Rapid Identification of Β-thalassemia Mutations by Mutagenically Separated Polymerase Chain Reaction

نویسنده

  • Waratip Sritong
چکیده

β-thalassemia is a syndrome characterized by a reduction or complete absence of the β-globin chain. More than 25 mutations within and flanking the β-globin gene have been identified as the cause of this syndrome in Thailand. To date, several PCRbased techniques have been employed to rapidly identify the types of β-thalassemia mutations. The recently introduced MS-PCR is among those techniques used for this particular purpose. However, the identification of the β-thalassemia mutations by this technique has never been carried out in Thailand. The main objective of this study was to demonstrate the potential of MS-PCR in the swift and accurate characterization of the βthalassemia mutations commonly seen in northern Thailand. The study was conducted in 60 β-thalassemic patients attending the Thalassemia Clinic, Department of Pediatrics, Maharaj Nakorn Chiang Mai Hospital. The direct DNA preparation from the buffy coat was accomplished using the Chelex method. The MS-PCR was performed in all samples with 3 primers including common, mutant and wild type. The cycle sequencing reaction using the Big Dye Nucleotide Sequencing kit was carried out to confirm the results obtained from the MS-PCR technique. The MS-PCR technique was proved to be acculately capable of detecting 6 common β-thalassemia mutations found in Thailand including codons (Cds) 41/42 (-TTCT), codon (Cd) 26 (G-A), codon (Cd) 17 (A-T), condons(Cds) 71/72 (+A), nt-28 (A-G) and IVS2 – nt 654. By employing the MS-PCR technique, 111 alleles including 54 Cds 41/42 (-TTCT), 30 Cd 26 (G-A), 20 Cd 17 (A-T) and 7 nt-28 (A-G) of the β-globin gene mutations were identified in 56 thalassemic patients. This included 93.3% of the β-thalassemic patients studied in this project. Only 9 alleles in 5 patients, or 6.7% of the studied subjects, were left unidentified and nucleotide sequencing was accomplished to complete the task. This study demonstrated the potential application of the MS-PCR technique in the detection of β-thalassemia mutations. It also indicated that the MS-PCR technique could be an alternative choice for the identification of β-thalassemia mutations which are commonly encountered in northern Thailand. Chiang Mai Med Bull 2004;43(4):133-141.

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تاریخ انتشار 2004