Detection of single-base mutations in DNA molecules using the solution melting method.

RNA molecules that differ by a single base pair in their highest melting domain can be separated by the solution melting method (Smith et al., 1988) due to sequence-specific melting properties of double-stranded (ds) RNA heteroduplexes. We now show that this method can be used to reliably detect single base pair differences in DNA molecules, and we define the upper limit of the high melting domain length that can be analyzed by this method for dsRNA and RNA-DNA molecules as about 250 bp and 130 bp, respectively. The usefulness of this method to detect point mutations in human genomic DNA is evaluated. X-linked genes are most amenable to study, because results are most easily interpretable when only a single allele is present. Using the human factor VIII gene as an example, we show this method is capable of detecting polymorphisms present in genomic DNA after amplification by the polymerase chain reaction. This technique should prove useful in the simultaneous rapid screening of multiple exons of X-linked genes for single-base mutations.