DNA detection by strand displacement amplification and fluorescence polarization with signal enhancement using a DNA binding protein.
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.
Walker
G T
GT
Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016, USA.
Linn
C P
CP
Nadeau
J G
JG
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins; D015246:Deoxyribonuclease EcoRI; D029968:Escherichia coli Proteins; D019793:Fluorescein; D005452:Fluoresceins; D005454:Fluorescence Polarization / Q000379:methods*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005452:Fluoresceins; C070851:Gln-111 protein, E coli; D015335:Molecular Probes; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D019793:Fluorescein