[Establishment of multiplex hybri-probe method--analysis using the polymorphism of UGT1A1 gene]

There are a number of methods for gene analysis of a point mutation and deletion/insertion of several nucleotides. In 2011, we reported an improved hybridization probe methods (Hybri-Probe method) that are highly sensitive and accurate, and excellent in cost and time effectiveness. Here, we have developed the Multiplex Hybri Probe method for several types of mutations or polymorphisms including the microsatellite polymorphisms, especially of palindromic sequence such as (TA)n and (GC)n. In addition, several mutations are analyzed at a time. In this research we focused on the three types of polymorphism on the Uridine diphosphate glucuronyltransferase (UGT) gene. Design of the probes for the detection of UGT1A1*6(211 G --> A G71R) and UGT1A1*27 (686 C --> A P229Q) was not difficult because the mutations were a single base substitution. However, UGT1A1*28 (A (TA)6TAA --> A (TA)7TAA) has tandem and palindromic sequence. Since the probes to detect the mutation in such sequence resulted in failure, we made several mismatches, i.e., TATATATATATA --> TATGTGTATATA. As a result, the probes designed for the three polymorphisms above did not overlap in the Tm and were separated by approximately 10 degree intervals between 63.2 degrees C and 37.5 degrees C. In this Multiplex Hybri-Probe method, the three kinds of probe are added tube into the PCR product in the same tube and followed by the measurement of the Tm using the LightCycler. Thus, it is very simple, and performed by one step for any mutation including the microsatellite polymorphisms, and it also has good cost performance and favorable time efficiency. It takes two hours including the running time of PCR for completion of the analysis. It may also be available to the detection of other gene abnormalities.