Screening human genes for small alterations performing an enzymatic cleavage mismatched analysis (ECMA) protocol.
Mol Biotechnol
; 37(3): 212-9, 2007 Nov.
Article
en En
| MEDLINE
| ID: mdl-17952667
ABSTRACT
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.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Análisis Mutacional de ADN
/
Disparidad de Par Base
/
Endonucleasas
/
Genes
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
/
Screening_studies
Límite:
Female
/
Humans
/
Male
Idioma:
En
Revista:
Mol Biotechnol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Año:
2007
Tipo del documento:
Article
País de afiliación:
Grecia