A novel pathogen detection system based on high-resolution CE-SSCP using a triblock copolymer matrix.
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.
Shin
Gi Won
GW
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Republic of Korea.
Hwang
Hee Sung
HS
Seo
Sang Woo
SW
Oh
Mi-Hwa
MH
Ryu
Chang Y
CY
Salvo
Charles J
CJ
Feldman
Shaina
S
Doh
Junsang
J
Jung
Gyoo Yeol
GY
D001424:Bacterial Infections / Q000175:diagnosis; D004247:DNA / Q000737:chemistry; D004269:DNA, Bacterial / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D023281:Genomics; D008823:Micelles; D015233:Models, Statistical; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry*; D018807:Polymorphism, Single-Stranded Conformational; D012336:RNA, Ribosomal, 16S / Q000378:metabolism
D004269:DNA, Bacterial; D008823:Micelles; D011108:Polymers; D012336:RNA, Ribosomal, 16S; D004247:DNA