Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. DNA Substrates and Oligonucleotides
- 5′-amine REEAD primer: 5′-/AmC6/CCAACCAACCAACCAAATAAGCGATCTTC
- ID Detection probe: 5′-/FAM/CCTCAATGCACATGTTTGGCTCC-3′
- T4 EcoRI PA substrate: 5′-/AmC6/ATTCACTgaattcAGCGCTTAGGAGTGCATATACGATGCACTGTGAAGATCGCTTATGCATCGTATATGCACTCCTAAGCGCT
- T4 EcoRI ID substrate: 5′-/AmC6/ATTTGACgaattcGTCGTATAGGAACTTCGAACGACTCGCCTCAATGCACATGTGGCTCCCGAGTCGTTCGAAGTTCCTATACGACgaattcGTCAAAT/AmC6/-3′
- T4 XhoI PA substrate: 5′-/AmC6/ATTCACTctcgagAGCGCTTAGGAGTGCATATACGATGCACTGTGAAGATCGCTTATGCATCGTATATGCACTCCTAAGCGCT
- T4 XhoI ID substrate: 5′-/AmC6/ATTTGACctcgagGTCGTATAGGAACTTCGAACGACTCGCCTCAATGCACATGTTTGGCTCC-CGAGTCGTTCGAAGTTCCTATAC
- Cre EcoRI PA substrate: 5′-/AmC6/GCGACgaattcAATTATACGAAGTTATTCGCA
- Cre XhoI PA substrate: 5′-/AmC6/GCGACctcgagAATTATACGAAGTTATTCGCA
- TACTGTGAAGATCGCTTATGAATAACTTCGTATAATTctcgagGTCGC/AmC6/-3′
2.3. Purification of T4 DNA Ligase
2.4. Purification of Cre Recombinase
2.5. Preparation of Bacterial Lysates
2.6. Preparation of Functionalized Glass Slide
2.7. T4 DNA Ligase Assisted RE Detection
2.8. Cre Assisted RE Detection
2.9. Statistics
3. Results and Discussion
3.1. T4 DNA Ligase Assisted Restriction Endonuclease Detection
3.2. Specific Detection of Purified RE Activities
3.3. Detection of REs in Cell Extracts
3.4. Specific End-Joining by Cre Recombinase
3.5. Detection of REs with Cre-Assisted RE Detection
4. Conclusions
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Petersen, K.V.; Tesauro, C.; Hede, M.S.; Pages, C.; Marcussen, L.B.; Keller, J.G.; Bugge, M.; Holm, K.; Bjergbæk, L.; Stougaard, M.; et al. Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts. Sensors 2022, 22, 7763. https://doi.org/10.3390/s22207763
Petersen KV, Tesauro C, Hede MS, Pages C, Marcussen LB, Keller JG, Bugge M, Holm K, Bjergbæk L, Stougaard M, et al. Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts. Sensors. 2022; 22(20):7763. https://doi.org/10.3390/s22207763
Chicago/Turabian StylePetersen, Kamilla Vandsø, Cinzia Tesauro, Marianne Smedegaard Hede, Camilla Pages, Lærke Bay Marcussen, Josephine Geertsen Keller, Magnus Bugge, Kasper Holm, Lotte Bjergbæk, Magnus Stougaard, and et al. 2022. "Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts" Sensors 22, no. 20: 7763. https://doi.org/10.3390/s22207763
APA StylePetersen, K. V., Tesauro, C., Hede, M. S., Pages, C., Marcussen, L. B., Keller, J. G., Bugge, M., Holm, K., Bjergbæk, L., Stougaard, M., Wejse, C., & Knudsen, B. R. (2022). Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts. Sensors, 22(20), 7763. https://doi.org/10.3390/s22207763