Gi Won Shin

Copy number variations associated with idiopathic autism identified by whole-genome microarray-based comparative genomic hybridization.

Objectives Autism spectrum disorder (ASD) has been thought to have strong genetic background, but major contributing genes or associated molecular–genetic pathways are yet to be identified. To explore the idiopathic ASD-associated copy number variations (CNVs), we conducted case–control study using whole-genome copy number analysis. Methods Whole-genome microarray-based comparative genomic hybridization was carried out on 28 children (24 boys and four girls) diagnosed as ASD and 62 Korean adults (45 males and 17 females) without any signs of abnormalities and family history of genetic disorders as normal controls. Fluorescence in-situ hybridization and capillary electrophoresis-single-strand conformational polymorphism were used for quantitative verification of the ASD-associated CNVs. Results Thirty-eight CNVs were identified. Among them, the distributions of copy number loss CNVs on 8p23.1 (odds ratio: 5.1, 95% confidence interval: 1.7–14.5, P=0.003) and on 17p11.2 (odds ratio: uncalculable because of zero cell, P=0.008) were found to be significantly different between ASD and control groups. DEFENSIN family occurs in a cluster at 8p23.1 region. Fluorescence in-situ hybridization and capillary electrophoresis-single-strand conformational polymorphism coherently showed reduced copy number of DEFENSIN in cases with 8p23.1 copy number loss CNV, which validated microarray-based comparative genomic hybridization results; but there are no known coding genes in the CNV on 17p11.2. Conclusion Our approach as well as results can help to elucidate the genetic mechanism of idiopathic ASD.

Simultaneous identification of seven foodborne pathogens and Escherichia coli (pathogenic and nonpathogenic) using capillary electrophoresis-based single-strand conformation polymorphism coupled with multiplex PCR.

The objective of this study was to develop a novel technique for parallel analysis of eight important foodborne microbes using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex PCR. Specific primers for multiplex PCR amplification of the 16S rRNA gene were designed, corresponding to eight species of bacteria, including Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus cereus, for the species-specific identification and optimal separation of their PCR products in subsequent analysis by CE-SSCP. Multiplex PCR conditions including annealing temperature, extension time, the number of PCR cycles, and primer concentrations were then optimized for simultaneous detection of all target foodborne bacteria. The diagnostic system using CE-SSCP combined with multiplex PCR developed here can be used for rapid investigation of causative agents of foodborne illness. The simplicity and high sensitivity of the method may lead to improved management of safety and illness related to food.

A two-step quantitative pathogen detection system based on capillary electrophoresis.

Rapid identification of bacterial pathogens is important for patient management and initiation of appropriate antibiotic therapy in the early stages of infection. Among the several techniques, capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis combined with small subunit rRNA gene-specific polymerase chain reaction (PCR) has come into the spotlight owing to its sensitivity, resolution, and reproducibility. Despite the advantages of the method, the design of PCR primers and optimization of multiplex PCR conditions remain to be studied so that as many pathogens as possible can be analyzed in a single run. Here we describe a novel two-step technique involving multiplex PCR pathogen detection by CE-SSCP analysis followed by singleplex PCR pathogen quantification by CE-SSCP. Specific PCR primers were designed for optimal separation of their products by CE-SSCP based on molecular weight. PCR conditions were then optimized for multiplex analysis of the targets. Subsequently, detected pathogens were quantified by PCR with specific primers. Eight clinically important strains were simultaneously identified under the optimized conditions. Each individual pathogen was then quantified at a level of sensitivity of tens of cells per milliliter. In conclusion, the two-step pathogen detection method based on CE-SSCP described here allows for sensitive detection of pathogens by multiplex PCR (first step) and quantification by specific PCR (second step). The results illustrate the potential of the method in clinical applications.

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.

Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP

Several methods based on screening for a 16S ribosomal RNA gene marker have been developed for rapid and sensitive detection of pathogenic microorganisms. One such method, CE‐based SSCP (CE‐SSCP), has enormous potential because the technique can separate sequence variants using a simple procedure. However, conventional CE‐SSCP systems have limited resolution and cannot separate most 16S ribosomal RNA gene‐specific markers unless combined with additional modification steps. A high‐resolution CE‐SSCP system that uses a poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this study, we developed a method based on a high‐resolution CE‐SSCP system using a poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) triblock copolymer that is capable of simultaneous and quantitative detection of 12 clinically important pathogens. Pathogen markers were amplified by PCR using universal primers and separated by CE‐SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing easy identification of pathogens. A series of experiments using different amounts of genomic pathogen DNA showed that the method had a limit of detection of 0.31–1.56 pg and a dynamic range of approximately 102. These results indicate that high‐resolution CE‐SSCP systems have considerable potential in the clinical diagnosis of bacteria‐induced diseases.

Multiplex quantitative foodborne pathogen detection using high resolution CE-SSCP coupled stuffer-free multiplex ligation-dependent probe amplification.

Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen‐specific genes are optimal markers. Although multiplex ligation‐dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length‐dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer‐free MLPA using high‐resolution CE‐SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE‐SSCP analysis was designed for species‐specific detection. After careful optimization of each MLPA step, to ensure that CE‐SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5–5 pg of genomic DNA, and more than 100‐fold increase could be quantitatively determined. Thus, MLPA‐CE‐SSCP is a sensitive and reliable technique for pathogen detection.

Recent developments in CE-based detection methods for food-borne pathogens

Rapid and sensitive detection of food‐borne pathogens is critical for food safety from the viewpoint of both the public health professionals and the food industry. Conventional method is, however, known to be labor‐intensive, time‐consuming, and expensive due to the separate cultivation and biochemical assay. Many relevant technologies, such as flow cytometry, MALDI‐MS, ESI‐MS, DNA microarray, and CE, have been intensively developed to date. Among them, CE is considered to be the most efficient and reproducible because of low sample loss and simple automation. CE‐based pathogen detection methods can be classified into three categories based on the separation targets: cell separation, nucleic‐acid‐based identification, and protein separation coupled with characterization. In this review, recent developments in each sphere of CE‐based technology are discussed. Additionally, the critical features of each approach and necessary future technical improvements are also reviewed.

A new single-step quantitative pathogen detection system: template-tagging followed by multiplex asymmetric PCR using common primers and CE-SSCP.

Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria‐induced disease. Among the existing techniques for identifying microbial, CE‐SSCP combined with 16S ribosomal RNA gene‐specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE‐SSCP can separate PCR products with high‐resolution, multiplex detection and quantification are complicated by primer‐dimer formation and non‐specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template‐tagging followed by multiplex asymmetric PCR and subsequent CE‐SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia‐inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE‐SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.

Stuffer‐free multiplex ligation‐dependent probe amplification based on conformation‐sensitive capillary electrophoresis: A novel technology for robust multiplex determination of copy number variation

Developing diagnostic tools based on the application of known disease/phenotype‐associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user‐friendly multiplex CNV detection method, termed stuffer‐free MLPA‐CE‐SSCP, that combines a variation of multiplex ligation‐dependent probe amplification (MLPA) with CE‐SSCP. In this variation, MLPA probes were designed without the conventionally required stuffer sequences. To separate the similar‐sized stuffer‐free MLPA products, we adopted CE‐SSCP rather than length‐dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X‐chromosome copy number (1–5) revealed that copy number determinations using stuffer‐free MLPA‐CE‐SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation.

Triblock copolymer matrix-based capillary electrophoretic microdevice for high-resolution multiplex pathogen detection

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE‐SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer as a sieving matrix for CE‐SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO‐PPO‐PEO copolymers, 255‐bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO‐PPO‐PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan™ gel. Due to enhanced dynamic coating and sieving ability, PEO‐PPO‐PEO copolymer displayed fourfold enhancement of resolving power in the CE‐SSCP to separate same‐sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high‐resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO‐PPO‐PEO triblock copolymer an excellent matrix in the CE‐SSCP analysis on the microdevice.