However, during the PCR amplification stage, by-products formation inhibits the product generation and consequently limits the application of conventional PCR in apatamer selection. Actually, efficient separation and amplification are both necessary for productive SELEX process. However, only few reported PCR amplification application in this area. Many studies showed that flow cytometry, capillary electrophoresis and other techniques are efficient methods for separation in apatamer selection –. For each round, it includes two stages: (1) separation of oligonucleotide-target complexes with free targets and non-bound oligonucleotides and (2) amplification of the bound sequences by polymerase chain reaction (PCR). The main strategy for obtaining aptamers is designated as SELEX (systematic evolution of ligands by exponential enrichment) which consists of several rounds of sequence selection that bind to specific target molecules. Aptamers can be obtained in vitro by directed selection from libraries of random DNA sequences. In particular, aptamers are very attractive as a replacement for antibodies in diagnostics and treatment of diseases. These oligonucleotides fragments share most properties with monoclonal antibodies and have been showed that they successfully replaced antibodies in ELISA and Western blot, even smaller, more stable, and easier to be chemically synthesized compared with antibodies –. For instance, aptamers often bind to the functional parts of proteins and inhibit their activity. In addition, targets characteristics will be affected after binding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.Īptamers are single-stranded DNA or RNA oligonucleotides capable of binding to other target molecules with high specificity, affinity and stability –. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This work was supported by a special Research Grant(XK 2007 23) for the key laboratory from the Department of Health, Jiangsu Province. Received: Accepted: AugPublished: September 15, 2011Ĭopyright: © 2011 Shao et al. PLoS ONE 6(9):Įditor: Reiner Albert Veitia, Institut Jacques Monod, France So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.Ĭitation: Shao K, Ding W, Wang F, Li H, Ma D, Wang H (2011) Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Here, we developed emulsion PCR for aptamer selection. Low efficiency is one of the limitations for conventional PCR amplification of random DNA sequence library in aptamer selection because of relative low products and high by-products formation efficiency. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Typically, they are selected from a large number of random DNA sequence libraries. Aptamers are short RNA or DNA oligonucleotides which can bind with different targets.
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