Solution-based scanning for single-base alterations using a double-stranded DNA binding dye and fluorescence-melting profiles

DNA molecules differing by as little as a single base substitution have traditionally been distinguished by gel electrophoresis-based methodologies that exploit differences in the sequence-specific properties of double-stranded DNA (dsDNA) such as melting temperature and secondary conformational configuration. By comparison, solution-based fluorescence methods using sequence-specific probes are limited to detecting mutations restricted to very short segments of DNA (similar to 20 bp). We describe a solution-based fluorescence method that discriminates between wild-type and mutant sequences using a dsDNA binding dye, and interrogates a region of > 200 nucleotides. This method is based on melting theory and entails fluorescence monitoring of the melting temperatures of GC-clamped amplicons subjected to gradual and progressive thermal denaturation in the presence of a constant concentration of urea. Heterozygous samples are easily identified by the lower melting temperatures of the less thermodynamically stable heteroduplex mismatches from the wild-type:mutant DNA hybrids as compared to the more stable wild-type Watson-Crick duplexes. All of the four possible sets of mismatches (A.G/T.C, T.G/A.C, G.G/C.C, and T.T/A.A) represented in 17 heterozygous mutations distributed throughout the length of 20 different amplicons (104 to 212 bp), were distinguished from the wild-type by their altered melting profiles. This methodology is advantageous in that it obviates gel electrophoresis or labeled oligonucleotide probes. Significantly, it expands the region of interrogation for detection of single-base changes using fluorescence-based methods in solution, and is amenable for automation and adaptation to high-throughput systems.