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J Clin Lab Anal. 2016 Sep;30(5):633-40. doi: 10.1002/jcla.21914. Epub 2016 Feb 18.

Scanning for α-Hemoglobin Variants by High-Resolution Melting Analysis.

Author information

1
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
2
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. sumalee.jin@mahidol.ac.th.

Abstract

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.

KEYWORDS:

DNA sequencing; hemoglobin typing; hemoglobin variant; high-resolution melting (HRM) analysis; quantitative PCR

PMID:
26892340
DOI:
10.1002/jcla.21914
[Indexed for MEDLINE]

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