Wei-Long Zhong1,2, Luo Wang3, Xia Wu1,4, Jie Zhang1, Xiao-Fan Chen3, Wei Zhang5, Xia Dou6,7, Bo Yu8,9. 1. Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China. 2. Division of Clinical Medicine, Shantou University Medical College, Shantou, Guangdong, P.R. China. 3. Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China. 4. Division of Clinical Medicine, Guangzhou Medical University, Guangzhou, Guangdong, P.R. China. 5. Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China. zhangweispace@yeah.net. 6. Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China. douxia@medmail.com.cn. 7. Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China. douxia@medmail.com.cn. 8. Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China. yubomd@hotmail.com. 9. Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China. yubomd@hotmail.com.
Abstract
BACKGROUND: Filaggrin gene (FLG) plays an important role in skin barrier function, and loss-of-function mutations of FLG have been shown to be a predisposing factor for atopic dermatitis (AD). The c.3321delA mutation is the most common FLG mutation in Chinese population. We aim to develop a rapid, cost-efficiency, and reliable closed-tube method that has not been described for the detection of c.3321delA mutation. METHODS: Recombinant wild-type and mutant plasmids of c.3321delA mutation were constructed, heterozygous mutant plasmids were prepared by mixing the mutant plasmids and wild-type plasmids at 1:1 ratio. High-resolution melting analysis (HRMA) coupled with an unlabeled DNA probe was employed to identify the shift in melting temperature of the probe-template complex, which reflects the presence of c.3321delA mutation. RESULTS: Unlabeled probe based HRMA was able to distinguish all three genotypes (wild-type, heterozygote, and mutant) of c.3321delA mutation. Then, we applied this method to genotype 1,317 clinical samples. Genotyping results obtained from unlabeled probe HRMA were 100% concordant with the results from direct sequencing. CONCLUSION: We developed a fast and high-throughput method to detect the c.3321delA mutation.
BACKGROUND:Filaggrin gene (FLG) plays an important role in skin barrier function, and loss-of-function mutations of FLG have been shown to be a predisposing factor for atopic dermatitis (AD). The c.3321delA mutation is the most common FLG mutation in Chinese population. We aim to develop a rapid, cost-efficiency, and reliable closed-tube method that has not been described for the detection of c.3321delA mutation. METHODS: Recombinant wild-type and mutant plasmids of c.3321delA mutation were constructed, heterozygous mutant plasmids were prepared by mixing the mutant plasmids and wild-type plasmids at 1:1 ratio. High-resolution melting analysis (HRMA) coupled with an unlabeled DNA probe was employed to identify the shift in melting temperature of the probe-template complex, which reflects the presence of c.3321delA mutation. RESULTS: Unlabeled probe based HRMA was able to distinguish all three genotypes (wild-type, heterozygote, and mutant) of c.3321delA mutation. Then, we applied this method to genotype 1,317 clinical samples. Genotyping results obtained from unlabeled probe HRMA were 100% concordant with the results from direct sequencing. CONCLUSION: We developed a fast and high-throughput method to detect the c.3321delA mutation.
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