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Interference of dye-associated fluorescence signals with quantitative analysis of chimerism by capillary electrophoresis

Leukemia volume 17, pages 221–223 (2003)Cite this article

Fluorescence-based detection of PCR products separated by capillary electrophoresis has become an important technical approach to the investigation of chimerism after allogeneic stem cell transplantation.1 Highly polymorphic loci including short tandem repeats (STRs)1,2,3,4,5and variable number of tandem repeats (VNTRs)6,7,8 can be analyzed with great precision. The fluorescence-labelled PCR fragments are electrophoretically separated, and signals are automatically sized and quantitated,1,9 thus eliminating the need for post-electrophoresis handling. This greatly reduces the hands-on time, and the risk of contamination by PCR products.

These signals are derived from exceedingly high specific signals that are off-scale (Figure 1a). They are generated by overlapping spectral emission of the fluorescent dyes used,10 and appear at the position of specific peaks in all color channels. For example, in Figure 1a, the high peak (arrow) detectable in the HEX-channel results from an off-scale FAM peak. The occurrence of pull-up peaks therefore plays a role in multiplex PCR assays using differently labelled primers.

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  1. Children's Cancer Research Institute, Vienna, Austria

    E Schraml & T Lion

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Schraml, E., Lion, T. Interference of dye-associated fluorescence signals with quantitative analysis of chimerism by capillary electrophoresis. Leukemia 17, 221–223 (2003). https://doi.org/10.1038/sj.leu.2402755

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