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, 6 (9), e24910

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

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

Keke Shao et al. PLoS One.

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Simulation diagrams of emulsion PCR amplification of a random DNA library.
Different compartment contained different templates marked with different colors, and some compartment are empty.
Figure 2
Figure 2. Conventional PCR amplification of a DNA random library versus emulsion PCR.
PAGE electropherograms of PCR reaction mixtures for amplification of a random DNA library with conventional PCR (A) and emulsion PCR (C). Microfluidic chip electrophoresis electropherograms of PCR reaction mixtures for amplification of a random DNA library with conventional PCR (B) and emulsion PCR (D). Dynamics of product and by-product concentrations in conventional PCR and emusion PCR (E). Conventional PCR amplification was performed with cycles ranging from 10 to 32; the template concentration was 0.01 pmol/ml; the primer concentration was 0.4 µmol/L; and the concentration of Taq DNA polymerase was 0.05 U/µl. Emulsion PCR amplification was performed with cycles ranging from 10 to 40; the template concentration was 0.01 pmol/ml; the primer concentration was 0.4 µmol/L; and the concentration of Taq DNA polymerase was 0.125 U/µl.
Figure 3
Figure 3. Influence of the number of template molecules.
Panel A indicates the PAGE electropherograms of PCR reaction mixtures for amplification of a random DNA library with different amounts of template molecules. Panel B indicates the concentration of dsDNA products and by-products obtained from microfluidic chip electrophoresis. Emulsion PCR amplification was carried out for 35 cycles; the annealing temperature was 65°C; the primer concentration was 0.4 µmol/L; and the concentration of Taq DNA polymerase was 0.125 U/µl.
Figure 4
Figure 4. Influence of the annealing temperature.
The concentration of the dsDNA products and by-products was obtained from microfluidic chip electrophoresis. Emulsion PCR amplification was carried out for 35 cycles; the number of template was 0.01 pmol/ml; the primer concentration was 0.4 µmol/L; and the concentration of Taq DNA polymerase was 0.125 U/µl.
Figure 5
Figure 5. Influence of DNA polymerase concentrations.
Emulsion PCR amplification was carried out for 35 cycles; the number of templates was 0.01 pmol/ml; the primer concentration was 0.4 µmol/L; and the annealing temperature was 65°C.
Figure 6
Figure 6. Influence of primer concentrations.
Emulsion PCR amplification was carried out for 35 cycles; the number of templates was 0.01 pmol/ml; the concentration of Taq DNA polymerase was 0.125 U/µl; and the annealing temperature was 65°C.
Figure 7
Figure 7. By-product forming.
Conventional PCR was carried out for 1 and 30 cycles with 5 µg/ml template. 1 and 6: PCR mixture only include template. 2 and 7: PCR mixture include all except primer. 3 and 8: PCR mixture include all except dNTP. 4 and 9: PCR mixture include all except DNA polymerase. 5 and 10: PCR mixture include all the masteries.

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