The specific structural features of stem-loop (hairpin) DNA constructs provide increased specificity of target recognition. Recently, several robust assays have been developed that exploit the potential of structurally constrained oligonucleotides to hybridize with their cognate targets. Here, I review new diagnostic approaches based on the formation of stem-loop DNA oligonucleotides: molecular beacon methodology, suppression PCR approaches and the use of hairpin probes in DNA microarrays. The advantages of these techniques over existing ones for sequence-specific DNA detection, amplification and manipulation are discussed.