Related Experiment Videos
Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA analyses.
G C Saunders1, J Dukes, H C Parkes
1Life Sciences Research, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, United Kingdom.
Clinical Chemistry
|January 10, 2001
Summary
Temperature calibration is crucial for reproducible polymerase chain reaction (PCR) results. Calibrated thermal cyclers significantly improve the consistency of random amplified polymorphic DNA (RAPD) assays compared to non-calibrated ones.
Area of Science:
- Molecular Biology
- Genetics
Background:
- Interlaboratory comparisons of PCR data necessitate assay reproducibility.
- Thermal cyclers significantly influence PCR performance, affecting interblock reproducibility and intrablock repeatability.
Purpose of the Study:
- To investigate the impact of various thermal cyclers on PCR performance.
- To assess the contribution of thermal cycler variability to assay reproducibility.
Main Methods:
- An interlaboratory study involving 18 UK labs and 33 thermal cyclers.
- Evaluation of two standardized PCR assays: a single-product PCR and a random amplified polymorphic DNA (RAPD) PCR.
- Analysis of assay repeatability based on temperature calibration, control mechanisms, cycler age, and accessory systems.
Main Results:
- All labs successfully amplified the single-product PCR target, with minor yield variations.
- RAPD reactions showed significant variability within and between thermal cyclers.
- Intrablock repeatability for RAPD was 88% in calibrated cyclers versus 63% in non-calibrated ones.
Conclusions:
- Temperature-calibrated thermal cyclers consistently yield more repeatable RAPD data.
- Instrument calibration is key for reliable PCR intercomparisons.
- Guidelines for optimizing and monitoring thermal cycler performance are provided.