title: 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.
authors.affiliation: School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.|Clinical Laboratory, YanCheng No. 1 People's Hospital, YanCheng, Jiangsu, People's Republic of China.
authors.forename: Keke
authors.initials: K
authors.lastname: Shao
authors.affiliation: Clinical Laboratory, YanCheng No. 1 People's Hospital, YanCheng, Jiangsu, People's Republic of China.
authors.forename: Xinhui
authors.initials: X
authors.lastname: Shi
authors.affiliation: Clinical Laboratory, YanCheng No. 1 People's Hospital, YanCheng, Jiangsu, People's Republic of China.
authors.forename: Xiangjun
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Clinical Laboratory, YanCheng No. 1 People's Hospital, YanCheng, Jiangsu, People's Republic of China.
authors.forename: Leilei
authors.initials: L
authors.lastname: Cui
authors.affiliation: School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Qixiang
authors.initials: Q
authors.lastname: Shao
authors.affiliation: Clinical Laboratory, YanCheng No. 1 People's Hospital, YanCheng, Jiangsu, People's Republic of China.
authors.forename: Da
authors.initials: D
authors.lastname: Ma
mesh_terms: D001710:Biotin / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000235:genetics*; D004655:Emulsions / Q000737:chemistry; D015723:Gene Library; D008024:Ligands; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D052156:SELEX Aptamer Technique; D019809:Streptavidin / Q000737:chemistry
chemical_list: D004277:DNA, Single-Stranded; D004655:Emulsions; D008024:Ligands; D001710:Biotin; D019809:Streptavidin
keywords: SELEX; aptamer; asymmetric emulsion PCR; random ssDNA library