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 PCRinitiallyfollowed by the optimization of asymmetric PCR. In the asymmetric PCR,optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg2+/dNTP concentrations and the amounts of Taq Polymerasewascarried out. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method, either via lambda exonuclease or biotin-streptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700 ± 11.3 and 820 ± 19.2nM 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.
Highlights
We have integrated an additional single-stranded DNA generation method via lambda exonuclease and biotin-streptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We propose this optimization pipeline as the first step that can be carried out before initiating any Systemic Evolution of Ligands via Exponential Enrichment (SELEX) experiments to augment the yield of ssDNA produced in each SELEX cycle
When the Polymerase Chain Reaction (PCR) cycle was further increased to 6 PCR cycles, there was no significant difference in the double-stranded DNA (dsDNA) band intensity between that of cycle 5 and 6
5 PCR cycles was selected as the optimum number of PCR cycles due to the much higher dsDNA band intensity than that of 4 PCR cycles (P < 0.0001)
Summary
Having the ability to bind to their targets with high affinity and specificity like antibodies, aptamers are dubbed as chemical antibodies. Aptamers fold into a myriad of three-dimensional (3D) structures, which are strengthened by hydrogen-bonding, van der Waals forces and electrostatic charges to forge interaction with their cognate targets for the formation of aptamer-target complex (Hermann and Patel 2000; Nomura, et al 2010; Piganeau and Schroeder 2003). Since their discovery in the early 1990 (Tuerk and Gold 1990; Ellington and Szostak 1990), aptamers have gradually emerged as a promising class of molecular recognition element (MRE) on par with antibodies due to the following features such as lower cost of synthesis, ability to undergo reversible denaturation and renaturation without the loss of binding capability and low-to-no immunogenicity. SELEX is made up of four major steps, which are incubation of the randomized single-stranded DNA/ RNA (ssDNA or RNA) library with the target, partitioning and recovery of the target-bound nucleic acids, amplification of the target-bound nucleic acid molecules via Polymerase Chain Reaction (PCR) for DNA SELEX or Reverse-Transcription Polymerase Chain Reaction (RT-PCR) for RNA SELEX and lastly the regeneration of ssDNA/ RNA for the subsequent round of selection process
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