TaqMan-based real-time PCR for genotyping common polymorphisms of haptoglobin (HP1 and HP2).

BACKGROUND: The haptoglobin gene (HP) has 2 common codominant alleles (HP(1) and HP(2)) that account for 3 phenotypes. HP(2) is generated by a 1.7-kb intragenic duplication of HP(1).
METHODS: We used the real-time TaqMan PCR system to develop an effective method for HP genotyping that allows us to evaluate the relative number of copies of the HP(2) allele-specific junctional region of the 1.7-kb gene duplication (HP2) by comparing the intensity of the amplification signals to those of the HP promoter region (HP5'), which was used as the internal control. The difference in threshold cycles (DeltaCt) between HP2 and HP5' was used to assess HP(2) copy number. In addition, the assay detects the HP deletion (HP(del)) at the same time.
RESULTS: The mean 2(-DeltaDeltaCt) values (the HP2/HP5' ratio) obtained from 123 samples of known HP genotypes clearly differentiated 2 nonoverlapping intervals that correspond to the HP genotypes. Ratios for HP(2)/HP(1) samples ranged from 0.34-0.50, HP(2)/HP(2) samples ranged from 0.79-0.98, and the absence of an HP(2) allele signal was defined as HP(1)/HP(1). We simultaneously detected HP(del). The assay produces results in <1 h.
CONCLUSIONS: The TaqMan-based real-time PCR method was successfully applied to HP genotyping. The method is easy to use in a molecular diagnosis laboratory, and its robustness and rapidity make it suitable for high-throughput analysis of large populations.