Comparison of different methods for generation of single-stranded DNA for SELEX processes

Abstract

Single-stranded DNA (ssDNA) generation is a crucial step in several molecular biology applications, such as sequencing or DNA chip and microarray technology. Molecules of ssDNA also play a key role in the selection of ssDNA aptamers through Systematic Evolution of Ligands by EXponential enrichment (SELEX). With particular interest for this application, herein we present a comparative study of the most used methods for generation of ssDNA used in SELEX, such as asymmetric PCR, enzyme digestion and magnetic separation with streptavidin beads. In addition, we evaluate a new technique that combines asymmetric PCR and enzyme digestion with the aim to achieve the maximum efficiency in ssDNA generation. The methods studied were compared in terms of quality of ssDNA using electrophoretic analysis and generated ssDNA yields were quantitatively measured using an Enzyme-Linked OligoNucleotide Assay (ELONA).

Imidazole-free purification of His 3 -tagged recombinant proteins using ssDNA aptamer-based affinity chromatography

“…Finally, the reamplified PCR product (5 × 50 L) was precipitated using isopropanol. To prepare the ssDNA for the next selection round, the PCR product was digested using exonuclease Lambda (Thermo Scientific) [27,28] according to the protocol supplied by the producer. The ssDNA was purified using a standard phenol/chloroform extraction procedure followed by isopropanol precipitation.…”

Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs

“…Asymmetric PCR (A-PCR) was consid-63 ered as a more favorable way for preparing ssDNAs in oligonucleotide 64 microarrays with its simplicity and low cost [10]. Svobodová and 65 coworkers also pointed out that A-PCR followed by enzyme digestion 66 produced the highest amount of ssDNAs with low cost in comparison 67 with other methodologies [11]. During the process of A-PCR, one 68 strand of the template DNA is amplified more than the other by using 69 an unequal molar ratio of forward and reverse primers; when the pri-70 mer presents at a markedly lower concentration (termed as limited 71 primer, with another termed as unlimited primer) and becomes 72 exhausted, ssDNA is produced in each PCR cycle [13].…”

“…However, this PCR-like process involved 100 60 cycles of reactions and, thus, was inefficient and time-consuming. 61 Various procedures for generating ssDNAs have been evaluated in 62 different applications [10][11][12]. Asymmetric PCR (A-PCR) was consid-63 ered as a more favorable way for preparing ssDNAs in oligonucleotide 64 microarrays with its simplicity and low cost [10].…”

“…150 Although PS has even higher proofreading activity, the mixture 151 and thermal conditions need to be further optimized for its higher 152 performance in A-PCR. 153 In comparison with other efficient strategies for ssDNA genera-154 tion such as A-PCR followed by enzyme digestion [11], lambda exo-155 nuclease, T7 gene 6 exonuclease, and streptavidin-coated magnetic 156 beads with alkaline denaturation [12], all of which were started 157 from a preamplified dsDNA product, in this study the ssDNAs were 158 directly amplified from the storage plasmid DNA via a one-step A-159 PCR procedure. However, the purification step based on the extrac- equally able to form homoduplex of S1F/S1R and heteroduplex of 174 S1F/S2R, whereas TE partially annealed the above strands.…”

An in Vitro Technique to Identify the RNA Binding-Site Sequences for RNA-Binding Proteins

“…The amplified DNA is then cloned, and its nucleotide sequence is analyzed. However, the size of the library is limited, it is difficult to determine the optimal conditions for selection, and repeating multiple cycles is time-consuming (8,9). …”