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, Mg2+/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 biotin-streptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700 ± 11.3 and 820 ± 19.2 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.
Subject(s)
Aptamers, Nucleotide/genetics , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/methods , Biotin , DNA, Single-Stranded , Gene Library , SELEX Aptamer Technique/methods , StreptavidinABSTRACT
Mosquito-borne diseases are a major threat to public health. The shortcomings of diagnostic tools, especially those that are antibody-based, have been blamed in part for the rising annual morbidity and mortality caused by these diseases. Antibodies harbor a number of disadvantages that can be clearly addressed by aptamers as the more promising molecular recognition elements. Aptamers are defined as single-stranded DNA or RNA oligonucleotides generated by SELEX that exhibit high binding affinity and specificity against a wide variety of target molecules based on their unique structural conformations. A number of aptamers were developed against mosquito-borne pathogens such as Dengue virus, Zika virus, Chikungunya virus, Plasmodium parasite, Francisella tularensis, Japanese encephalitis virus, Venezuelan equine encephalitis virus, Rift Valley fever virus and Yellow fever virus. Intrigued by these achievements, we carry out a comprehensive overview of the aptamers developed against these mosquito-borne infectious agents. Characteristics of the aptamers and their roles in diagnostic, therapeutic as well as other applications are emphasized.
