Nieuwe ontwikkelingen in de automatisering van moleculaire diagnostiek: mutatie-analyse

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Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes).

Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554-559, 2002

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

Generation of DNA probes for detection of microorganisms by polymerase chain reaction fingerprinting

Identification of medically relevant microorganisms is important for diagnosis, treatment and prevention of infectious diseases. This has initiated the development of a large number of identification and typing techniques based on phenotypic and genetic characteristics. In general, these last mentioned nucleic acid-mediated techniques provide more detailed and consistent information on strain-specific characteristics. However, the development of clinically useful microbial DNA/RNA probes requires nucleotide sequence information and a set of well defined reference organisms for test validation in comparison with the current gold standard. This is a requirement for the development of accurate nucleic acid hybridisation and/or amplification tests. Recently, it has been demonstrated that polymerase chain reaction (PCR)-mediated genetic typing of microorganisms can lead to the immediate isolation of species-specific DNA probes by comparison of DNA fingerprints. This combines the sensitivity of PCR with the specificity of DNA probing without the need to generate nucleic acid sequence information prior to probe development. The implications of this procedure for clinical microbiology and epidemiological surveillance will be discussed. It is shown that specific probes can be developed for various taxonomic levels and that detection and identification can be combined into a single, fast procedure. The versatility and widely applicable principles of this procedure will be highlighted and exemplified by some newly developed tests and a review of the current literature