Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization

In order to increase the discrimination of single nucleotide polymorphisms in DNA hybridization, artificial mismatches are inserted into probe oligonucleotides using the base analog 3-nitropyrrole. Differences in thermal stability (delta Tm) between hybrids formed with normal and single-nucleotide-variant DNA targets are increased by as much as 200% over conventional hybridization, and are strongly dependent upon the spacing between mismatches. The increased specificity is demonstrated by hybridization analysis and allele-specific amplification within the HLA-DRB locus.