SYTO9 and SYBR GREEN1 with high resolution melting analysis for molecular confirmatory testing of the common Southeast Asian beta(0)-thalassemia mutations

Hemoglobin
Unthika ChamrasSakorn Pornprasert

Abstract

Gel electrophoresis and ethidium bromide staining are routine methods in molecular laboratories. However, they are not ideally suited to large scale analyses in clinical laboratories. We used SYTO9 and high resolution melting (HRM) analyses for identification of the common beta(0)-thalassemia (beta(0)-thal) in Southeast Asia including the codons 17 (A>T), 41/42 (-TCTT) and 71/72 (+A) mutations. Multiplex amplification refractory mutation system-polymerase chain reaction (MARMS-PCR) was performed on 102 blood samples that had Hb A(2) levels between 3.1 and 9.9%. The SYTO9 HRM analysis showed specific characteristic peaks of each investigated type of beta(0)-thal mutation. Results of MARMS-PCR followed by SYTO9 HRM analyses were completely consistent with results of those analyzed by gel electrophoresis. Moreover, the beta(0)-thal 3.5 kb gene deletion could be identified in two samples with Hb A(2) levels of >4% by GREEN1 HRM analysis. The SYTO9 and SYBR GREEN1 HRM analysis is simple and rapid. This approach will facilitate laboratory diagnosis and genetic counseling for regions with a high prevalence of beta-thal.

References

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Citations

Sep 22, 2010·Hemoglobin·Sakorn PornprasertKanyakan Sukunthamala
Jun 21, 2011·American Journal of Clinical Pathology·Sitthichai PanyasaiSakorn Pornprasert
Aug 21, 2010·European Journal of Haematology·Sakorn Pornprasert, Kanyakan Sukunthamala
Jul 10, 2016·Clinical Chemistry and Laboratory Medicine : CCLM·Pitchaporn RungseeSakorn Pornprasert

Related Concepts

SYBR Green I
Fluorochromes
RNA Denaturation
Organic Chemicals
Thalassemia Minor
Nucleic Acid Amplification Tests
Molecular Diagnostic Testing
Freezing Point Temperature
Laboratory Diagnosis
Gene Deletion

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