The smears of DNA fragments are transferred onto a thin sheet of nylon by blotting and then the sheet is bathed with a radioactively-labeled FMR1 gene probe. The probe singles out the matching FMR1 gene fragments and binds tightly. Exposure of the nylon sheet to X-ray film results in short black horizontal lines on the film corresponding to the positions of the patient's FMR1 gene fragments. Some examples of these patterns are shown in the diagram entitled 'Fragile X Analysis by Southern Blot' at the end of this article. Many unusual patterns not illustrated here can also be encountered.
Caution is required when interpreting a pattern suggesting a small premutation.
Slight separation of two unmethylated bands may be seen when two normal alleles or a normal and a 'grey zone' allele differ significantly in size. After separation on a gel, pieces differing in length by as little as one CGG repeat can be distinguished. This is why quite accurate determination of CGG repeat number for normal alleles and premutations is possible by PCR analysis. X-ray film exposure results in one, two or no bands. The diagram entitled 'Fragile X Analysis by PCR' at the end of this article shows the appearance of these bands and gives several examples of results.