The interaction between oligonucleotides and long DNA fragments was analyzed by force curve measurements using atomic force microscopy (AFM). DNA fragments (150-base or 406-base) from the mitochondrial ATPase and cytochrome oxidase subunit III genes that contained a mismatch of one to three bases among Tunnus species were immobilized on glass slides. The statistical distribution of disruption forces between oligonucleotide probes (21-mer or 29-mer) and single stranded DNA fragments (150-base or 406-base) were analyzed by 40 or 180 force curve measurements. Histograms plotting the frequencies of disruption forces showed a wide distribution with a highest peak. The highest mean values in disruption force were obtained when DNA fragments with perfectly match sequences were employed. These results demonstrated that the specific sequence differences between long DNA fragments can be measured using force-based detection. A single base mismatch yielded a statistically significant 10% decrease in disruption force, furthermore, 2-base and 3-base mismatches provided approximately 20 and 25-30% decreases, respectively. Our results indicated that force-based detection potentially can be applied toward many other mismatched DNA detection techniques besides species-specific identification of tuna. © 2006 Elsevier B.V. All rights reserved.