Cantilever-Based Optical Deflection Assay for Discrimination of DNA Single-Nucleotide Mismatches

• Published in: Analytical chemistry (Washington) Vol. 73; no. 7; pp. 1567 - 1571
• Main Authors: Hansen, Karolyn M, Ji, Hai-Feng, Wu, Guanghua, Datar, Ram, Cote, Richard, Majumdar, Arunava, Thundat, Thomas
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.04.2001
• Subjects: atomic force microscopy; Base Pair Mismatch; Biochemistry; Biological and medical sciences; Deoxyribonucleic acid; Diverse techniques; DNA; DNA - isolation & purification; Fundamental and applied biological sciences. Psychology; Genomics; Molecular and cellular biology; Nucleic Acid Hybridization; Polymorphism, Single Nucleotide; Silicon; single nucleotide polymorphisms; Molecular hybridization; Base mismatching; DNA; Optical method; Molecular probe; Technique; Single nucleotide polymorphism; Detection
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0012748

Characterization of single-nucleotide polymorphisms is a major focus of current genomics research. We demonstrate the discrimination of DNA mismatches using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling. Gold-coated silicon AFM cantilevers were functionalized with thiolated 20- or 25-mer probe DNA oligonucleotides and exposed to target oligonucleotides of varying sequence in static and flow conditions. Hybridization of 10-mer complementary target oligonucleotides resulted in net positive deflection, while hybridization with targets containing one or two internal mismatches resulted in net negative deflection. Mismatched targets produced a stable and measurable signal when only a four-base pair stretch was complementary to the probe sequence. This technique is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization.