Detection of single-base changes using a bioluminometric primer extension assay.
A rapid bioluminometric technique for real-time detection of known single-base changes is presented. The concept relies on the measurement of the difference in primer extension efficiency by a DNA polymerase of a matched over a mismatched 3' terminal. The rate of the DNA polymerase-catalyzed primer extension is measured by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (P. Nyrén (1987) Anal. Biochem. 167, 235-238). The PPi formed in the polymerization reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the single-base detection assay, immobilized single-stranded DNA fragments are used as template. Two detection primers differing with one base at the 3' end are designed, one precisely complementary to the nonmutated DNA sequence and the other precisely complementary to the mutated DNA sequence. The primers are hybridized with the 3'-termini over the base of interest and the primer extension rates are, after incubation with DNA polymerase and deoxynucleotides, measured with the ELIDA. We show that the relative mismatch extension efficiency is strongly decreased by substituting the alpha-thiotriphosphate analog for the next correct natural deoxynucleotide after the 3'-mismatch termini. The possibility of using the technique for studies of mismatch extension kinetics for two polymerases lacking exonucleolytic activity is shown.
Nyrén
P
P
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Karamohamed
S
S
Ronaghi
M
M
D001681:Biological Assay / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D008163:Luminescent Measurements; D009841:Oligonucleotides / Q000032:analysis; D016133:Polymerase Chain Reaction; D011687:Purines / Q000032:analysis*; D011743:Pyrimidines / Q000032:analysis*; D012680:Sensitivity and Specificity
D009841:Oligonucleotides; D011687:Purines; D011743:Pyrimidines; D004259:DNA-Directed DNA Polymerase