We constructed a plasmid for the in vitro synthesis of a competitor RNA for use as an internal exogenous control during reverse transcriptase-PCR (RT-PCR) detection of epidermal growth factor receptor (EGFR) expression. The competitor RNA harbors a 32-base deletion compared 234 PCR Methods andApplicutions
Hereditary hemochromatosis (HH) is a very common autosomal recessive disorder of iron metabolism and frequently associated with mutations in the HFE gene. Molecular genetic testing for HFE mutations is considered valuable for carrier identification, as well as for early diagnosis of the disease, allowing simple treatment by phlebotomy and normal survival of patients. We have developed a reverse-hybridization assay for the routine diagnosis of eight previously described and one novel (E168Q) HFE point mutations. The test is based on multiplex DNA amplification and ready-to-use membrane teststrips, which contain oligonucleotide probes for each wild-type and mutated allele immobilized as an array of parallel lines. The procedure is rapid and accessible to automation on commercially available equipment, and by adding new probes the teststrip can easily be adapted to cover an increasing number of mutations.
An earlier study of reference values of iron parameters in Portugal showed significant differences between populations from northern and southern villages. This study addresses the question of the geographical distribution in Portugal of the two main mutations (C282Y and H63D) of the hereditary hemochromatosis gene, HFE. For that purpose, a stratified sample of 640 anonymous dried blood spot samples was randomly selected from the major regions of Portugal: North, Center, Lisbon and the Tagus Valley, Alentejo and Algarve. Differences in the geographical distribution of these two mutations were observed thus confirming the presumed differences between the age of the two mutations which is compatible with the postulated Celtic/Nordic origin of the C282Y mutation. The finding of a significantly higher allelic frequency of the C282Y mutation in the North (0.058) than in the South (0.009) could also point to an effect of differential selective forces acting in the different geographical areas of the country. Data on archaeological, ethnographic and linguistic records and on the North/South distribution of Portuguese cattle breeds of European or African origin have also been reported. In addition to their interest for population genetics, the results represent a reminder of the need to take into account regional differences in the design of strategies for population screening of hereditary hemochromatosis. European Journal of Human Genetics (2001) 9, 843 ± 848.
We have developed and evaluated a test for HLA-B*27 based on PCR and DNA hybridization in microtiter plates. A region within exon 2 of the HLA-B gene is amplified and labeled by PCR and the amplification product is hybridized to a group-specific HLA-B*27 and a generic control oligonucleotide probe in two separate cavities of the plate. Bound sequences are detected using an ELISA-like protocol. The assay has been evaluated on 254 DNA samples routinely received for B27 testing in parallel with serological and SSP-PCR typing. Results were concordant in typing 102 HLA-B27-positive and 152 HLA-B27-negative individuals except for two samples containing HLA-B*73, which stained B27 positive in the microwell test. The new procedure is rapid and simple to perform, and the microwell format is particularly suitable for automation.
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