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Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
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Highly specific DNA detection employing ligation on suspension bead array readout.
Anja Mezger1, Malte Kühnemund2, Mats Nilsson1
1Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden.
New Biotechnology
|February 15, 2015
Summary
Monomerized rolling circle amplification (RCA) products enable direct detection on Luminex bead arrays, eliminating PCR. This method enhances signal-to-noise for sensitive, reproducible DNA detection and multiplexing.
Area of Science:
- Molecular Biology
- Biotechnology
- Genomics
Background:
- Rolling Circle Amplification (RCA) is a method for amplifying nucleic acids.
- Luminex suspension bead arrays offer multiplexed detection capabilities.
- Current methods often require additional amplification steps like PCR.
Purpose of the Study:
- To demonstrate direct detection of monomerized RCA products using Luminex bead arrays.
- To improve signal-to-noise ratio for enhanced detection sensitivity.
- To validate the method for detecting pathogenic DNA sequences.
Main Methods:
- Utilized monomerized RCA products for direct detection.
- Employed padlock probes for DNA targeting.
- Integrated detection oligonucleotide ligation to barcode sequences on Luminex beads.
- Optimized washing steps and increased measurement temperature.
Main Results:
- Achieved direct detection of monomerized RCA products without PCR.
- Demonstrated a higher signal-to-noise ratio through optimized ligation and detection conditions.
- Successfully detected pathogenic DNA sequences with high reproducibility and sensitivity.
- Exhibited a dynamic detection range spanning four orders of magnitude.
Conclusions:
- Monomerized RCA products can be directly detected on Luminex bead arrays.
- The developed method offers a sensitive, reproducible, and highly multiplexable approach for DNA detection.
- This technique holds promise for advanced applications in DNA targeting and readout.
