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

Abstract

Construction of a random ssDNA sublibrary is an important step of the aptamer screening process. The available construction methods include asymmetric PCR, biotin–streptavidin separation, and lambda exonuclease digestions, in which PCR amplification is a key step. The main drawback of PCR amplification is overamplification increasing nonspecific hybridization among different products and by‐products, which may cause the loss of potential high‐quality aptamers, inefficient screening, and even screening failure. Cycle number optimization in PCR amplification is the main way to avoid overamplification but does not fundamentally eliminate the nonspecific hybridization, and the decreased cycle number may lead to insufficient product amounts. Here, we developed a new method, “asymmetric emulsion PCR,” which could overcome the shortcomings of conventional PCR. In asymmetric emulsion PCR, different templates were separated by emulsion particles, allowing single‐molecule PCR, in which each template was separately amplified, and the nonspecific hybridization was avoided. Overamplification or formation of by‐products was not observed. The method is so simple that direct amplification of 40 or more cycles can provide a high‐quality ssDNA library. Therefore, the asymmetric emulsion PCR would improve the screening efficiency of systematic evolution of ligands by exponential enrichment.

On-Flow Synthesis of Co-Polymerizable Oligo-Microspheres and Application in ssDNA Amplification

“…Functionally active nucleic acids, including aptamers [ 1 ] and single-strand oligonucleotide ligands (single stranded DNA, ssDNA), have been extensively used as target recognition elements due to their intrinsic properties. To especially carry out the ssDNA analysis, PCR amplification should be followed by the conversion of dsDNA to ssDNA using the extra steps such as asymmetric PCR [ 2 ], biotin-streptavidin separation using magnetic beads [ 3 ], enzymatic degradation, and denaturation of dsDNA by heating or by alkaline treatment, etc. [ 4 – 7 ].…”

Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

“…The main disadvantage of conventional PCR amplification during unequal length primer PCR is that overamplification increases nonspecific hybridization among different products and by-products, which may cause the loss of potential specific aptamers, inefficient selection, and even selection failure. It was reported [ 35 ] that emulsion PCR could overcome the shortcomings of conventional PCR. During emulsion PCR, different templates are separated by emulsion particles, allowing single-molecule PCR, and avoiding nonspecific hybridization.…”

A Comparison of Methods for the Production of Kilobase-Length Single-Stranded DNA

“…Performance of aPCR may be further enhanced by a method described by Tolnai et al wherein 3 phosphorylated primers ("blockers") are added to standard aPCR mixtures to minimize byproduct formation and thereby increase production yield [33]. Finally, performing aPCR in an emulsion state (a mixture of the aqueous reaction and oil) isolates DNA molecules from each other, mimicking single-molecule aPCR, and thereby reducing the formation of byproducts and increasing yield [44]. Finally, the dsDNA generated during the initial amplification cycles of aPCR can be used as a template for further ssDNA generation by aPCR, strand-specific exonuclease digestion, or chemical denaturation.…”