1. Designing a fluorescence padlock probe-based biosensor and colorimetric assay for the detection of G12D KRAS mutation
- Author
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Mohammad Hossein Ghahremani, Gholamreza Tavoosidana, Fatemeh Mahmoudian, Mostafa Akbariqomi, Reza Heidari, Samaneh Fathi, Nader Roshan, Fatemeh Karimi, Samira Bahrami, Moloud Absalan, and Mahdi Adabi
- Subjects
Detection limit ,Chromatography ,business.industry ,Point mutation ,Biochemistry (medical) ,Clinical Biochemistry ,medicine.disease_cause ,Target concentration ,Fluorescence ,Rolling circle replication ,Drug Discovery ,medicine ,KRAS ,business ,Biosensor ,Kras mutation - Abstract
Aim: Cell-free DNA in the plasma is known to be a potential biomarker for noninvasive diagnosis of oncogenic mutations. The authors aimed to design an optimized padlock probe-based hyperbranched rolling circle amplification biosensor to detect the KRAS G12D mutation using fluorescence and colorimetric methods. Methods: Single-factor experiments, Plackett–Burman design and response surface methodology were applied to optimize the padlock probe-based hyperbranched rolling circle amplification reaction. Results: The maximum fluorescence intensity was achieved at a padlock probe concentration of 1.5 pM and target concentration of 9 pM at 38°C ligation temperature. The proposed biosensor has a low detection limit of 60 fM of target DNA and a linear response in the concentration range of 60 fM to 0.2 pM. Conclusion: The results indicated the power of these assays to detect KRAS point mutations in liquid state reactions.
- Published
- 2021
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