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Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories
1Plant Research Centre, Central Experimental Farm, Agriculture Canada, Ottawa, Ontario.
PCR Methods and Applications
|May 1, 1993
Summary
Random amplified polymorphic DNA (RAPD) analysis is a cost-effective DNA fingerprinting method. However, inter-laboratory reproducibility of RAPD results is challenging due to variations in fragment amplification and temperature profiles.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Random amplified polymorphic DNA (RAPD) analysis offers a cost-effective alternative to restriction fragment-length polymorphism (RFLP) for DNA fingerprinting.
- Concerns exist regarding the inter-laboratory reproducibility of RAPD results, hindering widespread adoption and comparison.
Purpose of the Study:
- To assess the inter-laboratory reproducibility of Random amplified polymorphic DNA (RAPD) analysis.
- To identify factors affecting the reproducibility of RAPD results across different laboratories.
Main Methods:
- Five primers were used to amplify polymorphic DNA fragments between two oat cultivars in one laboratory.
- These amplified fragments were then tested for reproducibility in six other North American laboratories using the same and their own protocols.
Main Results:
- Four out of six participating laboratories showed poor amplification or no fragments using the original protocol.
- Variations in amplified fragment size ranges and inconsistent annealing temperatures significantly impacted reproducibility.
- Reproducible results were achieved with four of the five primers, but not the fifth, under identical conditions.
Conclusions:
- Inter-laboratory reproducibility of RAPD analysis is achievable but requires stringent standardization of protocols, particularly temperature profiles.
- Optimizing annealing temperatures and fragment size detection is crucial for reliable RAPD data comparison across different labs.