Contribution of the TA repeats on melting temperature (T(m)) in a double strand DNA: Comparison of two methods and implications in molecular diagnostics

The present paper is aimed to discuss and revise a not recent molecular laboratory protocol describing the behavior of UGT1A1 gene melting curves. Since some problems in the standardization of this already published molecular protocol were found, we tried to improve this method in order to correctly set up the amplification and melting profile steps. Under our standardized conditions, we were able to perfectly distinguish the three different UGT1A1 genotypes since each one showed a peculiar melting behavior, as compared with those previously published by another group. Finally, some suggestions and indications are provided for laboratory specialists interested in Gilbert syndrome diagnostics. Based on these results, we underline the need of more standardized protocols above all when they are used for clinical diagnostics.