Open AccessScanning for α‐Hemoglobin Variants by High‐Resolution Melting Analysis
Author(s) -
Yimniam Walaiporn,
Jindadamrongwech Sumalee
Publication year - 2016
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.21914
Subject(s) - high resolution melt , exon , primer (cosmetics) , microbiology and biotechnology , genetics , amplicon , biology , polymerase chain reaction , hemoglobin variants , point mutation , hemoglobin , dna sequencing , sequence analysis , primer dimer , mutation , dna , gene , chemistry , multiplex polymerase chain reaction , biochemistry , organic chemistry
Background Definitive detection of hemoglobin (Hb) variants requires DNA sequencing. High‐resolution melting (HRM) analysis of polymerase chain reaction (PCR) amplicons was applied to detect and discriminate among uncommon α‐Hb variants found in Thailand. Methods Uncommon suspected α‐Hb variants observed in Hb typing were identified by sequencing of DNA from whole blood samples. Three pairs of PCR primers covering the mutation regions in the three α‐globin exons then were used for PCR coupled with difference in HRM analysis to subtract out the concomitant melting profile of the normal allele in the heterozygous state. Results DNA sequencing identified six heterozygous α‐Hb variants, namely, Hb G‐Waimanalo (HBA2: exon 2, codon 64 G>A), Hb J‐Buda (HBA1: exon 2, codon 61 G>T), Hb Kurosaki (HBA2: exon 1; codon 7 A>G), Hb O‐Indonesia (HBA1: exon 3 codon 116 G>A), Hb Q‐India (HBA1:exon 2, codon 64 G>C), and Hb Q‐Thailand (HBA1: exon 2 codon 74 G>C). Difference HRM analysis showed one temperature melting profile using exon 1 primer pair, four different profiles with exon 2 primer pair, and one profile with exon 3 primer pair. Conclusions PCR‐HRM analysis was effective in detecting and discriminating among single point mutations causing six uncommon α‐Hb variants in heterozygous individuals. The method can be applied for routine screening due to its simplicity and relatively low cost.