Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons

Background: Our increasing knowledge of the genetic basis of inheritable di seases requires the development of automated reliable methods for high-thro ughput analyses. Methods: We investigated the combination of semiautomated DNA extraction fr om blood using a robotic workstation, followed by automated mutation detect ion using highly specific fluorescent DNA probes, so-called molecular beaco ns, which can discriminate between alleles with as little as one single-bas e mutation. We designed two molecular beacons, one recognizing the wild-typ e allele and the other the mutant allele, to determine genotypes in a singl e reaction. To evaluate this procedure, we examined the C677T mutation in t he methylenetetrahydrofolate reductase (MTHFR) gene, which is associated wi th an increased risk for cardiovascular disease and neural tube defects. DN A was isolated from 10 muL of fresh EDTA-blood samples by use of a robotic workstation. The DNA samples were analyzed using molecular beacons as well as conventional methods. Results: Both methods were compared, and no differences were found between outcomes of genotyping. Conclusions: The described assay enables robust and automated extraction of DNA and analysis of up to 96 samples (10 muL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high -throughput analyses. (C) 2001 American Association for Clinical Chemistry.