COLORIMETRIC SOLID-PHASE MINISEQUENCING ASSAY ILLUSTRATED BY DETECTION OF ALPHA(1)-ANTITRYPSIN Z-MUTATION

In solid-phase minisequencing, a defined point mutation is detected in microtiter plate-immobilized DNA by a single nucleotide primer extens ion reaction. We have here developed the method into a colorimetric as say and applied it to the detection of the Z mutation of the alpha1-an titrypsin gene. We used novel nucleoside triphosphates modified with d initrophenyl (DNP) hapten, permitting detection by anti-DNP-alkaline p hosphatase conjugate, with p-nitrophenyl phosphate as substrate. The Z mutation is detected in two reactions: DNP-labeled dCTP is incorporat ed when the template is normal, DNP-dUTP when the Z mutation is presen t, Both modified nucleotides were incorporated with high specificity a nd with an efficiency similar to that of unmodified nucleotides. The t est results are measured by spectrophotometry, yielding quantitative a bsorbance values. Calculation of the ratio of C to U signal permitted unambiguous distinction of normal homozygous, ZZ homozygous, and ZM he terozygous genotypes. The colorimetric minisequencing assay is rapid, standardized, and automatable, and thus provides an accurate and simpl e alternative for the analysis of known point mutations.