Single-stranded DNA (ssDNA) production in DNA aptamer generation

Abstract

The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.

Aptamer-Based Technology for Food Analysis

“…Different applications of aptamer-based assays have been already described for diagnostics, environmental or food quality testing, with analytical performances rivaling those of antibody-based assays. The analytical studies are now demonstrating that some of the limitations of many conventional assays can be circumvented by alternative recognition reagent such as aptamers [2]. Although the application of aptamers to poisonous and harmful materials in food is now in the initial stage, existing studies and findings provide theoretical basis and technical support for the actual application of aptamer technology (represented by the aptamer-based technology in Table 2) in the detection of food.…”

“…The aptamer technology is still under evolution and investigation due to its complexity and stability [67]. The utilization of the DNA or RNA aptamer in any applications requires the aptamer to be stable and resistant to the enzymatic activities of nucleases [2]. Aptamers are more stable at high temperature, and they can be regenerated easily and quickly after denaturation, and aptamers are in general more stable than antibodies, so they have a longer shelf life than antibodies.…”

“…Aptamers are more stable at high temperature, and they can be regenerated easily and quickly after denaturation, and aptamers are in general more stable than antibodies, so they have a longer shelf life than antibodies. RNA molecules have the drawback of susceptibility to enzymatic degradation, so that the DNA aptamer is advantageous over the RNA aptamer due to its well stability with the absence of the hydroxyl group at the 2′-end of the DNA molecule, making it widely used in a number of applications [2]. It is noted that the lackness of RNA stability can be overcome by modifying the 2′ position of the ribose ring in the RNA backbone with amino, fluoro, or O-methyl functional groups used in the SELEX process [128].…”

“…Aptamers are functional nucleic acids binding to specific target molecules, including DNA or RNA aptamer [2]. The aptamer selection technology is called systematic evolution of ligands by exponential enrichment (SELEX) (Fig.…”

An electrochemical biosensor for double-stranded Wnt7B gene detection based on enzymatic isothermal amplification

“…Moreover, the content of target in real samples is low. To solve this difficulty, some detection methods based on polymerase chain reaction (PCR) derived techniques are developed, such as asymmetric PCR (Campuzano et al, 2011;Chen et al, 2013), magnetic separation (Stevenson et al, 2008;Ma et al, 2012), size separation on denaturing urea polyacrylamide gel electrophoresis (Marimuthu et al, 2012;Liang et al, 2015) and toehold-mediated DNA strand displacement (Khodakov et al, 2013). Although these methods can generate ssDNA or singlestranded toehold, there are also different weaknesses to limit their use in bioanalysis.…”