Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Abstract

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.

Essential strategies to optimize asymmetric PCR conditions as a reliable method to generate large amount of ssDNA aptamers

“…This study aimed to investigate the optimum conditions to achieve the highest yield of highly pure ssDNA using asymmetric PCR. Carrying out of various studies to optimize asymmetric PCR suggests that this process is somewhat difficult, and also it is not possible to consider a single protocol for all random DNA libraries . The first step to get success in asymmetric PCR is the quality of designed library.…”

“…It is referred to the high purity of ssDNA template used at the first round. It seems that dsDNA remaining in the templates of round 2 and others can play a role similar to reverse primer and prepare the conditions for maximum activity of forward primer . While the primary library used in the first round is single stranded and lacks any complementary sequence, so reverse primers should make the template for PCR procedure at early cycles.…”

Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies

“…aPCR is one of the most extensively used methods for the direct production of short ssDNA aptamers [ 90 , 93 , 94 , 95 , 96 ]. Due to its highly specific reaction conditions and several limitations, this method was originally limited to the production of ssDNA with ten to a few hundred bases [ 94 , 97 ].…”

Strategies for the preparation of non-amplified and amplified genomic dengue gene samples for electrochemical DNA biosensing applications

“…67,68 Modied RT-PCR amplication, known as asymmetric PCR, coupled with a DNA sensor has been extensively used due to its higher sensitivity, as it generates an excess amount of the ssDNA target for direct hybridization with an immobilized-DNA-probe-modied electrode. [69][70][71][72][73][74] The reverse-to-forward (R/F) concentration ratio is an important part of the asymmetric PCR protocol for creating excess ssDNA production. 75 This is because in asymmetric PCR, the primer with a lower concentration is involved in the production of dsDNA, whereas the primer with a higher concentration (which does not bind to any template) is responsible for the production of ssDNA.…”