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Low fluorescence background electroblotting membrane for DNA sequencing
T J Chu1, K D Caldwell, R B Weiss
1Department of Materials Science, University of Utah, Salt Lake City.
Electrophoresis
|March 1, 1992
Summary
A new polypropylene membrane offers improved DNA binding for fluorescence-based DNA sequencing. Surface modification enhances its capacity for radioisotope-labeled DNA, proving stable through multiple cycles.
Area of Science:
- Biotechnology
- Materials Science
- Molecular Biology
Background:
- Polypropylene (PP) membranes are crucial for electroblotting in DNA sequencing.
- Existing membranes may have limitations in DNA binding capacity and stability for multiplex applications.
Purpose of the Study:
- To develop a low fluorescence background PP membrane for enhanced DNA sequencing.
- To improve DNA binding capacity and stability for multiplex processing.
Main Methods:
- Surface modification of PP membrane using radio frequency plasma discharge (RFPD) in ammonia gas.
- Evaluation of RFPD parameters, nitrogen content, and DNA binding capacity.
- Characterization of surface morphology (SEM), mechanical, and electrical properties.
- Assessment of DNA covalent attachment for multiplex hybridization/stripping cycles.
Main Results:
- RFPD treatment significantly improved DNA binding capacity compared to unmodified PP.
- Modified membrane exhibited comparable or slightly better binding of electroblotted DNA than GeneScreen membrane.
- The modified membrane demonstrated stability through tenfold probing cycles with minimal signal loss.
- Membrane-bound DNA remained stable during storage for one month with no detectable loss in probing efficiency.
Conclusions:
- The developed low fluorescence PP membrane is suitable for electroblotting in DNA sequencing.
- Surface modification via RFPD enhances DNA binding and stability for multiplex applications.
- The modified membrane offers a promising alternative for sensitive and robust DNA analysis.