DETECTION OF SINGLE-BASE CHANGES USING A BIOLUMINOMETRIC PRIMER EXTENSION ASSAY

A rapid bioluminometric technique for real-time detection of known sin gle-base changes is presented. The concept relies on the measurement o f the difference in primer extension efficiency by a DNA polymerase of a matched over a mismatched 3' terminal. The rate of the DNA polymmer aseccatayzee primer extension is measured by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (P. Nyren (1987 ) Anal. Biochem. 167, 235-238). The PPi formed in the polymerization r eaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the single-bas e detection assay, immobilized single-stranded DNA fragments are used as template. Two detection primers differing with one base at the 3' e nd are designed, one precisely complementary to the nonmutated DNA seq uence and the other precisely complementary to the mutated DNA sequenc e. The primers are hybridized with the 3'-termini over the base of int erest and the primer extension rates are, after incubation with DNA po lymerase and deoxynucleotides, measured with the ELIDA. We show that t he relative mismatch extension efficiency is strongly decreased by sub stituting the alpha-thiotriphosphate analog for the next correct natur al deoxynucleotide after the 3'-mismatch termini. The possibility of u sing the technique for studies of mismatch extension kinetics for two polymerases lacking exonucleolytic activity is shown. (C) 1997 Academi c Press