RESUMO
BACKGROUND: A simple and practical screening method, allowing the mass detection of targeted DNA mutations, was developed by combined use of allele specific PCR (ASP) and subsequent fluorogenic intercalation to the amplicon. METHODS: Crude DNAs were extracted from dried blood spots (DBS) by a simple boil method. Highly specific polymerase chain reaction (PCR) amplification was achieved by adopting Taq DNA polymerase (Amersham Pharmacia) modified with TaqStart Antibody (Clontech). A fluorogenic DNA intercalator, SYBR Green I (Molecular Probes), was directly added to the PCR products and the resultant fluorescence was measured by a conventional fluorometric microplate reader, microfluorometry (MFL). RESULTS: The most common mutation in cystic fibrosis (CF), del F508, was successfully detected with clear differentiation as homozygotes (n=4) and heterozygotes (n=9) from control subjects (n=18). Fluorescence intensities, with mean+/-S.D. in arbitrary unit, were 895+/-249, 900+/-184 and 257+/-53, respectively. Those from control newborns (n=352) were 250+/-27 with the range of 188-475. CONCLUSIONS: The proposed ASP/MFL provides a simple, objective and economical detection of known mutations or single nucleotide polymorphisms (SNPs). The usefulness of this method was clearly shown in the detection of del F508 in CF.
