Comparison Between Three Molecular Diagnostics for the Identification of Heterozygous Hemoglobin E

Pak J Biol Sci. 2020 Jan;23(1):17-26. doi: 10.3923/pjbs.2020.17.26.


Background and objectives: Hemoglobin E is a variant hemoglobin caused due to the base substitution G→A at codon 26 in the β-globin-coding gene that is followed by the alteration of glutamic acid (GAG) to lysine (AAG). Various types of molecular analysis methods such as tetra-primer amplification refractory mutation system (T-ARMS-PCR), Tm-shift real-time polymerase chain reaction (Tm-shift qPCR) and high-resolution melting analysis (HRMA) are commonly used to detect several mutations in the β-globin-coding gene. This study was conducted to compare the detection result of Cd 26 (G→A) mutation in the β-globin-coding gene of heterozygous HbE between the above-mentioned methods.

Materials and methods: DNA samples were isolated from blood archive of heterozygous HbE and analyzed for the detection of the mutation using HRMA and Tm-shift on a real-time PCR instrument, whereas T-ARMS analysis was performed on a conventional PCR equipment. High resolution melt v3.1 software and Bio-Rad CFX Manager software were used to analyze the result of HRMA and Tm-shift qPCR, whereas the T-ARMS-PCR result was analyzed by observing the number and size of DNA bands on gel electrophoresis.

Results: Among 21 samples, the Cd 26 mutation was detected in numbers 18, 19 and 21 by HRMA, Tm-shift qPCR and T-ARMS-PCR. DNA Sequencing confirmed Cd 26 mutation on 5 ambiguous samples and revealed two homozygous mutation.

Conclusion: The Cd 26 (G→A) mutation was detected in proportions 100, 91 and 86% by T-ARMS-PCR, Tm-shift qPCR and HRMA, respectively.

Keywords: Cd 26 (G→A); HRMA; HbE mutation; T-ARMS-PCR; Tm-shift qPCR.

MeSH terms

  • Base Sequence
  • Hemoglobin E / genetics*
  • Hemoglobin E / metabolism
  • Heterozygote
  • Homozygote
  • Humans
  • Mutation
  • Pathology, Molecular / methods*
  • Polymerase Chain Reaction


  • Hemoglobin E