DEVELOPING OF MULTIPLEX PCR AND RFLP FOR RAPID GENOTYPING OF POLYMORPHIC HFE GENE

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BAKAYEV V.V.; JAZAYERI M.; ZALI MOHAMMAD REZA; ZBOROVSKY S.S.; IGNATIEV I.V.

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Journal: JOURNAL OF RESEARCH IN MEDICAL SCIENCES (JRMS) Year:2003 | Volume:7 | Issue:4 Page(s): 332-335

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Title

DEVELOPING OF MULTIPLEX PCR AND RFLP FOR RAPID GENOTYPING OF POLYMORPHIC HFE GENE

Author(s)

Keywords

HEMOCHROMATOSISQ4

HFE GENEQ4

POLYMORPHISMQ2

MULTUPLEX PCRQ3

RFLPQ3

Abstract

Introduction: Alterations in the HFE protein compromise its function and are likely to induce the development of hereditary HEMOCHROMATOSIS (HH). Although most HH subjects homozygous for a Cys282Tyr POLYMORPHISM the His63Asp variant of the HFE GENE accounts for a fraction of the HH. In this study, We have developed a new assay that combines a multiplex polymerase chain reaction (PCR) and restriction fragment length POLYMORPHISM (RFLP) analysis for genotyping suspected HH patients.Methods: The allelic variants of the HFE GENE in genomic DNA from 24 subjects were analysed for assay validation. Two pairs of the HFE GENE specific primers were pooled into a multiplex reaction whose products were subtyped by digestion with two restriction endonucleases. Employing optimized PCR buffer, primers ratio and thermocycling regimen prevented PCR products under representing, caused by preferential amplification of the shorter fragment under suboptimal conditions. Results: The selected conditions for multiplex PCR-RFLP allowed uniform, reproducible DNA amplification and restriction endonuclease digestion of both DNA targets within one tube per sample that minimized labor and cost requirements and probability of PCR product carry- over. The results were reproducibly concordant with the data obtained by standard PCR analysis proving that the proposed method is reliable and could be of value in genotypig application.Discussion: The assay is robust and readily adaptable to the clinical molecular diagnostic laboratory.

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APA: Copy

BAKAYEV, V.V., JAZAYERI, M., ZALI, MOHAMMAD REZA, ZBOROVSKY, S.S., & IGNATIEV, I.V.. (2003). DEVELOPING OF MULTIPLEX PCR AND RFLP FOR RAPID GENOTYPING OF POLYMORPHIC HFE GENE. JOURNAL OF RESEARCH IN MEDICAL SCIENCES (JRMS), 7(4), 332-335. SID. https://sid.ir/paper/26672/en

Vancouver: Copy

BAKAYEV V.V., JAZAYERI M., ZALI MOHAMMAD REZA, ZBOROVSKY S.S., IGNATIEV I.V.. DEVELOPING OF MULTIPLEX PCR AND RFLP FOR RAPID GENOTYPING OF POLYMORPHIC HFE GENE. JOURNAL OF RESEARCH IN MEDICAL SCIENCES (JRMS)[Internet]. 2003;7(4):332-335. Available from: https://sid.ir/paper/26672/en

IEEE: Copy

V.V. BAKAYEV, M. JAZAYERI, MOHAMMAD REZA ZALI, S.S. ZBOROVSKY, and I.V. IGNATIEV, “DEVELOPING OF MULTIPLEX PCR AND RFLP FOR RAPID GENOTYPING OF POLYMORPHIC HFE GENE,” JOURNAL OF RESEARCH IN MEDICAL SCIENCES (JRMS), vol. 7, no. 4, pp. 332–335, 2003, [Online]. Available: https://sid.ir/paper/26672/en

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