Development of Unlabeled Probe Based High-Resolution Melting Analysis for Detection of Filaggrin Gene Mutation c.3321delA.
J Clin Lab Anal
; 30(6): 892-896, 2016 Nov.
Article
em En
| MEDLINE
| ID: mdl-27040325
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 11 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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Análise Mutacional de DNA
/
Deleção de Sequência
/
Doença de Alzheimer
/
Proteínas de Filamentos Intermediários
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Female
/
Humans
/
Male
Idioma:
En
Revista:
J Clin Lab Anal
Assunto da revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Ano de publicação:
2016
Tipo de documento:
Article