Secondary structure prediction and structure-specific sequence analysis ofsingle-stranded DNA

DNA sequence analysis by oligonucleotide binding is often affected by inter ference with the secondary structure of the target DNA. Here we describe an approach that improves DNA secondary structure prediction by combining enz ymatic probing of DNA by structure-specific 5'-nucleases with an energy min imization algorithm that utilizes the 5'-nuclease cleavage sites as constra ints. The method can identify structural differences between two DNA molecu les caused by minor sequence variations such as a single nucleotide mutatio n. It also demonstrates the existence of long-range interactions between DN A regions separated by > 300 nt and the formation of multiple alternative s tructures by a 244 nt DNA molecule. The differences in the secondary struct ure of DNA molecules revealed by 5'-nuclease probing were used to design st ructure-specific probes for mutation discrimination that target the regions of structural, rather than sequence, differences. We also demonstrate the performance of structure-specific 'bridge' probes complementary to non-cont iguous regions of the target molecule. The structure-specific probes do not require the high stringency binding conditions necessary for methods based on mismatch formation and permit mutation detection at temperatures from 4 to 37 degreesC. Structure-specific sequence analysis is applied for mutati on detection in the Mycobacterium tuberculosis katG gene and for genotyping of the hepatitis C virus.