Structural Destabilization of Intramolecular Duplexes Improves the Results of DNA Hybridization Analysis

Abstract

This study tested a method designed to correctly identify single nucleotide polymorphisms in DNA sequences that are capable of forming a hairpin. Fragments of the angiotensin (AGT) and cytochrome (CYP2C19) genes with rs699 (T>C) and rs4986893 (G>A), respectively, were chosen as examples. DNA probes complementary to the polymorphic sites formed hairpin structures with a loop of 6 nt in the case of rs699 (AGT) or 4 nt in the case of rs4986893 (CYP2C19). Fluorophore-labeled target DNA was obtained via two-round multiplex PCR with simultaneous incorporation of a fluorescent label in the second round. When target DNA was hybridized to a corresponding pair of probes immobilized in gel pads of a biochip, dramatically low, if any, fluorescent signals were detected from the pads. A replacement of one nucleotide in the DNA probes prevented the formation of intramolecular structures, as was confirmed by melting curves. However, the DNA probes completely lost their complementarity to target DNA as a result of the replacement. To restore the complementary interaction with the DNA probe, corresponding nucleotide replacements were introduced in target DNA via site-directed mutagenesis. The approach significantly increased the specific fluorescent signals from biochip pads, thus allowing correct genotyping of rs699 and rs4986893.