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Optimizing DNA Detection
Andrew Dubitsky
Pall Corporation, 25 Harbor Park Drive, Port Washington, NY
The performance of DNA detection systems can be optimized by adjusting process
parameters. Examples are presented to demonstrate that the desired combination of high
sensitivity and low background can be achieved by adjusting key steps.
Sensitivity in a DNA detection system can be affected by a variety of factors:
- Use of alkaline gel and neutral transfer buffer can increase sensitivity;
- Choice of membrane and fixation conditions must be optimized for each detection system;
- Concentration of solutions used for blocking steps, including pre-hybridization,
hybridization and conjugate steps, should be selected so that minimum concentrations
resulting in acceptable background are used;
- Probe concentration should be adjusted for highest sensitivity;
- Autorad exposure time can be adjusted for best signal to noise ratio, or maximum
sensitivity;
Background signal level can also be improved, as follows:
- Different membrane chemistries result in varying levels of background, due to the
membrane's specific affinity for reagents used in a given system;
- Probe agglomerates can be eliminated by centrifuging probe preparations before use;
- Insufficient block of the membrane can be overcome by increasing blocking
effectiveness-by the choice and concentration of blocking agent;
- Bacterial enzymes contaminating buffer solutions can also cause background signal.
Proper filtration of solutions before use will remove bacterial sources of enzyme.
Laboratories starting with a new detection system should perform pilot experiments to
determine the best combination of sensitivity and background. After pilot tests are
completed, a full scale usage test is run to confirm the performance of the system.
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