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Cell selex emulsion pcr11/19/2023 Second, they are relatively small in terms of their molecular weight (10–30 kDa), which allows them to reach epitopes that antibodies (~ 150 kDa) cannot target due to steric hindrance 1. Due to chemical synthesis, they can be easily modified with chemical groups and fluorescence dyes in a targeted manner for immobilization and signalling purposes. First, aptamers can be readily synthesized in large quantities at relatively low costs and with low batch-to-batch variations. In contrast to their antibody-counterparts, aptamers offer many inherent advantages, which make them appealing alternatives in diverse analytical applications. Furthermore, the applied monitoring and assessment techniques provide insight into the selection process and can be highly useful to study and improve experimental cell-SELEX designs to increase selection efficiency.ĭNA aptamers are short synthetic oligonucleotides (20–100 nucleotides) that can bind to a molecular target by their unique three-dimensional structure with high affinity and specificity. Our results demonstrate that this combined approach enabled the rapid and efficient identification of an aptamer with both high affinity and high specificity. faecalis from 20 different Enterococcus and non- Enterococcus spp. faecalis cells (K D-value: 37 nM) and successfully discriminated E. Among these, aptamer EF508 exhibited high binding affinity to E. Based on NGS-derived data, we identified 16 aptamer candidates. During the selection, we applied qPCR-based analyses to evaluate the ssDNA pool size and remelting curve analysis of qPCR amplicons to monitor changes in pool diversity and sequence enrichment. faecalis), which served as target in eleven rounds of cell-SELEX with multiple subtractive counter-selections against non-target species. This approach is demonstrated on Enterococcus faecalis ( E. Here we describe the selection and identification of DNA aptamers for bacterial cells using a combined approach based on cell-SELEX, state-of-the-art applications of quantitative real-time PCR (qPCR), next-generation sequencing (NGS) and bioinformatic data analysis. DNA aptamers generated by cell-SELEX against bacterial cells have gained increased interest as novel and cost-effective affinity reagents for cell labelling, imaging and biosensing.
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