Current Research and Scholarly Interests
Highly parallel analysis of complex genetic mixture
Precise expression profiling by stuffer-free multiplex ligation-dependent probe amplification.
2013; 85 (19): 9383-9389
In systems biological studies, precise expression profiling of functionally important gene sets is crucial. Real-time polymerase chain reaction is generally used for this purpose. Despite its widespread acceptance, however, this method is not suitable for multiplex analysis, resulting in an inefficient assay process. One alternative technology in the spotlight is multiplex ligation-dependent probe amplification (MLPA). But MLPA depends on length-based discrimination of amplified products, which complicates probe design and compromises analysis results. Here, we devised a variation of MLPA that utilizes conformation-sensitive capillary electrophoresis, and demonstrated the simplicity of the probe-design process and improved precision of the assay in analyses of 33 Escherichia coli metabolic genes and 16 Caenorhabditis elegans longevity-related genes. The results showed that relative expression could be quantitatively measured over a relevant dynamic range by using similar-sized probes. Importantly, the improved precision compared to conventional MLPA promises a wider application of this method for various biological systems.
View details for DOI 10.1021/ac402314h
View details for PubMedID 24000818
Effect of temperature gradients on single-strand conformation polymorphism analysis in a capillary electrophoresis system using Pluronic polymer matrix
ANALYTICA CHIMICA ACTA
2013; 793: 114-118
Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is a prominent bioseparation method based on the mobility diversity caused by sequence-induced conformational differences of single-stranded DNA. The use of Pluronic polymer matrix has opened up new opportunities for CE-SSCP, because it improved the resolution for various genetic analyses. However, there still exists a challenge in optimizing Pluronic-based CE-SSCP, because the physical properties of Pluronic solutions are sensitive to temperature, particularly near the gelation temperature, where the viscoelasticity of Pluronic F108 solutions sharply changes from that of a Newtonian fluid to a hydrogel upon heating. We have focused on a set of experiments to control the ambient temperature of the CE system with the aim of enhancing the reliability of the CE-SSCP analysis by using the Applied Biosystems ABI 3130xl genetic analyzer with Pluronic F108 solution matrix. The ambient temperature control allowed us to vary the inlet and outlet portion of the capillary column, while the temperature of the column was kept at 35°C. The resolution to separate 2 single-base-pair-differing DNA fragments was significantly enhanced by changing the temperature from 19 to 30°C. The viscoelastic properties of the F108 solution matrix upon heating were also investigated by ex situ rheological experiments with an effort to reveal how the development of gels in Pluronic solutions affects the resolution of CE-SSCP. We found that the column inlet and outlet temperatures of the capillary column have to be controlled to optimize the resolution in CE-SSCP by using the Pluronic matrix.
View details for DOI 10.1016/j.aca.2013.07.048
View details for Web of Science ID 000323855400015
View details for PubMedID 23953214
Multiplex ligase-based genotyping methods combined with CE.
In this genomic era, the ability to assay multiple genomic hot spots that have strong clinical implications is greatly desired. Conventional PCR-based methods suffer from frequent false-positive detections, particularly when a multiplex analysis is desirable. As an alternative to the error-prone conventional methods, multiplex ligase-based genotyping methods combined with CE have a strong potential. In this review, both previously developed methods and emerging methods are described to reveal the specificity, sensitivity, and simplicity of the ligase-based methods. For each step (ligation, amplification, and separation), the principles of several alternative methods are discussed along with their applications to explore the future development of ligase-based diagnostic methods.
View details for DOI 10.1002/elps.201300361
View details for PubMedID 24123070
A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP
2013; 138 (22): 6969-6976
Aberrant DNA methylation is a potential diagnostic marker for complex diseases, such as cancer. With the increase in the number of genes known to exhibit disease-associated aberrant methylation, the need for accurate multiplex assays for quantifying DNA methylation has increased. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is one method that has been highlighted in this context. However, two limitations make the custom design of MS-MLPA assays impractical: the need for long probes containing stuffer sequences and a reliance on only one restriction enzyme. Here, we developed a variation of MS-MLPA that employs a simpler probe-design process. To overcome the above-mentioned limitations, we used stuffer-free MS-MLPA probes that are subsequently analyzed using high-resolution capillary electrophoresis-based single-strand conformational polymorphism (CE-SSCP) instead of conventional length-dependent CE. Moreover, multiple methylation-sensitive restriction enzymes (HhaI, HpaII, and AciI) were used simultaneously; thus, probes satisfying desired criteria were available for all targets. Using this assay concept, we analyzed 17 genes associated with hepatocellular carcinoma. Our results showed that the custom-designed assay based on MS-MLPA-CE-SSCP provided robust multiplex quantification of DNA methylation levels.
View details for DOI 10.1039/c3an01178j
View details for Web of Science ID 000325819300034
View details for PubMedID 24089148
Stuffer-free multiplex ligation-dependent probe amplification based on conformation-sensitive capillary electrophoresis: A novel technology for robust multiplex determination of copy number variation
2012; 33 (19-20): 3052-3061
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.
View details for DOI 10.1002/elps.201200334
View details for Web of Science ID 000310289600014
View details for PubMedID 22965760
Precise H1N1 swine influenza detection using stuffer-free multiplex ligation-dependent probe amplification in conformation-sensitive capillary electrophoresis
2012; 424 (1): 54-56
The H1N1 influenza virus has spread worldwide to become pandemic. Here, we developed a new method to discriminate various types of influenza A, including H1N1, using stuffer-free multiplex ligation-dependent probe amplification based on a conformation-sensitive separation method, namely capillary electrophoresis-single-strand conformation polymorphism. Unlike conventional methods, our approach precisely detects five relevant gene markers permitting confirmation of infection.
View details for DOI 10.1016/j.ab.2012.02.009
View details for Web of Science ID 000302842700009
View details for PubMedID 22342882
Strategy for high-fidelity multiplex DNA copy number assay system using capillary electrophoresis devices
2011; 32 (14): 1837-1843
Structural variation of human genome such as duplications and deletions, collectively termed copy number variation (CNV), is one of the major genetic variations. Reliable and efficient measurement of CNV will be essential to develop diagnostic tools for CNV-related diseases. We established a strategy based on multiplex PCR and capillary electrophoresis (CE) for reliable CNV assay. Multiplex-PCR was performed using five primer sets for target loci and a diploid control (DC). We designed primers satisfying three conditions: different size of each PCR product for CE separation, unified annealing temperature for multiplex PCR, and suitability for quantitative PCR (qPCR). We defined the accurate PCR cycles for quantification of copy numbers at which the amplifications for all targets were supposed to be exponential, named maximum doubling cycle. CE was carried out with PCR product and the ratio of the peak areas (target/diploid control) was calculated. Our multiplex PCR-CE analysis reliably determined copy numbers of X chromosome with variable copies ranging from 1 to 5 and showed higher reliability than qPCR (correlation coefficient 0.996 versus 0.898). When measuring the six randomly selected autosomal CNV targets using our multiplex PCR-CE, the results agreed with those from qPCR. In addition, our strategy was validated for the broad application to commonly used CE devices. Taken together, this assay will be useful for accurate analysis of multiple disease-associated CNVs in a clinical setting.
View details for DOI 10.1002/elps.201100093
View details for Web of Science ID 000292987800008
View details for PubMedID 21688278
Recent developments in CE-based detection methods for food-borne pathogens
2010; 31 (13): 2137-2153
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.
View details for DOI 10.1002/elps.200900682
View details for Web of Science ID 000280226900006
View details for PubMedID 20593389
Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP
2010; 31 (14): 2405-2410
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 10(2). These results indicate that high-resolution CE-SSCP systems have considerable potential in the clinical diagnosis of bacteria-induced diseases.
View details for DOI 10.1002/elps.201000091
View details for Web of Science ID 000280709700014
View details for PubMedID 20568262