RESUMEN
Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD), and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations, which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases where other techniques have failed.
Asunto(s)
Hiperplasia Suprarrenal Congénita/genética , Bioensayo , Fibrosis Quística/genética , Enfermedad de Fabry/genética , Distrofia Muscular de Duchenne/genética , Hiperplasia Suprarrenal Congénita/diagnóstico , Alelos , Fibrosis Quística/diagnóstico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Distrofina/genética , Endonucleasas/química , Enfermedad de Fabry/diagnóstico , Pruebas Genéticas/métodos , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Mutación , Proteínas de Plantas/química , Polimorfismo Genético , Esteroide 21-Hidroxilasa/genética , alfa-Galactosidasa/genéticaRESUMEN
Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed.
Asunto(s)
Disparidad de Par Base , Análisis Mutacional de ADN/métodos , Endonucleasas/metabolismo , Genes , Hiperplasia Suprarrenal Congénita/genética , Disparidad de Par Base/genética , Secuencia de Bases , Fibrosis Quística/genética , Análisis Mutacional de ADN/economía , Enfermedad de Fabry/genética , Femenino , Pruebas Genéticas/economía , Pruebas Genéticas/métodos , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Linaje , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodosRESUMEN
Although the facioscapulohumeral muscular dystrophy (FSHD) locus was mapped to 4q35 chromosomal region in 1990, no gene transcript has been as yet identified. Molecular diagnosis is based mainly on the detection of deletions of a 3.3 kb-tandem repeat array in the locus. This procedure offers almost 95% accuracy but is quite complicated and therefore a simpler test would be preferable. We describe a convenient non-radioactive protocol which requires a simple PCR probe synthesis and labelling procedure, thus facilitating and accelerating the standard Southern blot based DNA test. 134 individuals (113 affected and 21 unaffected relatives) were studied and a causal deletion was detected in 72.
