Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons.
BACKGROUND
Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses.


METHODS
We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction. To evaluate this procedure, we examined the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with an increased risk for cardiovascular disease and neural tube defects. DNA was isolated from 10 microL 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 microL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high-throughput analyses.
Smit
M L
ML
University Medical Centre St. Radboud, 424 Department of Paediatrics, 6500 HB Nijmegen, The Netherlands.
Giesendorf
B A
BA
Vet
J A
JA
Trijbels
F J
FJ
Blom
H J
HJ
D001322:Autoanalysis; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D005456:Fluorescent Dyes; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics; D015203:Reproducibility of Results; D012371:Robotics
D015342:DNA Probes; D005456:Fluorescent Dyes; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)