Received on June 20, 2008
Accepted on August 19, 2008

Molecular Diagnostics and Genetics

TaqMan-Based Real-Time PCR for Genotyping Common Polymorphisms of Haptoglobin (HP¹ and HP²)

¹ Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume, Japan

^* To whom correspondence should be addressed. E-mail: ykoda{at}med.kurume-u.ac.jp.

BACKGROUND: The haptoglobin gene (HP) has 2 common codominant alleles (HP¹ and HP²) that account for 3 phenotypes. HP² is generated by a 1.7-kb intragenic duplication of HP¹.

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 HP² 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 (Ct) between HP2 and HP5' was used to assess HP² copy number. In addition, the assay detects the HP deletion (HP^del) at the same time.

RESULTS: The mean 2^-Ct 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 HP²/HP¹ samples ranged from 0.34–0.50, HP²/HP² samples ranged from 0.79–0.98, and the absence of an HP² allele signal was defined as HP¹/HP¹. 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.