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Cytogenetics and Molecular Genetics

Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats

Leukemia volume 25, pages 1467–1470 (2011)Cite this article

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Abstract

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30 000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for ‘small-scale’ DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.

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Acknowledgements

We thank the European Union for support of the EuroChimerism Consortium (project QLRT-2001-01485).

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Authors and Affiliations

  1. Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

    M van der Burg, B H Barendregt & J J M van Dongen

  2. Department of Pediatrics, Frankfurt, Germany

    H Kreyenberg, A Willasch & P Bader

  3. Department of Pediatrics, Erasmus MC, Rotterdam, The Netherlands

    B H Barendregt

  4. Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria

    S Preuner, F Watzinger & T Lion

  5. Division of Hematology, Department of Internal Medicine, Geneva University Hospitals and University of Geneva, Switzerland

    E Roosnek

  6. H & I Laboratory, National Health Service Blood and Transplantation, Filton, Bristol, UK

    J Harvey

  7. Department of Hematology, Hospital Clinico Universitario, Salamanca, Spain

    M Alcoceba & M G Díaz

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  1. M van der Burg
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  2. H Kreyenberg
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  3. A Willasch
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  5. S Preuner
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  7. T Lion
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  8. E Roosnek
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  9. J Harvey
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  10. M Alcoceba
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  11. M G Díaz
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  12. P Bader
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  13. J J M van Dongen
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on behalf of the EU-supported EuroChimerism Consortium (project QLRT-2001-01485)

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Correspondence to M van der Burg.

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van der Burg, M., Kreyenberg, H., Willasch, A. et al. Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats. Leukemia 25, 1467–1470 (2011). https://doi.org/10.1038/leu.2011.118

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  • DOI: https://doi.org/10.1038/leu.2011.118

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