Myeloid Leukemia

86 Branford and Hughes
Fig. 2. Impact of a polymorphism at the reverse primer binding site on quantitation
of BCR-ABL transcript levels. In a subset of patients with the b2a2 BCR-ABL transcript,
the initial quantitative levels were up to 10-fold lower than expected. Sequencing
revealed a polymorphism located at the reverse primer hybridization site. The
single base mismatch significantly affected the binding of the primer to the template
and thus the accuracy of quantitation. Re-designing the primer to avoid the polymorphism
significantly improved the results. A represents the b2a2 BCR-ABL transcript
level of 10 patients who did not have the polymorphism. The white columns represent
the value using the original primer that hybridized at the polymorphic site, and the
shaded columns represent the value using the re-designed primer that did not hybridize
at the polymorphic site. There was no difference in the results with either primer. B
represents the b2a2 BCR-ABL transcript level of 10 patients who did have the polymorphism.
There was up to a 10-fold lower quantitative value when the original primer
was used. In patient 10, the value was falsely negative. The graphs demonstrate that
the accuracy of quantitation may be compromised when there is a single base mismatch
at a primer site.