amount of starting template. Despite its advantages, careful optimization of the asymmetric PCR is still warranted to optimize the yield of ssDNA. In this present study, we have developed an extensive optimization pipeline that involves the optimization of symmetric PCR initially followed by the optimization of asymmetric PCR. In the asymmetric PCR, optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg 2+ /dNTP concentrations and the amounts of Taq Polymerase was carried out. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method, either via lambda exonuclease or biotinstreptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700 ± 11.3 and 820 ± 19.2 nM for A-PCR-lambda exonuclease and A-PCR-biotin-streptavidin-based separation, respectively. We urge to develop a separate optimization pipeline of asymmetric PCR for each different randomized ssDNA library before embarking on any SELEX studies.

Optimized Lambda Exonuclease Digestion or Purification Using Streptavidin-Coated Beads: Which One Is Best for Successful DNA Aptamer Selection?

“…A starting SELEX library consists of numerous sequences, composed of variable regions with more or less complexity, making aptamer amplification difficult and problematic. The annealing temperature was chosen based on the maximum dsDNA yield as it is reported to have little influence on the presence of non-specific products [ 18 ]. We observed a maximum amount of dsDNA at 58 °C ( Figure 1 b), which was the theoretical annealing temperature of the primers, as determined in silico.…”

“…At a low annealing temperature, primers can hybridize non-specifically to the complementary sequences present in the variable regions, reducing the efficiency of the PCR [ 19 ]. High annealing temperature weakens the hybridization of primers at their binding sites, also decreasing the PCR efficiency [ 18 ]. The annealing temperature can be set to 58 °C for aptamer selection with our library.…”

“…Several ssDNA generation methods have been reported for SELEX, including asymmetric PCR, strand separation under denaturing conditions, magnetic separation with streptavidin-coated beads and lambda exonuclease digestion [ 16 , 17 ]. Asymmetric PCR is often considered a robust and low-cost method [ 16 , 18 ]. However, this method is time-consuming as it is strongly recommended to adjust the PCR conditions for each round of SELEX.…”

“…Several optimizations can be performed, such as the modification of primers concentration and/or ratio, number of PCR cycles, annealing temperature, dNTP, and MgCl 2 concentrations [ 19 , 20 ]. In addition, asymmetric PCR produces ssDNA, as well as dsDNA [ 18 , 21 ]. As a result, asymmetric PCR is often combined with a gel purification step or other ssDNA generation methods [ 22 ].…”

Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications

“…Firstly, the RCA reaction can obtain long products, named RCA products (RCPs), with a large number of tandem repeat sequences that are complementary to the circular template. Therefore, by elaborately manipulating sequences of the template, the RCPs can be customized with functional nucleic acid sequences, such as DNA aptamers, 30,31 DNAzymes [32][33][34] and restriction enzyme sites. [35][36][37] Accordingly, functions such as specific recognition, cleavage, and catalysis can be flexibly integrated into RCPs.…”