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Strategy for controlling preferential amplification and avoiding false negatives in PCR typing
T Weissensteiner1, J S Lanchbury
1UMDS, London, England, UK. tweissen@hgmp.mrc.ac.uk
Biotechniques
|December 1, 1996
Summary
This study introduces a two-step method to prevent false negatives in Polymerase Chain Reaction (PCR) testing. By optimizing PCR conditions and using additives, reliable multiplex PCR results can be achieved for difficult targets.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Polymerase Chain Reaction (PCR) is widely used for routine testing, often involving multiplex assays.
- Preferential amplification of certain products in multiplex PCR can lead to false negatives, especially under suboptimal conditions.
- Stable double-stranded DNA (dsDNA) regions in amplified sequences can cause false negatives, even with optimized PCR parameters.
Purpose of the Study:
- To develop a strategy to overcome false negatives in multiplex PCR assays.
- To improve the reliability of PCR typing for challenging targets.
- To enhance the accuracy of both qualitative and quantitative multiplex PCR applications.
Main Methods:
- A two-step strategy was employed to address false negatives in PCR.
- Step 1: Titration of PCR with NaCl to identify the critical range for false negatives.
- Step 2: Titration with a dsDNA-destabilizing additive (betaine) to equalize amplification efficiencies.
Main Results:
- The proposed two-step strategy effectively prevents false negatives in multiplex PCR.
- Betaine was identified as a novel and efficient cosolute for stabilizing PCR reactions.
- Relative amplification efficiencies of co-amplified fragments were successfully adjusted.
Conclusions:
- The presented method ensures reliable PCR typing, particularly for difficult targets.
- This approach is valuable for various qualitative and quantitative multiplex PCR applications.
- Optimization using NaCl titration and dsDNA-destabilizing additives enhances PCR accuracy.