SOLID-PHASE MINISEQUENCING CONFIRMED BY FISH ANALYSIS IN DETERMINATION OF GENE COPY NUMBER

The solid-phase minisequencing method (Syvanen et al. 1990) allows acc urate quantative determination of the ratio between two DNA or RNA seq uences that are present as a mixture in a sample and differ from each other only by a single nucleotide. Here, we present another applicatio n of the minisequening method, the determination of the gene copy numb er in a genome. The copy number of a marker gene aspartyl glucosaminid ase (AGA) located at 4qter, was determined in three patients with a ch romosomal alteration involving the distal region of 4q. For the minise quencing assay an equal amount of DNA from a patient homozygous for a mutation in the AGA gene was added to the DNA samples concerned. The r elative amount of the normal sequence determined in each combined samp le gives the copy number of the AGA gene. Fluorescence in situ hybridi zation (FISH), applied in parallel as a control, produced concordant r esults with solid-phase minisequencing in each case. As the potential of the minisequencing lies in automation, it could be a useful tool in the screening of monosomies, trisomies or loss of heterozygosity in d iagnostics.