Southern and northern blot fixing by microwave oven.

Southern and Northern blot techniques are powerful procedures for studying gene structure and function. These methods involve the separation of nucleic acids according to size on an agarose or polyacrylamide gel and subsequent transfer of the nucleic acid to suitable nitrocellulose or nylon-based membranes. DNA or RNA molecules bound to the membrane are then fixed and hybridized with a radioactive probe, specific for the gene under study. The fixing step is particularly important for quantitatively reproducible results, especially if membranes are to be rehybridized several times. The traditional fixing procedure involved baking the membrane for 2 h at 80°C (1). More recently, it has been shown that efficient cross-linking of nucleic acids to the membrane could be obtained by 30 s to 3 min exposure of UV irradiation (2). Earlier studies described the use of a microwave oven to fix bacterial colonies to nylon membranes (3) or to fix DNA within paraffin tissue blocks (4). We demonstrate here that a 2 min treatment of Southern or Northern blots in a microwave oven efficiently fixes nucleic acids to nylon membranes which can withstand repeated reuse in hybridization reactions

A novel double nucleotide substitution in the HMG box of the SRY gene associated to Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient's brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal

ABSENCE OF ASSOCIATION BETWEEN A VARIANT OF THE MAST CELL CHYMASE GENE AND ATOPIC DERMATITIS IN AN ITALIAN POPULATION

Atopic dermatitis (AD) is a chronic dermatitis which belongs to the group of atopy-related diseases together with asthma and rhinitis. lgE and mast cell chymase (MCC) play a key role in atopic or allergic inflammation of the skin. An association between AD and a genetic variant of the MCC has been reported in a Japanese population, but failure of confirmation has rendered this association questionable. We have tested for genetic association to an MCC variant in relation to AD in an ltalian population. No significant association was found between AD and MCC genotypes. These data suggest that BstXl MCC polymorphism may not be involved in AD