Huh Ji Young, Park Geon, Jang Sook Jin, Moon Dae Soo, Park Young Jin
Department of Laboratory Medicine, CHA Bundang Medical Center, Gyeonggi-do, South Korea.
Ann Clin Lab Sci. 2011 Fall;41(4):340-5.
ABO is the most clinically important blood group system in transfusion and transplantation medicine. The popular ABO genotyping methods, such as the sequencing of exons 6 and 7 and sequence-specific primer (SSP)-PCR, often lead to ambiguous typing results. Long PCR-sequencing method was designed to analyze two regulatory regions (promoter and CBF/NF-Y enhancer regions) and all genomic sequences (except for intron 1) of the ABO gene. Using rapid DNA polymerase with high-fidelity, we amplified 6.3 kb and 7.3 kb for sequencing of enhancer-exon 1 and exons 2-7, respectively. ABO genotyping was performed using this technique in the peripheral blood of three unrelated families. The time requirements of the PCR amplification and purification processes were about 2.0 hours and 15 minutes, respectively. Five different ABO alleles (ABO A102, ABO A105, ABO O01, ABO O02, and ABO B101) with allele-specific CBF/NF-Y minisatellite repeats from three families were analyzed. All genotyping results agreed with serologic findings and results expected by Mendelian inheritance. Compared to conventional PCR-direct sequencing for ABO genotyping, this method proves simple and fast for the analysis of ABO genotypes. Therefore, it might be valuable in clinical transfusion or forensic applications.
ABO血型系统是输血和移植医学中临床上最重要的血型系统。常用的ABO基因分型方法，如外显子6和7测序以及序列特异性引物（SSP）-PCR，常常导致分型结果不明确。长PCR测序方法旨在分析ABO基因的两个调控区域（启动子和CBF/NF-Y增强子区域）以及所有基因组序列（除内含子1外）。使用具有高保真度的快速DNA聚合酶，我们分别扩增了6.3 kb和7.3 kb用于增强子-外显子1和外显子2-7的测序。使用该技术对三个无关家庭的外周血进行ABO基因分型。PCR扩增和纯化过程的时间要求分别约为2.0小时和15分钟。分析了来自三个家庭的具有等位基因特异性CBF/NF-Y微卫星重复序列的五种不同ABO等位基因（ABO A102、ABO A105、ABO O01、ABO O02和ABO B101）。所有基因分型结果均与血清学结果以及孟德尔遗传预期结果一致。与传统的ABO基因分型PCR直接测序相比，该方法被证明在分析ABO基因型方面简单快速。因此，它在临床输血或法医应用中可能具有价值。
