Soejima Mikiko, Koda Yoshiro
Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume, Japan.
Clin Chem. 2008 Nov;54(11):1908-13. doi: 10.1373/clinchem.2008.113126. Epub 2008 Sep 11.
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).
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.
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.
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.
触珠蛋白基因（HP）有2个常见的共显性等位基因（HP(1)和HP(2)），可产生3种表型。HP(2)是由HP(1)的1.7 kb基因内重复产生的。
我们使用实时TaqMan PCR系统开发了一种有效的HP基因分型方法，通过将扩增信号强度与用作内部对照的HP启动子区域（HP5'）的信号强度进行比较，来评估1.7 kb基因重复的HP(2)等位基因特异性连接区域（HP2）的相对拷贝数。HP2和HP5'之间的阈值循环差异（DeltaCt）用于评估HP(2)拷贝数。此外，该检测方法可同时检测HP缺失（HP(del)）。
从123个已知HP基因型的样本中获得的平均2(-DeltaDeltaCt)值（HP2/HP5'比值）清楚地区分了对应于HP基因型的2个不重叠区间。HP(2)/HP(1)样本的比值范围为0.34 - 0.50，HP(2)/HP(2)样本的比值范围为0.79 - 0.98，未检测到HP(2)等位基因信号被定义为HP(1)/HP(1)。我们同时检测到了HP(del)。该检测方法在<1小时内得出结果。
基于TaqMan的实时PCR方法成功应用于HP基因分型。该方法易于在分子诊断实验室中使用，其稳健性和快速性使其适用于对大量人群的高通量分析。
