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Updated: Jul 19, 2026
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Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51
Published on: February 13, 2019
Real-time, sequence-specific detection of nucleic acids during strand displacement amplification
J G Nadeau1, J B Pitner, C P Linn
1Department of Life Sciences, Becton Dickinson Technologies, 21 Davis Drive, Research Triangle Park, North Carolina 27709, USA.
Analytical Biochemistry
|December 22, 1999
Summary
New fluorogenic probes enable real-time, sequence-specific detection during Strand Displacement Amplification (SDA). This method allows for sensitive quantification of nucleic acid targets in a closed-tube format.
Area of Science:
- Molecular Biology
- Biotechnology
- Nucleic Acid Amplification
Background:
- Strand Displacement Amplification (SDA) is a key isothermal nucleic acid amplification technique.
- SDA relies on restriction enzyme nicking and exonuclease-deficient polymerase activity.
- Real-time detection of amplified targets is crucial for monitoring and quantification.
Purpose of the Study:
- To develop novel fluorogenic reporter probes for real-time, sequence-specific detection during SDA.
- To enable sensitive and quantitative monitoring of nucleic acid amplification.
- To create a closed-tube detection system minimizing contamination risks.
Main Methods:
- Design of fluorogenic probes incorporating a BsoBI recognition sequence, fluorophore, and quencher.
- Utilizing probe cleavage by BsoBI upon target binding to release fluorescence.
- Implementing a two-color competitive SDA format for quantification.
Main Results:
- Demonstrated real-time, sequence-specific detection of SDA targets.
- Achieved detection of as few as 10 target copies within 30 minutes.
- Validated RNA target detection in SDA mixtures with reverse transcriptase.
- Showcased accurate quantification using a two-color competitive format.
Conclusions:
- The developed fluorogenic probes provide a sensitive and specific method for real-time SDA monitoring.
- The closed-tube, real-time format enhances safety by preventing carry-over contamination.
- This technology offers a robust platform for nucleic acid quantification and detection.
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