Abstract
▼The DNA intercalative dye ethidium bromide is commonly added directly to agarose gels and buffer prior to the electrophoresis of restricted DNA to allow visualization of migrated DNA after electrophoresis. Our laboratory routinely used this technique but we recently experienced a significant problem with the electrophoretic migration of specific DNA fragments in the presence of ethidium bromide. Our laboratory employs a simple Southern blot assay for the detection of the CGG triplet repeat expansion associated with the fragile X syndrome (FRAXA). The restriction enzyme PstI is used to cut isolated patient genomic DNA which is run out on a 1% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0), supplemented with 5 µg/ml ethidium bromide. Electrophoresed DNA is next depurinated with 0.75 N HCl for 15 min, denatured with 1 N NaOH for 30 min and finally transferred by vacuum to a nylon membrane. Fragments are detected using a 440 bp BamHI/XhoI subclone of PE5.1 (Ref. 1) which hybridizes downstream of the CGG expansion site and slightly upstream of the second PstI cutting site. This yields restriction fragments containing the expansion region of 1−1.1 kb in normal individuals, greater than 1.7 kb in phenotypically affected individuals and 1.1−1.7 kb among ‘phenotypically normal’ carriers. A typical autoradiogram obtained before any problems arose is shown (Fig. 1a).
Published Version
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