SYSTEMATIC USE OF AUTOMATED FLUORESCENCE-BASED SEQUENCE-ANALYSIS OF AMPLIFIED GENOMIC DNA FOR RAPID DETECTION OF POINT MUTATIONS

Several approaches are now available for screening populations for kno wn mutations in a given gene. However, for detection of multiple mutat ions in a population that has not been characterized or for detection of new mutations, the value and efficiency of these screening procedur es decreases. Although more than 100 different beta-thalassemia mutati ons have so far been described, the spectrum of mutations in the Easte rn Mediterranean and Israel has not been defined in detail. We have us ed automated fluorescence-based DNA sequence analysis of PCR-amplified genomic DNA employing a cycle-sequencing strategy coupled with advanc ed analysis software to rapidly detect beta-thalassemia mutations in I sraeli patients. This method enabled rapid identification of eight dif ferent mutations in 10 patients, including two rare mutations, one of which has never been described in this geographic region. Our results show that automated fluorescence-based DNA sequence analysis of amplif ied genomic DNA is a rapid and reliable method for detection of point mutations and small deletions or insertions in both heterozygous and h omozygous states. This approach is particularly effective for a relati vely small gene such as beta-globin, but it can also be used for rapid detection of mutations in large genes by first sequencing clusters of exons and intron/exon borders. (C) 1994 Wiley-Liss, Inc.