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

Authors
  • Walker, G T
  • Linn, C P
  • Nadeau, J G
Type
Published Article
Journal
Nucleic acids research
Publication Date
Jan 15, 1996
Volume
24
Issue
2
Pages
348–353
Identifiers
PMID: 8628661
Source
Medline
License
Unknown

Abstract

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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