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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
PubMed
Summary
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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.

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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.