Simple and precise detection of UGT1A1 polymorphisms with a modified loop-hybrid mobility shift assay using Cy5-labeled loop probes

Abstract

Background: Irinotecan, an inhibitor of topoisomerase I, has been widely used as an important anti-cancer therapeutic drug. Deleterious effects of the drug in hypersensitive patients are known to be associated with genetic polymorphisms of the UGT1A1 gene, namely the polymorphic variants, *28 and *6.

Methods: A modified form of loop-hybrid mobility shift assay using a Cy5-tagged loop-hybrid probe was proposed as a precise and easy method of determining TA repeat polymorphisms at the *28 locus.

Results: In this modified method, only loop-hybrid bands were detected by a Cy5-fluorescent signal, despite several irregular electrophoretic bands due to TA repeats in the PCR product.

Conclusions: When a loop-hybrid using a Cy5-tagged probe for the *28 locus and *6 locus were combined and used for mobility shift assay, simultaneous typing of the *28 and *6 variants was achieved in a single lane.