A luminescent probe of mismatched DNA hybridization: Location and number of mismatches.

The human genome is susceptible to change; base mismatches can arise from damaged DNA, replication errors, and spontaneous mutation, and have the potential to cause apoptosis, carcinogenesis, and mutagenesis. Many techniques have been developed for DNA mismatch detection, but many of these methods have complex, time-consuming procedures and are limited to the detection of specific types of DNA mismatches. In this work, we present a general method for the simple and sensitive nucleobase-sensitized luminescent detection of mismatches in double-stranded DNA (dsDNA) using terbium ions. Terbium ions luminesce differently depending on the site of coordination in DNA due to the proximity effect of the energy transfer process that occurs from excited, non-hydrogen bonded nucleobases in single-stranded DNA (ssDNA) regions to the terbium ions. We examined the effect of location and number of mismatches on the sensitivity and selectivity of this probe in both synthetic oligonucleotides containing mismatches and natural calf thymus DNA exposed to UV light to induce reduced base pairing due to damage. This method shows good sensitivity for the determination of DNA mismatches, with limit of detection and limit of quantification of 1 and 3 mismatches, respectively, per dsDNA sequence.