One single-tube PCR assay to detect both CD17 (A>T) and IVS-II nt-654 (C>T) homozygous mutations of β-thalassemia
One single-tube PCR assay to detect both CD17 (A>T) and IVS-II nt-654 (C>T) homozygous mutations of β-thalassemia
Aim: Several methods can be used to detect gene mutations associated with β-thalassemia, but it is difficult to achieve reliable and reproducible results. In this study, we introduce a new method, a single-tube multiplex polymerase chain reaction (PCR) assay, to detect both CD17 (A>T) and IVS-II nt-654 (C>T) homozygous mutations. Materials and methods: This new method designs specific primers to diagnose homozygous mutations and normal controls. Results: After PCR amplification, homozygous mutations produce different fragments from normal controls. Conclusion: This study represents an important step towards the development of a novel protocol to diagnose β-thalassemia and other diseases that target numerous mutations.
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- 1. Bunn HF, Forger BG. Hemoglobin: Molecular, Genetic and Clinical Aspects, 1st ed. Philadelphia: WB Saunders; 1986.
- 2. Batebi A, Pourreza A, Esmailian R. Discrimination of betathalassemia minor and iron deficiency anemia by screening test for red blood cell indices. Turk J Med Sci 2012; 42: 27580.
- 3. Orkin SH, Kazazian HH Jr. The mutation and polymorphism of the human beta-globin gene and its surrounding DNA. Annu Rev Genet 1984; 18: 13171.
- 4. Antonarakis SE, Irkin SH, Cheng TC, Scott AF, Sexton JP, Trusko SP et al. β-Thalassemia in American Blacks: novel mutations in the TATA box and an acceptor splice site. Proc Natl Acad Sci USA 1984; 81: 11548.
- 5. Kazazian HH Jr, Orkin SH, Antonarakis SE, Sexton JP, Boehm CD, Goff SC et al. Molecular characterization of seven betathalassemia mutations in Asian Indians. EMBO J 1984; 3: 593 6.
- 6. Cheng TC, Orkin SH, Antonarakis SE, Potter MJ, Sexton JP, Markham AF et al. β-Thalassemia in Chinese: use of in vivo RNA analysis and oligonucleotide hybridization in systematic characterization of molecular defects. Proc Natl Acad Sci USA 1984; 81: 28215.
- 7. Chan V, Chan TK, Chebab FF, Todd D. Distribution of betathalassemia mutations in south China and their association with haplotypes. Am J Hum Genet 1987; 41: 67885.
- 8. Xu XM, Zhou YQ, Luo GX, Liao C, Zhou M, Chen PY et al. The prevalence and spectrum of alpha and beta thalassaemia in Guangdong Province: implications for the future health burden and population screening. J Clin Pathol 2004; 57: 51722.
- 9. Lin M, Zhu JJ, Wang Q, Xie LX, Lu M, Wang JL et al. Development and evaluation of a reverse dot blot assay for the simultaneous detection of common alpha and beta thalassemia in Chinese. Blood Cells Mol Dis 2012; 48: 8690.
- 10. Cao A, Galanello R. Beta-thalassemia. Genet Med 2010; 12: 6176.
- 11. Vrettou C, Traeger-Synodinos J, Tzetis M, Malamis G, Kanavakis E. Rapid screening of multiple beta-globin gene mutations by real-time PCR on the LightCycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes. Clin Chem 2003; 49: 76976.
- 12. Ye BC, Zhang Z, Lei Z. Molecular analysis of alpha/betathalassemia in a southern Chinese population. Genet Test 2007; 11: 7583.
- 13. Clark BE, Thein SL. Molecular diagnosis of haemoglobin disorders. Clin Lab Haematol 2004; 26: 15976.
- 14. Xiong F, Sun M, Zhang X, Cai R, Zhou Y, Lou J et al. Molecular epidemiological survey of haemoglobinopathies in the Guangxi Zhuang Autonomous Region of southern China. Clin Genet 2010; 78: 13948.
- 15. Xu XM, Zhou YQ, Luo GX, Liao C, Zhou M, Chen PY et al. The prevalence and spectrum of α- and β-thalassemia in Guangdong Province: implications for the future health burden and population screening. J Clin Pathol 2004; 57: 51722.