Abstract
The Haemoglobin Constant Spring (HbCS) mutation is one of the non-deletion variants with the highest frequency causing Alpha-Thalassaemia disease. High Resolution Melting analysis was developed to detect HbCS mutation on HbA2 gene effectively to decrease the alpha-thalassaemia hereditary for offsprings. Two healthy non-pregnant DNA samples and seven HbCS positive DNA samples was collected and detected by a real-time PCR SYRBGreen assay. The Realtime PCR assay was optimized based on the concentration of PCR reagents like DNA template, DMSO, Mg2+. The DNA fragment containing alleles of the mutation is amplified from the assay and will be analyzed by melting curve. According to the normalized melting curve and the temperature shifted different plot, the difference in melting curve shapes present and the genotype of each individual which is related to the disease will be indicated clearly. The HRM method was optimized and developed successfully. The condition with 5% of DMSO and 2.5 mM of MgCl2 is the best PCR condition and gave out the best separated melting curve shape in discriminating three various genotypes. This study showed that the HRM analysis is a reliable, incomplex tool and effective cost method for identifying HbCS variant in alpha globin genes and could be applied in clinical diagnostics.
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References
David Weatheral J, Clegg JL (2001) The Thalassaemia Syndromes, 4th. Wiley-Blackwell, Oxford
Derry S et al (1994) Hematologic and biosynthetic studies in homozygous hemoglobin Constant Spring. J Clin Invest 73(6):1673–1682
Fucharoen S, Winichagoon P (2007) Prevention and control of thalassemia in Asia. Asian Biomed 1(1):1–6
Gundry CN et al (2003) Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem 49(3):396–406
Harteveld CL, Higgs DR (2010) Alpha-Thalassaemia. Orphanet J Rare Dis 5:13
Hà LTT, Ngọc ND et al (2010) Ứng dụng kỹ thuật sinh hoc phân tử trong chẩn đoán trước và sau sinh bệnh Alpha Thalassemia tại Bệnh Nhi Trung Ương. Nhi Khoa 3:337–342
Kazazian HH Jr (1990) The thalassemia syndromes: molecular basis and prenatal diagnosis in 1990. Semin Hematol 27(3):209–228
Liew M et al (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50(7):1156–1164
Norambuena PA et al (2009) Diagnostic method validation: high resolution melting (HRM) of small amplicons genotyping for the most common variants in the MTHFR gene. Clin Biochem 42(12):1308–1316
Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8(6):597–608
Roche (2008). LightCycler 480 Gene Scanning Software. Roche Diagn GmbH Roche Appl Sci
Tan AS et al (2001) A rapid and reliable 7-deletion multiplex polymerase chain reaction assay for alpha-thalassemia. Blood 98(1):250–251
Waye JS, Chui DH (2001) The alpha-globin gene cluster: genetics and disorders. Clin Invest Med 24(2):103–109
Wee YC et al (2009) Molecular characterisation of Haemoglobin Constant Spring and Haemoglobin Quong Sze with a Combine-Amplification Refractory Mutation System. Malays J Med Sci 16(3):21–28
Wittwer CT et al (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49(6):853–860
Zhou L et al (2005) High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem 51(10):1770–1777
Acknowledgements
The authors thank to Dr Nguyen Khac Han Hoan, and staffs of Genetics Department, Tu Du Hospital for supporting in sample collecting. Thank to patients who agree to join the study. Thanks to OUCRU staffs for supporting in providing suitable facility for this study.
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Tran, N.B., Nguyen, H.T. (2018). Method for Identifying HbCS Variant in Alpha Globin Genes Causing Alpha-Thalassaemia Disease. In: Vo Van, T., Nguyen Le, T., Nguyen Duc, T. (eds) 6th International Conference on the Development of Biomedical Engineering in Vietnam (BME6) . BME 2017. IFMBE Proceedings, vol 63. Springer, Singapore. https://doi.org/10.1007/978-981-10-4361-1_30
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DOI: https://doi.org/10.1007/978-981-10-4361-1_30
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