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

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.

Construction and optimization of an efficient amplification method of a random ssDNA library by asymmetric emulsion PCR

“…Then, the dsDNA library was converted into an ssDNA library 16. During the PCR amplification, the product yield was low, and the formation of by‐products could not be avoided 10. Musheev and Krylov 9 reduced the quantity of by‐products by lowering the number of cycles and optimizing the enzyme concentration.…”

“…The primers were prone to react with ssDNA, and their higher concentration enhanced the formation of nonspecific hybridization products, as described earlier 10. Therefore, we first optimized the concentration of upstream primer.…”

“…The random ssDNA library was constructed as 90‐mer oligonucleotides, which were composed of two 19‐bp fixed sequences on both sides and the 52‐bp central random sequence: 5′‐GAACATTGGCGTCCGTGAG‐N 52 ‐ACTTCCTCAAACGCCCAA‐3′ 10. The upstream primer was 5′‐GAACATTGGCGTCCGTGAG‐3′ and the downstream primer was 5′‐TTGGGCGTTTGAGGAAGTG‐3′.…”

“…Therefore, aptamers have an application potential as substitutes of antibodies in clinical diagnosis and disease treatment 1, 2, 3, 4, 5, 6, 7, 8. However, in fundamental research and clinical diagnosis, relevant applications are still dominated by monoclonal antibodies, and aptamer diagnostics is in the experimental stage, because the aptamer screening efficiency of the classical systematic evolution of ligands by exponential enrichment (SELEX) technology is relatively low 9, 10, and high‐quality aptamers are not easily available.…”

DNA marker techniques in study and selection of plant

“…PCR nhũ tương là phản ứng PCR sử dụng hỗn hợp phản ứng PCR thông thường và hỗn hợp phản ứng PCR dạng nhũ tương nước trong dầu [158]. Hệ thống Roche 454 đầu tiên chỉ đọc được đoạn 100 bp, hiện nay do cải tiến có thể đọc đoạn đến 800 bp.…”