DNA DETECTION BY STRAND DISPLACEMENT AMPLIFICATION AND FLUORESCENCE POLARIZATION WITH SIGNAL ENHANCEMENT USING A DNA-BINDING PROTEIN

Strand displacement amplification (SDA) is an isothermal in vitro meth od of amplifying a DNA sequence prior to its detection. We have combin ed SDA with fluorescence polarization detection. A 5'-fluorescein-labe lled oligodeoxynucleotide detector probe hybridizes to the amplificati on product that rises in concentration during SDA and the single- to d ouble-strand conversion is monitored through an increase in fluorescen ce polarization. Detection sensitivity can be enhanced by using a dete ctor probe containing an EcoRI recognition sequence at its 5'-end that is not homologous to the target sequence. During SDA the probe is con verted to a fully double-stranded form that specifically binds a genet ically modified form of the endonuclease EcoRI which lacks cleavage ac tivity but retains binding specificity. We have applied this SDA detec tion system to a target sequence specific for Mycobacterium tuberculos is.