Minisequencing with acyclonucleoside triphosphates tethered to lanthanide(III) chelates

Four acyclic nucleoside triphosphates (derivatives of cytosine, thymine, 7-deazaadenine, and 7-deazaguanine) labeled with nonluminescent europium, terbium, dysprosium, and samarium chelates of 2,2',2'',2'''-[[4-(4-isothiocyanatophenyl)ethyl]pyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetic acid) were applied to minisequencing using two mutations (Delta F 508 and 1717-1 G to A) of cystic fibrosis as a model system. When synthetic targets were used, all four alleles involved could be analyzed in a single reaction using four terminating substrates labeled with four different lanthanide(III) chelates and DELFIA technology for detection. Blood spot samples without DNA isolations were used for PCR amplification and genotyping the target mutations by minisequencing. The single- and dual-labeled minisequencing assays were robust, while the four-label assay still requires further optimization of the multiplexed PCR amplification.