Molecular analysis
Genomic DNA was extracted from peripheral blood leukocytes using a
standard protocol of phenol-chloroform extraction. Alpha-globin
genotyping was performed by a single-tube multiplex gap polymerase chain
reaction (Gap-PCR) for detecting seven common α-globin deletions
(–SEA, –THAI, -(α)20.5, –FIL, –MED-α3.7, -α4.2)
and a single-tube multiplex amplification refractory mutation system
(ARMS-PCR) for screening six common non-deletional α-globin mutations in
Thailand: initiation codon (ATGA-G), codon 30 (ΔGAG), codon 59 (GGCGAC),
codon 125 (CTGCCG) or Hb QuangSze, termination codon (TAACAA) or Hb
Constant Spring, and a termination codon (TAATAT) or Hb
Paksé.31 Beta-globin genotyping was performed by
ARMS-PCR for detecting 16 common beta-globin mutations (-28, CD8/9,
CD17, CD19, CD26 (Hb E), CD26 G>T (stop codon), CD27/28,
IVSI-I, IVSI-5, CD35, CD41, CD41/42, CD43, CD71/72, CD95, and
IVSII-654).32 A single-tube multiplex Gap-PCR and
enzymatic amplification was used for common beta-globin gene deletions
(3.48 kb, 619 bp, Filipino (β)°, SEA HPFH (β)°, ChineseGγ( Aγδβ)°, Thai (δβ)°, Hb Lepore,
HPFH-6 Gγ( Aγδβ)°, Siriraj-thalGγ( Aγδβ)°, Asian Indian type A, and
Asian Indian type B).33,34 Hemoglobin E testing was
studied by restriction fragment length polymorphism (RFLP)-PCR
utilizing Mnl I restriction enzyme.35