DNA Detection by Strand Displacement Amplification and Fluorescence Polarization With Signal Enhancement Using a DNA Binding Protein

• Published in: Nucleic acids research Vol. 24; no. 2; pp. 348 - 353
• Main Authors: Walker, G. Terrance, Linn, C. Preston, Nadeau, James G.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15-01-1996
• Subjects: Base Sequence; Deoxyribonuclease EcoRI; DNA - analysis; DNA, Bacterial - analysis; DNA-Binding Proteins; Escherichia coli Proteins; Fluorescein; Fluoresceins; Fluorescence Polarization - methods; Molecular Probes; Molecular Sequence Data; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Nucleic Acid Amplification Techniques
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/24.2.348

Strand displacement amplification (SDA) is an isothermal in vitro method of amplifying a DNA sequence prior to its detection. We have combined SDA with fluorescence polarization detection. A 5′-fluorescein-labelled oligodeoxynucleotide detector probe hybridizes to the amplification product that rises in concentration during SDA and the single-to double-strand conversion is monitored through an increase in fluorescence polarization. Detection sensitivity can be enhanced by using a detector probe containing an EcoRI recognition sequence at its 5′-end that is not homologous to the target sequence. During SDA the probe is converted to a fully double-stranded form that specifically binds a genetically modified form of the endonuclease EcoRI which lacks cleavage activity but retains binding specificity. We have applied this SDA detection system to a target sequence specific for Mycobacterium tuberculosis.