A novel pathogen detection system based on high‐resolution CE‐SSCP using a triblock copolymer matrix

Abstract

Although CE-SSCP analysis combined with 16S ribosomal RNA gene-specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE-SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE-SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE-SSCP analysis. A poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE-SSCP analysis using the PEO-PPO-PEO triblock copolymer as the polymer matrix and four same-sized DNA fragments of similar sequence content. Among 48 commercially available PEO-PPO-PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO(137)PPO(43)PEO(137) exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE-SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO-PPO-PEO triblock copolymer may serve as an ideal matrix for high-resolution CE-SSCP analysis.

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Consequently, CE-SSCP analysis using conventional polymer matrices is not ideal. To address this problem, a high resolution CE-SSCP system using a polyethyleneoxidepolypropyleneoxide-polyethyleneoxide (PEO-PPO-PEO) triblock copolymer matrix was recently developed to effectively separate highly similar PCR products [62][63][64]. This PEO-PPO-PEO matrix displayed four-fold higher resolving power than linear polyacrylamide and commercial GeneScan gel for the separation of same-sized DNA molecules.…”

Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F‐108 tri‐block copolymer and capillary electrophoresis

“…Capillary electrophoresis‐single‐strand conformation polymorphism (CE‐SSCP) allows for the separation of DNA fragments of the same fragment length based on their intrinsic DNA sequence differences as they migrate through a native, non‐denaturing polymer . Recently, F‐108 triblock copolymer, poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide), has been used as a sieving matrix for CE‐SSCP . The increase in resolution is due to the PPO and PEO interactions that result in a high‐density sieving matrix .…”

“…Recently, F‐108 triblock copolymer, poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide), has been used as a sieving matrix for CE‐SSCP . The increase in resolution is due to the PPO and PEO interactions that result in a high‐density sieving matrix . The advantage of the F‐108 polymer when coupled with CE‐SSCP analysis is that it allows for the separation of longer fragments (200–450 bp) and can distinguish the intrinsic sequence polymorphisms in most cases .…”

F‐108 polymer and capillary electrophoresis easily resolves complex environmental DNA mixtures and SNPs

“…Previous study by Shin et al. 2010 discovered that the PEO chain length increased the resolution due to the hydrophilic mesh that interacts with DNA and, together, it results in a high‐density sieving matrix .…”