EXPAND LONG PCR FOR FRAGILE-X MUTATION DETECTION

Fragile X mutation detection by DNA analysis enables accurate diagnosi s of the fragile X syndrome. The mutation involves the expansion of CG G repeats in the FMR1 gene and has been primarily detected by the Sout hern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutate d and full-mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by eth idium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. Th e amplified premutations and full mutations were identified by hybridi zation with a digoxigenin-labeled 5'-(CGG)(5)-3' probe, followed by ch emiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this pape r we propose a new strategy for molecular diagnosis of the fragile X s yndrome in which our Expand Long PCR assay is used as the first screen ing test for fragile X mutation detection.