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PLoS One. 2011;6(9):e24910. doi: 10.1371/journal.pone.0024910. Epub 2011 Sep 15.

Emulsion PCR: a high efficient way of PCR amplification of random DNA libraries in aptamer selection.

Author information

1
Clinical Laboratory, Yancheng City NO.1 People's Hospital, Yancheng, China.

Abstract

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.

PMID:
21949784
PMCID:
PMC3174225
DOI:
10.1371/journal.pone.0024910
[Indexed for MEDLINE]
Free PMC Article

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