title: Screening human genes for small alterations performing an enzymatic cleavage mismatched analysis (ECMA) protocol.
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.
authors.affiliation: Department of Medical Genetics, University of Athens, Athens, Greece.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Vogiatzakis
authors.forename: Kyriaki
authors.initials: K
authors.lastname: Kekou
authors.forename: Christalena
authors.initials: C
authors.lastname: Sophocleous
authors.forename: Sophia
authors.initials: S
authors.lastname: Kitsiou
authors.forename: Ariadni
authors.initials: A
authors.lastname: Mavrou
authors.forename: Chrisa
authors.initials: C
authors.lastname: Bakoula
authors.forename: Emmanouel
authors.initials: E
authors.lastname: Kanavakis
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D004720:Endonucleases / Q000378:metabolism*; D000795:Fabry Disease / Q000235:genetics; D005260:Female; D005796:Genes; D005820:Genetic Testing / Q000191:economics / Q000379:methods; D006801:Humans; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D010375:Pedigree; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D009692:Nucleic Acid Heteroduplexes; D004720:Endonucleases