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Nucleic Acids Research, Vol 24, Issue 2 348-353, Copyright © 1996 by Oxford University Press

ARTICLES

DNA detection by strand displacement amplification and fluorescence polarization with signal enhancement using a DNA binding protein

GT Walker, CP Linn and JG Nadeau
Becton Dickinson Research Center, Research Triangle Park, NC 27709- 2016, USA.

Strand displacement amplification (9SDA) 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.
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