Browsing by Author "Wu, Shou-Mei"

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A "turn on/off" scorpion biosensor targeting point mutation of SMN genes for diagnosis of spinal muscular atrophy
(2014)
Chen, Chung-An
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Wang, Chun-Chi
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Chen, Yen-Ling
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Jong, Yuh-Jyh
;
Wu, Shou-Mei
A "turn on/off\'\' biosensor for diagnosis of exon 7 of the SMN1 gene was developed by employing a "scorpion primer\'\'. This scorpion primer was based on the principle of fluorescence resonance energy transfer using a fluorophore, a blocker and a quencher. It was successfully applied to detect 10 volunteer samples, and not only to in vitro testing.
Genotyping of exons 1 to 20 in Duchenne muscular dystrophy by universal multiplex PCR and short-end capillary electrophoresis
(2014-12-01)
Syu, Jing-Rou
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Wang, Chun-Chi
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Jong, Yuh-Jyh
;
Wu, Shou-Mei
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生物科技學院
One rapid CE method was established to diagnose Duchenne muscular dystrophy (DMD). DMD is a severe recessive inherited disorder frequently caused by gene deletions. Among them, exons 1-20 account for nearly 30% of occurrences. In this study, the universal multiplex PCR was used to enhance the fluorescently labeling efficiency, which was performed only by one universal fluorescent primer. After PCR, a short-end injection CE (short-end CE) speeded up the genotyping of the DMD gene. This method involved no extra purification, and was completed within 9 min. The CE conditions contained a polymer solution of 1.5% hydroxylethylcellulose in 1 x TBE buffer at 6 kV for separation. This method was applied to test six DMD patients and one healthy male person. The results showed good agreement with those of multiplex ligation-dependent probe amplification. This method can be applied for clinical diagnosis of DMD disease. Accurate diagnosis of the DMD gene is the best way to prevent the disease.
Label-Free Fluorescent Copper Nanoclusters for Genotyping of Deletion and Duplication of Duchenne Muscular Dystrophy
(2015-06-16)
Chen, Chung-An
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Wang, Chun-Chi
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Jong, Yuh-Jyh
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Wu, Shou-Mei
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生物科技學院
Real applications in clinical diagnosis of label-free fluorescent copper nanoclusters (CuNCs) are demonstrated. Double-strand DNA (dsDNA) can act as an effective template for the formation of CuNCs, which can be used to distinguish the deletion or duplication genotypes of Duchenne muscular dystrophy (DMD) due to different fluorescent intensities. After PCR, the DMD amplicons reacted with copper ion by reduction of ascorbic acid and generated fluorescence. The exons of the DMD gene were taken as the model analytes for genetic diagnosis. In this sensing system, the deletion type does not show fluorescence; on the other hand, the duplication type emits higher fluorescence than normal type. Parameters of this sensing system were optimized, including PCR conditions, levels of copper ion and ascorbate, and reaction time. The DMD-dominated exons 45, 46, and 47 were detected, and the method was applied to six samples of DMD patients. The results were consistent with those of the multiplex ligation-dependent probe amplification method. This strategy was feasible to detect all exons of this disease.
Universal fluorescent tri-probe ligation equipped with capillary electrophoresis for targeting SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy
(2014-06-23)
Wang, Chun-Chi
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Shih, Chi-Jen
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Jong, Yuh-Jyh
;
Wu, Shou-Mei
;
生物科技學院
This is the first ligase chain reaction used for diagnosis of spinal muscular atrophy (SMA). Universal fluorescent tri-probe ligation (UFTPL), a novel strategy used for distinguishing the multi-nucleotide alternations at single base, is developed to quantitatively analyze the SMN1/SMN2 genes in diagnosis of SMA. Ligase chain reaction was performed by adding three probes including universal fluorescent probe, connecting probe and recognizing probe to differentiate single nucleotide polymorphisms in UFTPL. Our approach was based on the two UFTPL products of survival motor neuron 1 (SMN1) and SMN2 genes (the difference of 9 mer) and analyzed by capillary electrophoresis (CE). We successfully determined various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects. By using the UFTPL-CE method, the SMN1 and SMN2 genes were fully resolved with the resolution of 2.16 +/- 0.37 (n = 3). The r values of SMN1 and SMN2 regression curves over a range of 1-4 copies were above 0.9944. Of the 48 DNA samples, the data of gene dosages were corresponding to that analyzed by conformation sensitive CE and denatured high-performance liquid chromatography (DHPLC). This technique was found to be a good methodology for quantification or determination of the relative genes having multi-nucleotide variants at single base. (C) 2014 Elsevier B.V. All rights reserved.