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Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection

Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Low efficiency is one of...

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Autores principales: Shao, Keke, Ding, Weifeng, Wang, Feng, Li, Haiquan, Ma, Da, Wang, Huimin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174225/
https://www.ncbi.nlm.nih.gov/pubmed/21949784
http://dx.doi.org/10.1371/journal.pone.0024910
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author Shao, Keke
Ding, Weifeng
Wang, Feng
Li, Haiquan
Ma, Da
Wang, Huimin
author_facet Shao, Keke
Ding, Weifeng
Wang, Feng
Li, Haiquan
Ma, Da
Wang, Huimin
author_sort Shao, Keke
collection PubMed
description Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). 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. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.
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spelling pubmed-31742252011-09-26 Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection Shao, Keke Ding, Weifeng Wang, Feng Li, Haiquan Ma, Da Wang, Huimin PLoS One Research Article Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). 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. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX. Public Library of Science 2011-09-15 /pmc/articles/PMC3174225/ /pubmed/21949784 http://dx.doi.org/10.1371/journal.pone.0024910 Text en Shao et al. http://creativecommons.org/licenses/by/4.0/ 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 properly credited.
spellingShingle Research Article
Shao, Keke
Ding, Weifeng
Wang, Feng
Li, Haiquan
Ma, Da
Wang, Huimin
Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection
title Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection
title_full Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection
title_fullStr Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection
title_full_unstemmed Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection
title_short Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection
title_sort emulsion pcr: a high efficient way of pcr amplification of random dna libraries in aptamer selection
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174225/
https://www.ncbi.nlm.nih.gov/pubmed/21949784
http://dx.doi.org/10.1371/journal.pone.0024910
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