Detection of Three Common, Deletional α‐Thalassemia Determinants in Southern China by a Single‐Tube Multiplex Polymerase Chain Reaction Method

Abstract

alpha-Thalassemia (thal) is one of the most common inherited disorders in the world and in Southern China. Three large deletions of the alpha-globin gene, namely the -alpha3.7 and -alpha4.2 (single gene deletions), and --SEA (Southeast Asian double gene deletion), are the main alpha-thal abnormalities in Southern China. We have developed a reliable, single-tube multiplex polymerase chain reaction (m-PCR) assay for these three most frequently observed determinants in Southern China. By using this assay, we detected 40 alpha-thal patients from Guangdong, Guangxi Province, and analyzed 116 blood samples from the Li ethnic group of Hainan Province. To our surprise, the combined incidence of -alpha3.7 and -alpha4.2 was found to be as high as 38.0% among the Li people, and the -alpha4.2 genotype is more frequent than -alpha3.7 in the Li people. No SEA deletions were found in the Li samples.

Molecular diagnosis of α-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis

“…6 Their genotypes were detected before by the single tube multiplex PCR. 5,6 PCR primers: The PCR primers were designed according to the principle of the gap-PCR. The sequence of the primers and the corresponding alleles of the positive PCR results were the same as the previous paper 5 except that the 1.9-Kb amplicon representing the ␣␣ (including ␣ T ␣) alleles was replaced by a 206-bp amplicon obtained by using the following primers (Fig 1): CD 2 =: 5=-CTGCGGGCCTGGG CCG-3= and C==: 5=-GCCCATCGGGCAGGAGGAAC-3=.…”

“…The mPCR/agarose gel electrophoresis: It is described in the previously published paper. 5 Cloning and sequencing: The gap-PCR products were cloned into the plasmid DNA and sequenced by using the routine procedures. 9…”

Improvement in the Detection of α0- and Deletional α-Thalassemia by Real-Time PCR Combined with Dissociation Curve Analysis

“…The primer sequences used for detection of the –α 3.7 deletion were the same as described previously [5]. The reaction mix contained 2.5 µl of 10× buffer (with NH 4 Cl), 2.5 µl of 7-deaza-dGTP/dNTP, 0.02 µl of β-mercaptoethanol, 5 µl of DMSO, 0.5 µl of betaine (final concentration 1.0 mol/l), 0.5 µl of SYBR Green 1, 1 µl of 2 U/µl Taq-Plus DNA Polymerase (Beijing Dingguo Biotech Ltd.).…”

“…The total reaction volume was 25 µl. The PCR conditions for the detection of the –α 4.2 allele were the same as for the –α 3.7 allele, with the exception of different primers [5]. …”

“…Tungwiwat et al [4] developed a real-time quantitative seminested PCR method for identifying fetal α0-thalassemia in maternal plasma to provide a noninvasive prenatal diagnosis. A multiplex PCR-based method [5, 6] and a real-time PCR method [7] have also been developed for detection of fetal α0-thalassemia. The method of Tungwiwat et al [4] requires gel electrophoresis, while the real-time PCR method requires 4 separate reactions [real-time PCR with SYBR Green 1 (SYBR-PCR)/dissociation curve analysis] for each sample to detect the –SEA, 3.7-kb deletion, 4.2-kb deletion and the nondeletional αα/ or α T α/ alleles [7].…”