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KMID : 0387720130240010071
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Korean Journal of Blood Transfusion
2013 Volume.24 No. 1 p.71 ~ p.78
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High-Resolution Melting Analysis for Genotyping Duffy Blood Group Antigens
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Lim Chae-Seung
Kim Kyung-Hee
Chung So-Young
Kim Yoon-Ho
Choi Jae-Yeol
Kim Il-Tae
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Abstract
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Background:Accurate typing of Duffy blood group is important because anti-Duffy antibodies cause hemolytic transfusion reaction and hemolytic disease of the newborn. The aim of this study was to evaluate a new genotyping method using high resolution melting (HRM) analysis, a rapid and inexpensive approach for high-throughput Duffy genotyping. Methods:A total of 20 unrelated Korean blood samples were obtained and an African-black sample was used for GATA control. Phenotyping was performed by hemagglutination (DiaMed AG, Switzerland). GATA and FYA/B PCR products were obtained by PCR-restriction fragment length polymorphism (RFLP) using Taq DNA polymerase (Promega, WI) and enzymes BanI and StyI (New England Biolab, UK). For HRM, PCR amplification was performed using LightCycler 480 ResoLight Dye (Roche, USA) and Lightcycer 480 (Roche, USA). Results:Phenotyping and genotyping data using PCR-RFLP and HRM analysis were compared. Different types of HRM curves were obtained according to genotypes, FYA/FYA, FYB/FYB, and FYA/FYB, and to GATA mutations, homozygote FYB-33T (T/T), heterozygote FYB-33T/33C (T/C), and homozygote FYB-33C (C/C). Phenotypes 18 Fy(a＋b?), 1 Fy(a＋b＋), 1 Fy(a?b＋), and 1 Fy(a?b?) showed complete concordance with genotyping methods. Fy(a?b?) sample was found to be a FYB-33C homozygote by both genotyping methods. Conclusion:Phenotyping and genotyping showed concordant results and both genotyping methods using PCR-RFLP and HRM analysis showed good agreement in finding mutation in GATA and FY gene coding regions. HRM analysis is suitable and reliable for high-throughput screening for Duffy genotyping.
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KEYWORD
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Duffy blood group, Duffy genotyping, High resolution melting analysis, PCR-restriction fragment length polymorphism
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