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Clinical Chemistry 54: 1908-1913, 2008. First published September 11, 2008; 10.1373/clinchem.2008.113126
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(Clinical Chemistry. 2008;54:1908-1913.)
© 2008 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

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

Mikiko Soejima1 and Yoshiro Koda1,a

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

aAddress correspondence to this author at: Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume 830-0011, Japan. 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 ({Delta}Ct) between HP2 and HP5' was used to assess HP2 copy number. In addition, the assay detects the HP deletion (HPdel) at the same time.

Results: The mean 2–{Delta}{Delta}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 HPdel. 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.






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Copyright © 2008 by the American Association for Clinical Chemistry.