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Extraction of High Molecular Weight DNA from Microbial Mats
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Microbial Redox Regulator-Enabled Pulldown for Rapid Analysis of Plasma Low-Molecular-Weight Biothiols
Jin Oh Lee1,2, Yoon-Mo Yang1, Jae-Hoon Choi1,2
1Department of Life Science , Hanyang University , Seoul 04763 , Republic of Korea.
Analytical Chemistry
|July 10, 2019
Summary
Analyzing plasma low-molecular-weight biothiols is now faster and more cost-effective. A new biosensor platform uses a specific protein and DNA to detect these disease indicators in blood.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Molecular Biology
Background:
- Low-molecular-weight (LMW) biothiols are crucial disease indicators in plasma.
- Analyzing LMW biothiols in extracellular environments is challenging due to oxidative stress and technical limitations.
Purpose of the Study:
- To develop a rapid, cost-effective affinity pulldown platform for analyzing plasma LMW biothiols.
- To enable simultaneous detection of free and total LMW biothiols in biological samples.
Main Methods:
- Utilized a Bacillus subtilis-derived organic hydroperoxide resistance regulatory (OhrRBS) protein and its operator dsDNA.
- Employed an affinity pulldown assay with anti-FLAG antibody-coated beads and FAM-labeled dsDNA.
- Leveraged S-thiolation of OhrRBS by LMW biothiols to trigger dsDNA dissociation and fluorescence signal generation.
Main Results:
- The platform demonstrated rapid and sensitive detection of LMW biothiols through fluorescence intensity changes.
- Successfully extended the method to simultaneously detect free and total LMW biothiols in mouse plasma using a reducing agent.
- Observed elevated total plasma LMW biothiols in ΔLDLR mice compared to normal mice.
Conclusions:
- The developed OhrRBS/dsDNA pulldown platform offers a rapid and cost-effective solution for plasma LMW biothiol analysis.
- This method is suitable for monitoring redox changes and studying oxidative stress and related diseases in blood.
- The platform facilitates immediate application in clinical diagnostics and biomedical research.
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