FLUORESCENCE-BASED DNA MINISEQUENCE ANALYSIS FOR DETECTION OF KNOWN SINGLE-BASE CHANGES IN GENOMIC DNA

We describe a rapid, automated method for direct detection of known single-base changes in genomic DNA. Fluorescence-based DNA minisequence analysis is employed in a template-dependent reaction which involves a single nucleotide extension of an oligonucleotide primer by the correct fluorescently-tagged dideoxynucleotide chain terminator. Detection following electrophoresis on denaturing acrylamide gels is facilitated by alkaline phosphatase treatment of reaction products after extension followed by isopropanol precipitation of the dye-tagged, single-base-extended primer to remove unincorporated deoxynucleotides. Fluorescence analysis of the incorporated dye tag reveals the identity of the template nucleotide immediately 3' to the primer site. This technique does not require radioactivity or biotinylated PCR product, relies on the incorporation of a single dideoxynucleotide terminator to extend the primer by one nucleotide and takes advantage of the sensitivity of fluorescent terminators developed for automated DNA sequence analysis. As a demonstration, we have applied the assay to human genomic DNA for detection of the sickle mutation in the beta-globin gene, and have also examined feasibility for simultaneous delineation using a multiplex-like strategy in a single gel-lane of some of the most common P-thalassemia mutations in the Mediterranean basin.