title: Single-primer-limited amplification: a method to generate random single-stranded DNA sub-library for aptamer selection.
abstract: The amplification of a random single-stranded DNA (ssDNA) library by polymerase chain reaction (PCR) is a key step in each round of aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX), but it can be impeded by the amplification of by-products due to the severely nonspecific hybridizations among various sequences in the PCR system. To amplify a random ssDNA library free from by-products, we developed a novel method termed single-primer-limited amplification (SPLA), which was initiated from the amplification of minus-stranded DNA (msDNA) of an ssDNA library with reverse primer limited to 5-fold molar quantity of the template, followed by the amplification of plus-stranded DNA (psDNA) of the msDNA with forward primer limited to 10-fold molar quantity of the template and recovery of psDNA by gel excision. We found that the amount of by-products increased with the increase of template amount and thermal cycle number. With the optimized template amount and thermal cycle, SPLA could amplify target ssDNA without detectable by-products and nonspecific products and could produce psDNA 16.1 times as much as that by asymmetric PCR. In conclusion, SPLA is a simple and feasible method to efficiently generate a random ssDNA sub-library for aptamer selection.
authors.affiliation: Department of Gastroenterology, First Affiliated Hospital of Nanchang University, Jiangxi Institute of Gastroenterology and Hepatology, Nanchang 330006, China.
authors.forename: Chao-Zhu
authors.initials: CZ
authors.lastname: He
authors.forename: Kun-He
authors.initials: KH
authors.lastname: Zhang
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.forename: Qin-Si
authors.initials: QS
authors.lastname: Wan
authors.forename: Piao-Ping
authors.initials: PP
authors.lastname: Hu
authors.forename: Mei-Di
authors.initials: MD
authors.lastname: Hu
authors.forename: De-Qiang
authors.initials: DQ
authors.lastname: Huang
authors.forename: Nong-Hua
authors.initials: NH
authors.lastname: Lv
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D052156:SELEX Aptamer Technique / Q000379:methods*
chemical_list: D052157:Aptamers, Nucleotide; D017931:DNA Primers; D004277:DNA, Single-Stranded
keywords: Aptamer; Methodology; Random ssDNA library; Single-primer-limited amplification; ssDNA generation