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Appendix: Method In Focus

Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms

Leukemia volume 17, pages 630–633 (2003)Cite this article

The quantitative method is based on a real-time PCR (Perkin-Elmer Applied Biosystems, ABI Prism 7700), with allele-specific primers for DNA sequences containing single nucleotide polymorphisms (SNPs) and target DNA-specific probes.1,2 The nonextendible hybridization probes are labeled with a reporter fluorescent dye at the 5′ end and a quencher at the 3′ end, which results in a fluorescence signal after cleavage by 5′–3′ nuclease activity of Taq polymerase. A charge-coupled device camera attached to ABI Prism 7700 detects target-specific signals at a threshold of 10 standard deviations above the baseline fluorescence. Normalized signals minus baseline signals of the reporter dyes (ΔRn) are plotted against PCR cycle numbers, which results in a logarithmic amplification function. The cycle number of DNA amplification that generates the first specific fluorescence signal (ΔRn) above the threshold is called cycle threshold (Ct). Therefore, Ct represents directly the relative amount of target DNA in the analyzed samples.

Allele-specific amplification revealed that each biallelic SNP acts as a specific marker in 24–50% of 80 sibling pairs. The discriminative capacity of each SNP marker depends on the frequency of the SNPs in the human population. Calculation of the percentage donor and recipient cells is based on the relation of specific amplification signals from both recipient and donor DNA to specific amplification signals obtained by calibration curves for both recipient and donor markers. The amount of input DNA is simultaneously calibrated by amplifying a DNA fragment of the albumin gene.

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Acknowledgements

Supported by the Dutch Cancer Foundation Grant KUN 96-1363.

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Authors and Affiliations

  1. Central Hematology Laboratory, University Medical Center St Radboud, Nijmegen, The Netherlands

    F Maas, S Kolen, A Zoetbrood, H Dolstra & E van de Wiel-van Kemenade

  2. Department of Hematology, University Medical Center St Radbod, Nijmegen, The Netherlands

    N Schaap, T de Witte & A Schattenberg

  3. Bone Marrow Transplantation Unit, Hosp. G.U. Gregorio Marañon, Madrid, Spain

    I Buño

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  1. F Maas
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  2. N Schaap
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  3. S Kolen
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  8. A Schattenberg
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  9. E van de Wiel-van Kemenade
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Maas, F., Schaap, N., Kolen, S. et al. Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms. Leukemia 17, 630–633 (2003). https://doi.org/10.1038/sj.leu.2402857

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