CAPILLARY ZONE ELECTROPHORESIS OF POLYMERASE CHAIN REACTION-AMPLIFIEDDNA FRAGMENTS IN POLYMER NETWORKS - THE CASE OF GATT MICROSATELLITES IN CYSTIC-FIBROSIS

In cystic fibrosis (CF), the most common mutation, delta F508 (a three -base-pair deletion) accounts for ca. 70% of mutations in the worldwid e population. The majority of other mutations (more than 350 reported so far to the Genetic Analysis Consortium) have been detected in singl e cases, thus rendering quite cumbersome a molecular diagnostic approa ch for the identification of CF chromosomes. As an alternative, linkag e analysis based on intragenic polymorphism can be useful for prenatal diagnosis and CF-carrier detection, provided that the heterozygosity of the allelic forms is very high. For this purpose, DNA microsatellit es, consisting of two to epta nucleotide repeat clusters, displaying a high degree of polymorphism, are being increasingly used as markers i n linkage studies. Two main allelic forms, one hexameric (111 bp) and one heptameric (115 bp), of a tetranucleotide (GATT) repeat polymorphi sm, at the junction of intron IVS6a and exon 6b, have been amplified b y PCR technology. These two alleles can be separated in a 10-20%T poly acrylamide gradient gel and detected by ethidium bromide staining. As an alternate procedure, these two fragments are efficiently separated by capillary zone electrophoresis in a viscous solution of 6%T linear polyacrylamide and detected by their intrinsic absorbance at 254 nm.